# Surface temperatures are influenced by handling stress independently of corticosterone levels in wild king penguins (Aptenodytes patagonicus)

Agnès Lewden, Chelsea Ward, Aude Noiret, Sandra Avril, Lucie Abolivier, Caroline Gérard, Tracey Hammer, Émilie Raymond, Jean-Patrice Robin, Vincent Viblanc, et al.

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Agnès Lewden, Chelsea Ward, Aude Noiret, Sandra Avril, Lucie Abolivier, et al. Surface temperatures are influenced by handling stress independently of corticosterone levels in wild king penguins (Aptenodytes patagonicus). Journal of Thermal Biology, 2024, 121, pp.103850. 10.1016/j.jtherbio.2024.103850. hal-04612230

HAL Id: hal-04612230

https://hal.univ-brest.fr/hal-04612230

Submitted on 14 Jun 2024

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

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<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Surface temperatures are influenced by handling stress independently of corticosterone levels in wild king penguins (Aptenodytes patagonicus)

Agnès Lewden, Chelsea Ward, Aude Noiret, Sandra Avril, Lucie Abolivier, Caroline Gérard, Tracey Hammer, Émilie Raymond, Jean-Patrice Robin, Vincent Viblanc, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Agnès Lewden, Chelsea Ward, Aude Noiret, Sandra Avril, Lucie Abolivier, et al. Surface temperatures are influenced by handling stress independently of corticosterone levels in wild king penguins (Aptenodytes patagonicus). Journal of Thermal Biology, 2024, 121, pp.103850. 10.1016/j.jtherbio.2024.103850. hal-04612230

HAL Id: hal-04612230

https://hal.univ-brest.fr/hal-04612230v1

Submitted on 14 Jun 2024

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

# Surface temperatures are influenced by handling stress independently of corticosterone levels in wild king penguins (*Aptenodytes patagonicus*)

Agnès Lewden<sup>a, b, **</sup>, Chelsea Ward<sup>c</sup>, Aude Noiret<sup>d</sup>, Sandra Avril<sup>e</sup>, Lucie Abolivier<sup>e</sup>, Caroline Gérard<sup>e</sup>, Tracey L. Hammer<sup>e</sup>, Émilie Raymond<sup>d, e</sup>, Jean-Patrice Robin<sup>e</sup>, Vincent A. Viblanc<sup>e</sup>, Pierre Bize<sup>c, f</sup>, Antoine Stier<sup>d, e, g, *</sup>

<figure><img src="image_4.png" /></figure>

## ARTICLE INFO

**Keywords:**

Thermal imaging

Stress

Corticosterone

Heterothermy

Bird

Beak

Eye

## ABSTRACT

Assessing the physiological stress responses of wild animals opens a window for understanding how organisms cope with environmental challenges. Since stress response is associated with changes in body temperature, the use of body surface temperature through thermal imaging could help to measure acute and chronic stress responses non-invasively. We used thermal imaging, acute handling-stress protocol and an experimental manipulation of corticosterone (the main glucocorticoid hormone in birds) levels in breeding king penguins (*Aptenodytes patagonicus*), to assess: 1. The potential contribution of the Hypothalamo-Pituitary-Adrenal (HPA) axis in mediating chronic and acute stress-induced changes in adult surface temperature, 2. The influence of HPA axis manipulation on parental investment through thermal imaging of eggs and brooded chicks, and 3. The impact of parental treatment on offspring thermal's response to acute handling.

Maximum eye temperature (*T*<sub>eye</sub>) increased and minimum beak temperature (*T*<sub>beak</sub>) decreased in response to handling stress in adults, but neither basal nor stress-induced surface temperatures were significantly affected by corticosterone implant. While egg temperature was not significantly influenced by parental treatment, we found a surprising pattern for chicks: chicks brooded by the (non-implanted) partner of corticosterone-implanted individuals exhibited higher surface temperature (both *T*<sub>eye</sub> and *T*<sub>beak</sub>) than those brooded by glucocorticoid-implanted or control parents. Chick's response to handling in terms of surface temperature was characterized by a drop in both *T*<sub>eye</sub> and *T*<sub>beak</sub> independently of parental treatment.

We conclude that the HPA axis seems unlikely to play a major role in determining chronic or acute changes in surface temperature in king penguins. Changes in surface temperature may primarily be mediated by the Sympathetic-Adrenal-Medullary (SAM) axis in response to stressful situations. Our experiment did not reveal a direct impact of parental HPA axis manipulation on parental investment (egg or chick temperature), but a potential influence on the partner's brooding behaviour.

## 1. Introduction

Measuring stress response is of central interest in wild animals to understand how they cope with environmental change (Ellenberg et al., 2007; Romero, 2004). Traditionally, ecologists have evaluated acute

stress by assessing the activation of the Sympathetic-Adrenal-Medullary (SAM) or Hypothalamic-Pituitary-Adrenal (HPA) axes, either at baseline levels or in response to acute disturbances in the environment, by metrics such as increased heart rate (Cabanac and Guillemette, 2001; Viblanc et al., 2012), ventilation rate (Carere and Oers, 2004) or glucocorticoid hormone (GC) secretion (Sapolsky et al., 2000). Stress

<footnote><sup>a</sup> Faculty of Biological Sciences, University of Leeds, Leeds, UK</footnote>

<footnote><sup>b</sup> Université de Brest - UMR 6539 CNRS/UBO/IRD/Ifremer, Laboratoire des Sciences de l'environnement Marin - IUEM, Rue Dumont D'Urville, 29280, Plouzané, France</footnote>

<footnote><sup>c</sup> School of Biological Sciences, University of Aberdeen, Aberdeen, UK</footnote>

<footnote><sup>d</sup> Univ Lyon, Université Claude Bernard Lyon 1, CNRS, ENTPE, UMR 5023 LEHNA, F-69622, Villeurbanne, France</footnote>

<footnote><sup>e</sup> Université de Strasbourg, CNRS, IPHC UMR 7178, F-67000, Strasbourg, France</footnote>

<footnote><sup>f</sup> Swiss Ornithological Institute, CH-6204, Sempach, Switzerland</footnote>

<footnote><sup>g</sup> Department of Biology, University of Turku, Turku, Finland</footnote>

<footnote>* Corresponding author. Université de Strasbourg, CNRS, IPHC UMR 7178, F-67000, Strasbourg, France</footnote>

<footnote>** Corresponding author. Faculty of Biological Sciences, University of Leeds, Leeds, UK.</footnote>

<footnote>E-mail addresses: agnes.lewden@univ-brest.fr (A. Lewden), amstie@utu.fi (A. Stier).</footnote>
<figure><img src="image_3.png" /></figure>

# Surface temperatures are influenced by handling stress independently of corticosterone levels in wild king penguins (*Aptenodytes patagonicus*)

Agnès Lewden<sup>a, b, **</sup>, Chelsea Ward<sup>c</sup>, Aude Noiret<sup>d</sup>, Sandra Avril<sup>e</sup>, Lucie Abolivier<sup>e</sup>, Caroline Gérard<sup>e</sup>, Tracey L. Hammer<sup>e</sup>, Émilie Raymond<sup>d, e</sup>, Jean-Patrice Robin<sup>e</sup>, Vincent A. Viblanc<sup>e</sup>, Pierre Bize<sup>c, f</sup>, Antoine Stier<sup>d, e, g, *</sup>

<figure><img src="image_4.png" /></figure>

## ARTICLE INFO

**Keywords:**

Thermal imaging

Stress

Corticosterone

Heterothermy

Bird

Beak

Eye

## ABSTRACT

Assessing the physiological stress responses of wild animals opens a window for understanding how organisms cope with environmental challenges. Since stress response is associated with changes in body temperature, the use of body surface temperature through thermal imaging could help to measure acute and chronic stress responses non-invasively. We used thermal imaging, acute handling-stress protocol and an experimental manipulation of corticosterone (the main glucocorticoid hormone in birds) levels in breeding king penguins (*Aptenodytes patagonicus*), to assess: 1. The potential contribution of the Hypothalamo-Pituitary-Adrenal (HPA) axis in mediating chronic and acute stress-induced changes in adult surface temperature, 2. The influence of HPA axis manipulation on parental investment through thermal imaging of eggs and brooded chicks, and 3. The impact of parental treatment on offspring thermal's response to acute handling.

Maximum eye temperature (*T*<sub>eye</sub>) increased and minimum beak temperature (*T*<sub>beak</sub>) decreased in response to handling stress in adults, but neither basal nor stress-induced surface temperatures were significantly affected by corticosterone implant. While egg temperature was not significantly influenced by parental treatment, we found a surprising pattern for chicks: chicks brooded by the (non-implanted) partner of corticosterone-implanted individuals exhibited higher surface temperature (both *T*<sub>eye</sub> and *T*<sub>beak</sub>) than those brooded by glucocorticoid-implanted or control parents. Chick's response to handling in terms of surface temperature was characterized by a drop in both *T*<sub>eye</sub> and *T*<sub>beak</sub> independently of parental treatment.

We conclude that the HPA axis seems unlikely to play a major role in determining chronic or acute changes in surface temperature in king penguins. Changes in surface temperature may primarily be mediated by the Sympathetic-Adrenal-Medullary (SAM) axis in response to stressful situations. Our experiment did not reveal a direct impact of parental HPA axis manipulation on parental investment (egg or chick temperature), but a potential influence on the partner's brooding behaviour.

## 1. Introduction

Measuring stress response is of central interest in wild animals to understand how they cope with environmental change (Ellenberg et al., 2007; Romero, 2004). Traditionally, ecologists have evaluated acute

stress by assessing the activation of the Sympathetic-Adrenal-Medullary (SAM) or Hypothalamic-Pituitary-Adrenal (HPA) axes, either at baseline levels or in response to acute disturbances in the environment, by metrics such as increased heart rate (Cabanac and Guillemette, 2001; Viblanc et al., 2012), ventilation rate (Carere and Oers, 2004) or glucocorticoid hormone (GC) secretion (Sapolsky et al., 2000). Stress

<footnote><sup>a</sup> Faculty of Biological Sciences, University of Leeds, Leeds, UK</footnote>

<footnote><sup>b</sup> Université de Brest - UMR 6539 CNRS/UBO/IRD/Ifremer, Laboratoire des Sciences de l'environnement Marin - IUEM, Rue Dumont D'Urville, 29280, Plouzané, France</footnote>

<footnote><sup>c</sup> School of Biological Sciences, University of Aberdeen, Aberdeen, UK</footnote>

<footnote><sup>d</sup> Univ Lyon, Université Claude Bernard Lyon 1, CNRS, ENTPE, UMR 5023 LEHNA, F-69622, Villeurbanne, France</footnote>

<footnote><sup>e</sup> Université de Strasbourg, CNRS, IPHC UMR 7178, F-67000, Strasbourg, France</footnote>

<footnote><sup>f</sup> Swiss Ornithological Institute, CH-6204, Sempach, Switzerland</footnote>

<footnote><sup>g</sup> Department of Biology, University of Turku, Turku, Finland</footnote>

<footnote>* Corresponding author. Université de Strasbourg, CNRS, IPHC UMR 7178, F-67000, Strasbourg, France</footnote>

<footnote>** Corresponding author. Faculty of Biological Sciences, University of Leeds, Leeds, UK.</footnote>

<footnote>E-mail addresses: agnes.lewden@univ-brest.fr (A. Lewden), amstie@utu.fi (A. Stier).</footnote>
Abbreviations

Adrenocorticotropic hormone ACTH

Ambient air temperature $T_a$

Corticosterone CORT

Glucocorticoid hormones GC

Egg surface temperature $T_{egg}$

Hypothalamic-pituitary-adrenal HPA

Linear mixed models LMMs

Maximum eye region surface temperature $T_{eye}$

Minimum beak surface temperature $T_{beak}$

Sympathetic-adrenal-medullary SAM

exposure is also known to trigger core body and surface temperature changes (Oka, 2018). As the latter can be measured using thermal imaging (McCafferty, 2013), it led to a recent burgeoning of thermal imaging studies to measure the stress response of captive and wild endotherms (McCafferty et al., 2021). Both the SAM and HPA axes can potentially influence changes in body surface temperature, but the importance of these two pathways in mediating the stress response(s) measured through thermal imaging remains largely unknown (but see Jerem and Romero (2023)). Considering that both HPA and SAM axes provide strong and very reactive responses to stress, teasing apart their

contribution to changes in peripheral temperature is challenging.

SAM axis responds within seconds to a stressor by releasing catecholamines hormones (adrenaline and noradrenaline) into the blood stream, which induces an immediate increase in heart rate (tachycardia) and vasoconstriction of peripheral blood vessels (Wingfield and Romero, 2015). This redistribution of peripheral blood flow to essential internal organs generally leads to a response called stress-induced hyperthermia along with a decrease of surface temperature (Cabanac and Guillemette, 2001; Herborn et al., 2015; Oka, 2018). The SAM axis response is followed by the slower activation of HPA axis that releases GC into the blood stream within a few minutes (Wingfield and Romero, 2015). GC are metabolic hormones that helps responding to a stressor by mobilizing energy resources and by triggering an increase in behavioural activity and metabolic rate (Wingfield and Romero, 2015). Circulating GC levels are usually considered at baseline (i.e. without an inducing stressor) or at stress-induced levels, which are often interpreted as mirroring chronic stress/metabolic demand and acute stress response respectively. Yet, the actual interpretation of circulating GC levels is likely more complex (MacDougall-Shackleton et al., 2019). GC-mediated changes in metabolism may cause an increase in internal heat production that, in turn, may lead to changes in body surface temperature (Oka, 2018). Additionally, previous evidence suggests that GC might be necessary for enabling the SAM axis to induce shivering, free fatty acid mobilization or vasoconstriction (Deavers and Musacchia, 1979).

<caption>Table 1</caption>

Summary table of avian studies testing the impact of acute stress exposure on changes in body surface temperatures measured with thermal imaging (studies related to heat and food stress are not included). Area corresponds to the body region being measured, response to the direction of the temperature response (=: no significant change, ↓: significant decrease and ↑: significant increase, Δ T°C to the amplitude of the temperature response, stressor the nature and duration of the experimental stressor. Some studies measured temperature responses at different time points, and when the effects of stress exposure change through time, the responses are shown with consecutive symbols (e.g. =, ↑: initial absence of change followed later on by an increase in surface temperature). Please note that the area of interest for $T_{eye}$ differs between studies (periorbital region vs. eyeball region) and that the surface temperature being measured can differ in nature between studies (i.e. maximum, average or minimum temperature of the area of interest).

<table><thead><tr><th>Species</th><th>Condition</th><th>Age</th><th>Stressor</th><th>Area</th><th>Response</th><th>Δ T°C</th><th>Ref.</th></tr></thead><tbody><tr><td>Blue tit<br>Cyaniste caerulescens</td><td>Wild</td><td>Adult</td><td>Trap closure + handling<br>(3min)</td><td>Eye</td><td>↓, ↑, =</td><td>-1.0, +1.0 °C, =</td><td>Jerem et al. (2019)</td></tr><tr><td>Budgerigars<br>Melopsittacus undulatus</td><td>Captive</td><td>Adult</td><td>Handling<br>(30 min)</td><td>Eye<br>Legs</td><td>↑, =<br>=</td><td>+0.7 °C, =<br>=</td><td>Ikkatai and Watanabe (2015)</td></tr><tr><td>Chicken<br>Gallus domesticus</td><td>Captive</td><td>Adult</td><td>Air puff<br>(10 min)</td><td>Eye</td><td>↓</td><td>-1.0 °C</td><td>Edgar et al. (2011)</td></tr><tr><td>Chicken<br>Gallus domesticus</td><td>Captive</td><td>Adult</td><td>Handling<br>(20 min)</td><td>Eye<br>Head<br>Comb</td><td>↓, =<br>=, ↑<br>↓, =</td><td>-0.5 °C, =<br>=, +1.0 °C<br>-1.5 °C, =</td><td>Edgar et al. (2013)</td></tr><tr><td>Chicken<br>Gallus domesticus</td><td>Captive</td><td>Chick<br>(9 weeks)</td><td>Air puff<br>(10 min)</td><td>Eye</td><td>↓</td><td>-1.0 °C</td><td>Edgar and Nicol (2018)</td></tr><tr><td>Chicken<br>Gallus domesticus</td><td>Captive</td><td>Adult</td><td>Handling: mild/severe<br>(20 min)</td><td>Eye<br>Face<br>Wattle<br>Comb</td><td>↓<br>=<br>↓<br>↓</td><td>-0.4 °C<br>=<br>-0.7/-1.3 °C<br>-0.5/-2.2 °C</td><td>Herborn et al. (2015)</td></tr><tr><td>Chicken<br>Gallus domesticus</td><td>Captive</td><td>Adult</td><td>Enrichment removal<br>(within 2 h)</td><td>Eye<br>Face<br>Comb</td><td>↓<br>↓<br>↓</td><td>-0.8 °C<br>-0.7 °C<br>-3.5 °C</td><td>Herborn et al. (2018)</td></tr><tr><td>Chicken<br>Gallus domesticus</td><td>Captive</td><td>Chick<br>(30 days)</td><td>Handling<br>(10 min)</td><td>Head<br>Feet</td><td>↑<br>↓</td><td>+0.76 °C<br>-0.45 °C</td><td>Moe et al. (2017)</td></tr><tr><td>Chicken<br>Gallus domesticus</td><td>Captive</td><td>Chick<br>(9 days)</td><td>Visual<br>(45 min)</td><td>Eye<br>Beak</td><td>=<br>=, ↑</td><td>+0.5 °C<br>-0.4 °C</td><td>Pijpers et al. (2022)</td></tr><tr><td>Domestic pigeon<br>Columbia livia domestica</td><td>Captive</td><td>Adult</td><td>Handling<br>(3.5 min)</td><td>Beak</td><td>↓</td><td>-2.1 °C</td><td>Tabh et al. (2021)</td></tr><tr><td>House sparrow<br>Passer domesticus</td><td>Captive</td><td>Adult</td><td>Handling and injection<br>(~1min)</td><td>Eye<br>Beak</td><td>↓<br>↓</td><td>-1.0 °C<br>-4.0 °C</td><td>Jerem and Romero (2023)</td></tr><tr><td>King penguin<br>Aptenodytes patagonicus</td><td>Wild</td><td>Adult</td><td>Handling<br>(5-10 min)</td><td>Eye<br>Beak</td><td>↑<br>↓</td><td>+0.8 °C<br>-1.5 °C</td><td>This study</td></tr><tr><td></td><td>Wild</td><td>Chick<br>(20 days*)</td><td>Handling<br>(5-10 min)</td><td>Eye<br>Beak</td><td>↓<br>↓</td><td>-0.9 °C<br>-3.3 °C</td><td></td></tr><tr><td>Little Auk Alle<br>Pheasant<br>Phasianus colchicus</td><td>Wild</td><td>Adult</td><td>Handling<br>(30 min)</td><td>Eye<br>Aggression</td><td>↑<br>↓</td><td>+1.7 °C<br>+0.6 °C</td><td>Jakubas et al. (2022)<br>Knoch et al. (2022)</td></tr><tr><td>Song sparrow<br>Melospiza melodia</td><td>Captive</td><td>Adult</td><td>Handling<br>(13 min)</td><td>Eye<br>Beak</td><td>↓, =<br>↓, ↑, =</td><td>-1.5 °C, =<br>-2.6 °C, +2.6 °C, =</td><td>Zuluaga and Danner (2023)</td></tr></tbody><tfoot><tr><td colspan="8">* king penguins are not thermally emancipated at this age</td></tr></tfoot></table>
Abbreviations

Adrenocorticotropic hormone ACTH

Ambient air temperature $T_a$

Corticosterone CORT

Glucocorticoid hormones GC

Egg surface temperature $T_{egg}$

Hypothalamic-pituitary-adrenal HPA

Linear mixed models LMMs

Maximum eye region surface temperature $T_{eye}$

Minimum beak surface temperature $T_{beak}$

Sympathetic-adrenal-medullary SAM

exposure is also known to trigger core body and surface temperature changes (Oka, 2018). As the latter can be measured using thermal imaging (McCafferty, 2013), it led to a recent burgeoning of thermal imaging studies to measure the stress response of captive and wild endotherms (McCafferty et al., 2021). Both the SAM and HPA axes can potentially influence changes in body surface temperature, but the importance of these two pathways in mediating the stress response(s) measured through thermal imaging remains largely unknown (but see Jerem and Romero (2023)). Considering that both HPA and SAM axes provide strong and very reactive responses to stress, teasing apart their

contribution to changes in peripheral temperature is challenging.

SAM axis responds within seconds to a stressor by releasing catecholamines hormones (adrenaline and noradrenaline) into the blood stream, which induces an immediate increase in heart rate (tachycardia) and vasoconstriction of peripheral blood vessels (Wingfield and Romero, 2015). This redistribution of peripheral blood flow to essential internal organs generally leads to a response called stress-induced hyperthermia along with a decrease of surface temperature (Cabanac and Guillemette, 2001; Herborn et al., 2015; Oka, 2018). The SAM axis response is followed by the slower activation of HPA axis that releases GC into the blood stream within a few minutes (Wingfield and Romero, 2015). GC are metabolic hormones that helps responding to a stressor by mobilizing energy resources and by triggering an increase in behavioural activity and metabolic rate (Wingfield and Romero, 2015). Circulating GC levels are usually considered at baseline (i.e. without an inducing stressor) or at stress-induced levels, which are often interpreted as mirroring chronic stress/metabolic demand and acute stress response respectively. Yet, the actual interpretation of circulating GC levels is likely more complex (MacDougall-Shackleton et al., 2019). GC-mediated changes in metabolism may cause an increase in internal heat production that, in turn, may lead to changes in body surface temperature (Oka, 2018). Additionally, previous evidence suggests that GC might be necessary for enabling the SAM axis to induce shivering, free fatty acid mobilization or vasoconstriction (Deavers and Musacchia, 1979).

<caption>**Table 1**</caption>

**Summary table of avian studies testing the impact of acute stress exposure on changes in body surface temperatures measured with thermal imaging**
(studies related to heat and food stress are not included). Area corresponds to the body region being measured, response to the direction of the temperature response (=: no significant change, ↓: significant decrease and ↑: significant increase, Δ T°C to the amplitude of the temperature response, stressor the nature and duration of the experimental stressor. Some studies measured temperature responses at different time points, and when the effects of stress exposure change through time, the responses are shown with consecutive symbols (e.g. =, ↑: initial absence of change followed later on by an increase in surface temperature). Please note that the area of interest for $T_{eye}$ differs between studies (periorbital region vs. eyeball region) and that the surface temperature being measured can differ in nature between studies (i.e. maximum, average or minimum temperature of the area of interest).

<table><thead><tr><th>Species</th><th>Condition</th><th>Age</th><th>Stressor</th><th>Area</th><th>Response</th><th>Δ T°C</th><th>Ref.</th></tr></thead><tbody><tr><td>Blue tit<br><i>Cyaniste caerulescens</i></td><td>Wild</td><td>Adult</td><td>Trap closure + handling<br>(3min)</td><td>Eye</td><td>↓, ↑, =</td><td>-1.0, +1.0 °C, =</td><td>Jerem et al. (2019)</td></tr><tr><td>Budgerigars<br><i>Melopsittacus undulatus</i></td><td>Captive</td><td>Adult</td><td>Handling<br>(30 min)</td><td>Eye</td><td>↑, =</td><td>+0.7 °C, =</td><td>Ikkatai and Watanabe (2015)</td></tr><tr><td>Chicken<br><i>Gallus domesticus</i></td><td>Captive</td><td>Adult</td><td>Air puff<br>(10 min)</td><td>Eye</td><td>↓</td><td>-1.0 °C</td><td>Edgar et al. (2011)</td></tr><tr><td>Chicken<br><i>Gallus domesticus</i></td><td>Captive</td><td>Adult</td><td>Handling<br>(20 min)</td><td>Eye</td><td>↓, =</td><td>-0.5 °C, =</td><td>Edgar et al. (2013)</td></tr><tr><td>Chicken<br><i>Gallus domesticus</i></td><td>Captive</td><td>Chick<br>(9 weeks)</td><td>Air puff<br>(10 min)</td><td>Head</td><td>=, ↑</td><td>=, +1.0 °C</td><td></td></tr><tr><td>Chicken<br><i>Gallus domesticus</i></td><td>Captive</td><td>Adult</td><td>Handling: mild/severe<br>(20 min)</td><td>Comb</td><td>↓, =</td><td>-1.5 °C, =</td><td>Edgar and Nicol (2018)</td></tr><tr><td>Chicken<br><i>Gallus domesticus</i></td><td>Captive</td><td>Adult</td><td>Enrichment removal<br>(within 2 h)</td><td>Eye</td><td>↓</td><td>-0.4 °C</td><td>Herborn et al. (2015)</td></tr><tr><td>Chicken<br><i>Gallus domesticus</i></td><td>Captive</td><td>Chick<br>(30 days)</td><td>Handling<br>(10 min)</td><td>Face</td><td>=</td><td>=</td><td></td></tr><tr><td>Chicken<br><i>Gallus domesticus</i></td><td>Captive</td><td>Chick<br>(9 days)</td><td>Visual<br>(45 min)</td><td>Wattle</td><td>↓</td><td>-0.7/-1.3 °C</td><td></td></tr><tr><td>Domestic pigeon<br><i>Columbia livia domestica</i></td><td>Captive</td><td>Adult</td><td>Handling<br>(3.5 min)</td><td>Comb</td><td>↓</td><td>-0.5/-2.2 °C</td><td></td></tr><tr><td>House sparrow<br><i>Passer domesticus</i></td><td>Captive</td><td>Adult</td><td>Handling and injection<br>(~1min)</td><td>Eye</td><td>↓</td><td>-0.8 °C</td><td>Herborn et al. (2018)</td></tr><tr><td>King penguin<br><i>Aptenodytes patagonicus</i></td><td>Wild</td><td>Adult</td><td>Handling<br>(5-10 min)</td><td>Head</td><td>↑</td><td>+0.76 °C</td><td>Moe et al. (2017)</td></tr><tr><td></td><td>Wild</td><td>Chick<br>(20 days*)</td><td>Handling<br>(5-10 min)</td><td>Feet</td><td>↓</td><td>-0.45 °C</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>Beak</td><td>=, ↑</td><td>+0.5 °C</td><td>Pijpers et al. (2022)</td></tr><tr><td></td><td></td><td></td><td></td><td>Eye</td><td>=</td><td>-0.4 °C</td><td>Tabh et al. (2021)</td></tr><tr><td></td><td></td><td></td><td></td><td>Beak</td><td>↓</td><td>-2.1 °C</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>Eye</td><td>↓</td><td>-1.0 °C</td><td>Jerem and Romero (2023)</td></tr><tr><td></td><td></td><td></td><td></td><td>Beak</td><td>↓</td><td>-4.0 °C</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>Eye</td><td>↑</td><td>+0.8 °C</td><td>This study</td></tr><tr><td></td><td></td><td></td><td></td><td>Beak</td><td>↓</td><td>-1.5 °C</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>Eye</td><td>↓</td><td>-0.9 °C</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td>Beak</td><td>↓</td><td>-3.3 °C</td><td></td></tr><tr><td>Little Auk<br><i>Alle</i></td><td>Wild</td><td>Adult</td><td>Handling (30 min)</td><td>Eye</td><td>↑</td><td>+1.7 °C</td><td>Jakubas et al. (2022)</td></tr><tr><td>Pheasant<br><i>Phasianus colchicus</i></td><td>Captive</td><td>Juvenile<br>(42-49 days)</td><td>Aggression<br>(20 s)</td><td>Head</td><td>↑</td><td>+0.6 °C</td><td>Knoch et al. (2022)</td></tr><tr><td>Song sparrow<br><i>Melospiza melodia</i></td><td>Captive</td><td>Adult</td><td>Handling<br>(13 min)</td><td>Eye</td><td>↓, =</td><td>-1.5 °C, =</td><td>Zuluaga and Danner (2023)</td></tr><tr><td></td><td></td><td></td><td></td><td>Beak</td><td>↓, ↑, =</td><td>-2.6 °C, +2.6 °C, =</td><td></td></tr></tbody><tfoot><tr><td colspan="8">* king penguins are not thermally emancipated at this age</td></tr></tfoot></table>
Therefore, both the SAM and HPA axes could trigger, independently from each other, or together, a change in body core and/or surface temperatures (Ouyang et al., 2021). The immediate activation of SAM axis could lead to a decrease of peripheral temperature independently of GC release by the HPA axis. On the other hand, the short to long-term elevation of baseline GC might lead to increased metabolic rate, heat production and, ultimately, higher peripheral temperature (to facilitate heat dissipation and maintain homeothermy) independently of the SAM axis. The use of thermal imaging as a non-invasive tool to measure stress in unmanipulated animals is actively developing, but the implication of the SAM and HPA axes in mediating changes in surface temperature remains uncertain (Jerem and Romero, 2023; Ouyang et al., 2021). For instance, data in captive and wild birds show that acute stress exposure often leads to a decrease in body surface temperature (reviewed in Table 1, but note some discrepancies between species and/or body parts), as predicted if these changes are driven by the SAM axis. However, evidence for an impact of chronic stressors on body surface temperature (Herborn et al., 2018; Robertson et al., 2020a, 2020b; Winder et al., 2020) or for a correlation between circulating GC levels and body surface temperature (Giloh et al., 2012; Jerem et al., 2018; Weimer et al., 2020) remains unclear.

In this study, we took up the challenge of distinguishing the role of HPA and SAM axes on changes in body surface temperatures using an experimental approach in adult king penguins (*Aptenodytes patagonicus*). Breeding king penguins were subcutaneously implanted with either a corticosterone (CORT, the main GC hormone in avian species) implant or a placebo implant. The use of CORT implants in king penguin (see methods and Fig. 1), as in many other bird species (see Torres-Medina et al., 2018), leads to higher baseline CORT levels, while inhibiting the acute CORT response in the days following implantation. Indeed, CORT-implanted birds no longer exhibit high blood CORT release in response to acute stress induced by a handling stress protocol because of the HPA negative feedback loop (Torres-Medina et al., 2018). Specifically, high circulating CORT levels are inhibiting the activity of the paraventricular nucleus in the hypothalamus through binding with glucocorticoid receptors. This inhibits the release of corticotrophin-releasing hormone and the afferent HPA cascade through

the pituitary (secreting the adrenocorticotropic hormone, ACTH) and adrenal (secreting CORT) glands (Smulders, 2021). Hence, the use of CORT implants in conjunction with handling stress allows for the separation of the contributions of the HPA and SAM axes to acute changes in body surface temperatures. Here, we therefore measured the effects of our CORT treatment, together with a handling stress protocol, on surface temperatures in the king penguin. If changes in surface temperatures are mainly driven by activating the HPA axis, we expect to find higher initial body surface temperatures in CORT-implanted individuals (with elevated baseline CORT) prior to capture and handling. However, because CORT implants shut-down the release of CORT in response to handling, we expect to find no change in body surface temperature in response to handling stress in CORT individuals if such changes are mediated by the HPA axis only. On the contrary, if changes in surface temperatures are mainly driven by the activity of the SAM axis, we expect our CORT treatment to have no effect on body surface temperatures (both before or after capture and handling).

In addition to direct effects on adults, parental stress exposure or increased CORT levels may also affect the following generation, for instance by affecting parental care (Thierry et al., 2013a) or by conveying information about a stressful environment (Brandl et al., 2022). The latter idea has been framed as the concept of family-transmitted stress by Noguera et al. (2017). Therefore, we also aimed to investigate whether thermal imaging could be used to quantify indirect effects of stress on king penguin's progeny. To this end, we measured egg surface temperature on the same day as the body surface temperatures of implanted parents, and the surface temperatures of their chicks. Based on experimental data in Adélie penguin (*Pygoscelis adeliae*) showing that elevated baseline CORT levels in adults led to lower parental care (Thierry et al., 2013a), we hypothesized that increasing CORT levels may lower the surface temperatures of eggs and/or chicks during incubation and brooding respectively, as parents may pay less attention in keeping the egg/chick covered and warm under their brood pouch.

## 2. Material and methods

### 2.1. Study site and species

The king penguin is a heterothermic bird whose body temperature largely varies between core and peripheral tissues (Lewden et al., 2017a, 2017b). Its thick body plumage provides a very efficient insulation, while counter-current vascular heat exchangers in the appendages allows for a decrease in peripheral temperature loss (Thomas et al., 2011). These adaptations allow birds to conserve heat when foraging in cold Antarctic waters (Handrich et al., 1997), and to rapidly recover to normothermia when exiting the water (Lewden et al., 2020).

The study was conducted in 2018–2019 in a king penguin colony of *ca.* 22,000 breeding birds at ‘La Baie du Marin’ on Possession Island (Barbraud et al., 2020), Crozet Archipelago, in the Southern Indian Ocean (46°26′ S, 51°52′ E). We followed 49 breeding pairs from courtship (early November) until the onset of the Austral winter (April). All adults were identified by a hair dye mark on the breast feathers. During the early breeding season, male and female alternate between periods on-land caring for their single-egg or chick, and periods foraging at sea (Weimerskirch et al., 1992). The male takes the first incubation shift while the female forages at sea. The female returns ~15 days later to relieve her partner, and the parents continue to alternate shifts throughout incubation (~53 days) and early chick brooding. Chicks become thermally emancipated from the parents around one month of age, allowing both parents to go back at sea simultaneously to forage (Weimerskirch et al., 1992). Twenty days after hatching, king penguin chicks are able to maintain their core body temperature under mild environmental conditions (>10 °C) but still rely on their parent’s brooding behaviour (Duchamp et al., 2002), and are able to express a HPA-dependent stress response (X et al. unpublished).

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Baseline and stress-induced plasma corticosterone levels of breeding king penguins implanted either with a Placebo, or a CORT subcutaneous implant (50 mg, 90 days release; n = 80 observations, N = 40 individuals) during incubation. Birds were blood sampled 6 days after implantation. CORT (red) vs. Placebo (blue) individual responses (raw data) are presented along with group and time specific mean ± SE. CORT levels were influenced by the interaction between handling stress (baseline: <4 min vs. stress-induced: 30 min of handling stress) and CORT treatment (LMM on log-transformed plasma CORT: $F_{1,37.2} = 62.4, p < 0.001$). Letters indicate significant differences according to post-hoc tests with Tukey adjustments ($p < 0.030$).</figcaption></figure>
Therefore, both the SAM and HPA axes could trigger, independently from each other, or together, a change in body core and/or surface temperatures (Ouyang et al., 2021). The immediate activation of SAM axis could lead to a decrease of peripheral temperature independently of GC release by the HPA axis. On the other hand, the short to long-term elevation of baseline GC might lead to increased metabolic rate, heat production and, ultimately, higher peripheral temperature (to facilitate heat dissipation and maintain homeothermy) independently of the SAM axis. The use of thermal imaging as a non-invasive tool to measure stress in unmanipulated animals is actively developing, but the implication of the SAM and HPA axes in mediating changes in surface temperature remains uncertain (Jerem and Romero, 2023; Ouyang et al., 2021). For instance, data in captive and wild birds show that acute stress exposure often leads to a decrease in body surface temperature (reviewed in Table 1, but note some discrepancies between species and/or body parts), as predicted if these changes are driven by the SAM axis. However, evidence for an impact of chronic stressors on body surface temperature (Herborn et al., 2018; Robertson et al., 2020a, 2020b; Winder et al., 2020) or for a correlation between circulating GC levels and body surface temperature (Giloh et al., 2012; Jerem et al., 2018; Weimer et al., 2020) remains unclear.

In this study, we took up the challenge of distinguishing the role of HPA and SAM axes on changes in body surface temperatures using an experimental approach in adult king penguins (*Aptenodytes patagonicus*). Breeding king penguins were subcutaneously implanted with either a corticosterone (CORT, the main GC hormone in avian species) implant or a placebo implant. The use of CORT implants in king penguin (see methods and Fig. 1), as in many other bird species (see Torres-Medina et al., 2018), leads to higher baseline CORT levels, while inhibiting the acute CORT response in the days following implantation. Indeed, CORT-implanted birds no longer exhibit high blood CORT release in response to acute stress induced by a handling stress protocol because of the HPA negative feedback loop (Torres-Medina et al., 2018). Specifically, high circulating CORT levels are inhibiting the activity of the paraventricular nucleus in the hypothalamus through binding with glucocorticoid receptors. This inhibits the release of corticotrophin-releasing hormone and the afferent HPA cascade through

the pituitary (secreting the adrenocorticotropic hormone, ACTH) and adrenal (secreting CORT) glands (Smulders, 2021). Hence, the use of CORT implants in conjunction with handling stress allows for the separation of the contributions of the HPA and SAM axes to acute changes in body surface temperatures. Here, we therefore measured the effects of our CORT treatment, together with a handling stress protocol, on surface temperatures in the king penguin. If changes in surface temperatures are mainly driven by activating the HPA axis, we expect to find higher initial body surface temperatures in CORT-implanted individuals (with elevated baseline CORT) prior to capture and handling. However, because CORT implants shut-down the release of CORT in response to handling, we expect to find no change in body surface temperature in response to handling stress in CORT individuals if such changes are mediated by the HPA axis only. On the contrary, if changes in surface temperatures are mainly driven by the activity of the SAM axis, we expect our CORT treatment to have no effect on body surface temperatures (both before or after capture and handling).

In addition to direct effects on adults, parental stress exposure or increased CORT levels may also affect the following generation, for instance by affecting parental care (Thierry et al., 2013a) or by conveying information about a stressful environment (Brandl et al., 2022). The latter idea has been framed as the concept of family-transmitted stress by Noguera et al. (2017). Therefore, we also aimed to investigate whether thermal imaging could be used to quantify indirect effects of stress on king penguin's progeny. To this end, we measured egg surface temperature on the same day as the body surface temperatures of implanted parents, and the surface temperatures of their chicks. Based on experimental data in Adélie penguin (*Pygoscelis adeliae*) showing that elevated baseline CORT levels in adults led to lower parental care (Thierry et al., 2013a), we hypothesized that increasing CORT levels may lower the surface temperatures of eggs and/or chicks during incubation and brooding respectively, as parents may pay less attention in keeping the egg/chick covered and warm under their brood pouch.

## 2. Material and methods

### 2.1. Study site and species

The king penguin is a heterothermic bird whose body temperature largely varies between core and peripheral tissues (Lewden et al., 2017a, 2017b). Its thick body plumage provides a very efficient insulation, while counter-current vascular heat exchangers in the appendages allows for a decrease in peripheral temperature loss (Thomas et al., 2011). These adaptations allow birds to conserve heat when foraging in cold Antarctic waters (Handrich et al., 1997), and to rapidly recover to normothermia when exiting the water (Lewden et al., 2020).

The study was conducted in 2018–2019 in a king penguin colony of *ca.* 22,000 breeding birds at ‘La Baie du Marin’ on Possession Island (Barbraud et al., 2020), Crozet Archipelago, in the Southern Indian Ocean (46°26′ S, 51°52′ E). We followed 49 breeding pairs from courtship (early November) until the onset of the Austral winter (April). All adults were identified by a hair dye mark on the breast feathers. During the early breeding season, male and female alternate between periods on-land caring for their single-egg or chick, and periods foraging at sea (Weimerskirch et al., 1992). The male takes the first incubation shift while the female forages at sea. The female returns ~15 days later to relieve her partner, and the parents continue to alternate shifts throughout incubation (~53 days) and early chick brooding. Chicks become thermally emancipated from the parents around one month of age, allowing both parents to go back at sea simultaneously to forage (Weimerskirch et al., 1992). Twenty days after hatching, king penguin chicks are able to maintain their core body temperature under mild environmental conditions (>10 °C) but still rely on their parent’s brooding behaviour (Duchamp et al., 2002), and are able to express a HPA-dependent stress response (X et al. unpublished).

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Baseline and stress-induced plasma corticosterone levels of breeding king penguins implanted either with a Placebo, or a CORT subcutaneous implant (50 mg, 90 days release; n = 80 observations, N = 40 individuals) during incubation. Birds were blood sampled 6 days after implantation. CORT (red) vs. Placebo (blue) individual responses (raw data) are presented along with group and time specific mean ± SE. CORT levels were influenced by the interaction between handling stress (baseline: <4 min vs. stress-induced: 30 min of handling stress) and CORT treatment (LMM on log-transformed plasma CORT: $F_{1,37.2} = 62.4, p < 0.001$). Letters indicate significant differences according to post-hoc tests with Tukey adjustments ($p < 0.030$).</figcaption></figure>
## 2.2. Use of sub-cutaneous CORT and SHAM implants

We divided the 49 breeding pairs into two groups of either female-treated pairs (N = 23) or male-treated pairs (N = 26). In the female-treated pairs, we implanted females with either a CORT (NG-111, 50 mg corticosterone, 90-days release, 8 mm diameter and 3 mm thickness; N = 12 females) or a Placebo (NC-111, same vehicle but no CORT; N = 11 females) implant. Sub-cutaneous implants were purchased from Innovative Research of America (Sarasota, FL, USA). Females were implanted during their first incubation shift, 3 days after returning from sea. Similarly, in the male-treated pairs, we implanted 13 males with a CORT and 13 males with a Placebo implant, 3 days after returning from sea. In males, the first incubation shift is immediately after egg laying, and males have already been fasting for ~15 days during courtship. Therefore, we chose to implant the males during their second incubation shift so that they would have comparable fasting times compared to females, and therefore both sexes would be in a comparable physiological state during measurements. Allocation of birds to specific groups was designed to avoid spatial or temporal biases within and between groups. Male and female partners in, respectively, the female-treated and male-treated pairs were not implanted. Hereafter, they are referred to as "Partner-CORT" or "Partner-Placebo" regardless of their sex. Partner penguins were only considered in the chick analysis (see below).

On the day of the implantation, adults were captured by hand while incubating their egg and restrained by one experimenter with a hood covering the head to keep them calm. While maintaining individuals on their territory within the colony to avoid risking reproductive abandonment, sub-cutaneous CORT or placebo implants were inserted in the upper part of the back by a veterinary surgeon under local anaesthesia (ca. 0.5 mg/kg xylocaine combined with 0.0001 mg/kg adrenalin, Aspen Pharma). Immediately after implantation, the small incision (ca. 15 mm) was closed using 3 sterile surgical staples (SurgiClose™ Skin Stapler), and a prophylactic dose of antibiotics (cephalexin ca. 50 mg/kg, Rilexine®, Virbac) was injected to prevent any risk of infection. The incision was checked 3 days later for implant rejection; no sign of infection or implant rejection were noticed and staples were removed 6 days after implantation.

To verify the efficacy of our CORT treatment, 6 days after implantation, 1 mL of blood was sampled from the flipper vein using a heparinized syringe in less than 4 min after being detected by the focal individual (baseline CORT) and after 30 min of standardized handling (stress-induced, see Stier et al. (2019) and Viblanc et al. (2018)). Blood samples were taken between 09:00 and 13:00 to limit variation linked to circadian variation in plasma CORT levels. Blood samples were centrifuged within 1 h of sampling at 3000 g for 10 min. The plasma fraction was then removed and plasma aliquots were stored at -20 °C until the end of the day, before being transferred to -80 °C until laboratory analyses in 2023. Plasma total CORT levels were measured from 25 µL of plasma by immunoassay according to guidelines provided by the manufacturer (Corticosterone Enzyme Immunoassay Kit, Arbor Assay, USA). Intra-plate coefficient of variation based on duplicates was 8.17 ± 0.90% (mean ± SE), and inter-plate coefficient of variation based on one repeated sample was 8.08%. We tested the effect of implant on log-transformed plasma CORT using a linear mixed model (LMM) including as fixed factors: Handling stress (Baseline vs. Handling stress), Treatment (CORT vs. Placebo) and their interaction, as well as penguin ID as random effect. Implants were successful in raising circulating baseline CORT levels and in inhibiting stress-induced CORT release (Fig. 1).

## 2.3. Thermal image collection and analysis

Because of their dense and watertight plumage, the use of thermal imaging in seabirds is usually restricted to measuring the eye (periodal region) and beak regions (Gauchet and Grémillet, 2022). These

regions are highly vascularised and provide valuable information on body surface temperatures. Thermal pictures of implanted adults (Fig. 2A) and their egg (Fig. 2B) were taken 3 days after implantation and thermal pictures of chicks (Fig. 2C) were taken at day 20 after hatching (mean ± SE: 49.8 ± 1.4 days after CORT or Placebo implant of the focal parent, i.e. within the 90-days release given by the CORT pellet manufacturer). Thermal pictures were taken at a distance between ca. 0.8 and 2 m using FLIR E8 thermal imaging camera (320 × 240 pixels) resulting in a spotsize between 1.95 and 4.88 mm (Playa-Montmany and Tattersall 2021). King penguins' beaks being approximately 120 mm long, and the eye region being approximately 18 mm of diameter, our target areas exceeded the 3 times larger spotsize recommended by Playa-Montmany and Tattersall (2021). When approaching the adults and/or chicks, we started a stopwatch when the focal individual showed the first signs of alarm behaviour (e.g. stopping activity, looking towards the experimenter) to measure the duration of our disturbance at the time the different pictures were taken. In adults, we took a first set of pictures (1–2 pictures per individual) before handling, on average (mean ± SE) 0.63 ± 0.07 min after the first sign of alarm behaviour at a distance of ca. 1.5–2 m from the focal individuals. Adults were then captured-restrained, incision healing was checked, and we took a second set of pictures (1–2 pictures per individual) after terminating handling stress (mean ± SE handling stress duration was 8.3 ± 0.3 min; a timeframe within which CORT is known to increase by ca. 100% above baseline levels in king penguins (Viblanc et al., 2018).

Just after capturing the adult, we removed the single-egg from the brood pouch and immediately (ca. within 10 s) took a thermal picture at a distance of ca. 0.8 m. We then measured egg length and breadth using a Vernier calliper (Mitutoyo; accuracy ± 0.02 mm) and estimated egg volume (cm³) following (Narushin, 2005). When chicks were 20 days old, we took a first set of pictures (1–2 pictures per individual) at a distance of ca. 0.8 m immediately (ca. within 10 s) after they were removed from the brood pouch (i.e. before handling: 0.73 ± 0.08 min after the first sign of alarm behavior from the parent) and a second set of pictures (1–2 pictures per individual) was taken after handling, 5.40 ± 0.30 min later. Thermal images of the chicks were only taken on non-rainy days, which reduced the sample size from 38 chicks that survived until day 20, to 16 chicks with thermal images available. Chick sex was unknown and body mass was not significantly influenced by the parental treatment (CORT: 1.14 ± 0.11 kg vs. Placebo 1.32 ± 0.09 kg, N = 8/group, t-test: t = -1.28, p = 0.22). Chicks were kept within the colony at ambient temperature (14.9 ± 0.9 °C) between the first and second set of pictures, a temperature at which they are known to be able to maintain their internal body temperature (Duchamp et al., 2002). In adults and chicks, we defined 'before handling' surface temperatures, the temperatures extracted from the first set of pictures taken in <1 min and 'after handling' surface temperatures, the temperatures extracted from the second set of pictures taken after >5 min of handling stress.

We analysed thermal images using the ThermoCAM TM Researcher Pro 2.10 software (Flir systems, Wilsonville, Oregon, USA). Only profile pictures were analysed to avoid surface temperature errors related to head orientation (Playà-Montmany and Tattersall, 2021; Tabh et al., 2021). For each image we set the emissivity at 0.98, reflected temperature at 20 °C and controlled in the analyses for daily variation in air temperature (T_a) and relative humidity using daily climatic measures from a permanent Météo France weather station located 2 km inland from the colony (https://rp5.ru/Archives_m%C3%A9t%C3%A9o_sur_la_base_Alfred-Faure). We extracted measures of surface temperatures on maximum periorbital temperature (T_eye) and minimum beak temperature (T_beach) in adults and chicks. We used maximum T_eye as recommended by Jerem et al. (2015 & 2019) as well as recorded in recent studies (e.g. Robertson et al., (2020a); Zuluaga and Danner (2023), and the minimum T_beach to gain insight on the maximum state of vasoconstriction for this body surface (Tattersall et al., 2009). Finally, we estimated egg surface temperature (T_egg) as the mean of average length and width surface temperature.
## 2.2. Use of sub-cutaneous CORT and SHAM implants

We divided the 49 breeding pairs into two groups of either female-treated pairs (N = 23) or male-treated pairs (N = 26). In the female-treated pairs, we implanted females with either a CORT (NG-111, 50 mg corticosterone, 90-days release, 8 mm diameter and 3 mm thickness; N = 12 females) or a Placebo (NC-111, same vehicle but no CORT; N = 11 females) implant. Sub-cutaneous implants were purchased from Innovative Research of America (Sarasota, FL, USA). Females were implanted during their first incubation shift, 3 days after returning from sea. Similarly, in the male-treated pairs, we implanted 13 males with a CORT and 13 males with a Placebo implant, 3 days after returning from sea. In males, the first incubation shift is immediately after egg laying, and males have already been fasting for ~15 days during courtship. Therefore, we chose to implant the males during their second incubation shift so that they would have comparable fasting times compared to females, and therefore both sexes would be in a comparable physiological state during measurements. Allocation of birds to specific groups was designed to avoid spatial or temporal biases within and between groups. Male and female partners in, respectively, the female-treated and male-treated pairs were not implanted. Hereafter, they are referred to as "Partner-CORT" or "Partner-Placebo" regardless of their sex. Partner penguins were only considered in the chick analysis (see below).

On the day of the implantation, adults were captured by hand while incubating their egg and restrained by one experimenter with a hood covering the head to keep them calm. While maintaining individuals on their territory within the colony to avoid risking reproductive abandonment, sub-cutaneous CORT or placebo implants were inserted in the upper part of the back by a veterinary surgeon under local anaesthesia (ca. 0.5 mg/kg xylocaine combined with 0.0001 mg/kg adrenalin, Aspen Pharma). Immediately after implantation, the small incision (ca. 15 mm) was closed using 3 sterile surgical staples (SurgiClose™ Skin Stapler), and a prophylactic dose of antibiotics (cephalexin ca. 50 mg/kg, Rilexine®, Virbac) was injected to prevent any risk of infection. The incision was checked 3 days later for implant rejection; no sign of infection or implant rejection were noticed and staples were removed 6 days after implantation.

To verify the efficacy of our CORT treatment, 6 days after implantation, 1 mL of blood was sampled from the flipper vein using a heparinized syringe in less than 4 min after being detected by the focal individual (baseline CORT) and after 30 min of standardized handling (stress-induced, see Stier et al. (2019) and Viblanc et al. (2018)). Blood samples were taken between 09:00 and 13:00 to limit variation linked to circadian variation in plasma CORT levels. Blood samples were centrifuged within 1 h of sampling at 3000 g for 10 min. The plasma fraction was then removed and plasma aliquots were stored at -20 °C until the end of the day, before being transferred to -80 °C until laboratory analyses in 2023. Plasma total CORT levels were measured from 25 µL of plasma by immunoassay according to guidelines provided by the manufacturer (Corticosterone Enzyme Immunoassay Kit, Arbor Assay, USA). Intra-plate coefficient of variation based on duplicates was 8.17 ± 0.90% (mean ± SE), and inter-plate coefficient of variation based on one repeated sample was 8.08%. We tested the effect of implant on log-transformed plasma CORT using a linear mixed model (LMM) including as fixed factors: Handling stress (Baseline vs. Handling stress), Treatment (CORT vs. Placebo) and their interaction, as well as penguin ID as random effect. Implants were successful in raising circulating baseline CORT levels and in inhibiting stress-induced CORT release (Fig. 1).

## 2.3. Thermal image collection and analysis

Because of their dense and watertight plumage, the use of thermal imaging in seabirds is usually restricted to measuring the eye (periodal region) and beak regions (Gauchet and Grémillet, 2022). These

regions are highly vascularised and provide valuable information on body surface temperatures. Thermal pictures of implanted adults (Fig. 2A) and their egg (Fig. 2B) were taken 3 days after implantation and thermal pictures of chicks (Fig. 2C) were taken at day 20 after hatching (mean ± SE: 49.8 ± 1.4 days after CORT or Placebo implant of the focal parent, i.e. within the 90-days release given by the CORT pellet manufacturer). Thermal pictures were taken at a distance between ca. 0.8 and 2 m using FLIR E8 thermal imaging camera (320 × 240 pixels) resulting in a spotsize between 1.95 and 4.88 mm (Playa-Montmany and Tattersall 2021). King penguins' beaks being approximately 120 mm long, and the eye region being approximately 18 mm of diameter, our target areas exceeded the 3 times larger spotsize recommended by Playa-Montmany and Tattersall (2021). When approaching the adults and/or chicks, we started a stopwatch when the focal individual showed the first signs of alarm behaviour (e.g. stopping activity, looking towards the experimenter) to measure the duration of our disturbance at the time the different pictures were taken. In adults, we took a first set of pictures (1–2 pictures per individual) before handling, on average (mean ± SE) 0.63 ± 0.07 min after the first sign of alarm behaviour at a distance of ca. 1.5–2 m from the focal individuals. Adults were then captured-restrained, incision healing was checked, and we took a second set of pictures (1–2 pictures per individual) after terminating handling stress (mean ± SE handling stress duration was 8.3 ± 0.3 min; a timeframe within which CORT is known to increase by ca. 100% above baseline levels in king penguins (Viblanc et al., 2018)).

Just after capturing the adult, we removed the single-egg from the brood pouch and immediately (ca. within 10 s) took a thermal picture at a distance of ca. 0.8 m. We then measured egg length and breadth using a Vernier calliper (Mitutoyo; accuracy ± 0.02 mm) and estimated egg volume (cm³) following (Narushin, 2005). When chicks were 20 days old, we took a first set of pictures (1–2 pictures per individual) at a distance of ca. 0.8 m immediately (ca. within 10 s) after they were removed from the brood pouch (i.e. before handling: 0.73 ± 0.08 min after the first sign of alarm behavior from the parent) and a second set of pictures (1–2 pictures per individual) was taken after handling, 5.40 ± 0.30 min later. Thermal images of the chicks were only taken on non-rainy days, which reduced the sample size from 38 chicks that survived until day 20, to 16 chicks with thermal images available. Chick sex was unknown and body mass was not significantly influenced by the parental treatment (CORT: 1.14 ± 0.11 kg vs. Placebo 1.32 ± 0.09 kg, N = 8/group, t-test: t = -1.28, p = 0.22). Chicks were kept within the colony at ambient temperature (14.9 ± 0.9 °C) between the first and second set of pictures, a temperature at which they are known to be able to maintain their internal body temperature (Duchamp et al., 2002). In adults and chicks, we defined 'before handling' surface temperatures, the temperatures extracted from the first set of pictures taken in <1 min and 'after handling' surface temperatures, the temperatures extracted from the second set of pictures taken after >5 min of handling stress.

We analysed thermal images using the ThermoCAM TM Researcher Pro 2.10 software (Flir systems, Wilsonville, Oregon, USA). Only profile pictures were analysed to avoid surface temperature errors related to head orientation (Playà-Montmany and Tattersall, 2021; Tabh et al., 2021). For each image we set the emissivity at 0.98, reflected temperature at 20 °C and controlled in the analyses for daily variation in air temperature (T_a) and relative humidity using daily climatic measures from a permanent Météo France weather station located 2 km inland from the colony (https://rp5.ru/Archives_m%C3%A9t%C3%A9o_sur_la_base_Alfred-Faure). We extracted measures of surface temperatures on maximum periorbital temperature (T_eye) and minimum beak temperature (T_beach) in adults and chicks. We used maximum T_eye as recommended by Jerem et al. (2015 & 2019) as well as recorded in recent studies (e.g. Robertson et al., (2020a); Zuluaga and Danner (2023), and the minimum T_beach to gain insight on the maximum state of vasoconstriction for this body surface (Tattersall et al., 2009). Finally, we estimated egg surface temperature (T_egg) as the mean of average length and width surface temperature.
<figure><img src="image_6.png" /><figcaption>Fig. 2. Infrared images of (A) breeding adult, (B) incubated egg and (C) 20 days-old chick.</figcaption></figure>

## 2.4. Ethical note

All the procedures were approved by the French Ethical Committee (APAFIS#16465–2018080111195526 v4) and the Terres Australes et Antartiques Françaises (Arrêté TAAF A-2018-118).

## 2.5. Statistical analysis

We ran separate analyses for adults, eggs and chicks. In adults, we investigated variations in $T_{eye}$ and $T_{beak}$ using two separated linear mixed models (LMMs) where Treatment (CORT vs. Placebo), Handling stress (before vs. after), Sex and $T_a$ were specified as fixed effects. Relative humidity was initially tested but removed from final models since it was never significant. All two-ways interactions were also initially included but removed from the final model if $p > 0.10$ using a backward stepwise procedure. The p-values (just before removal in the backward stepwise procedure) for the focal interaction between Treatment and Handling stress are reported in tables. Bird identity was included as a random intercept to control for repeated measures. We investigated variation in $T_{egg}$ by entering parental CORT Treatment (CORT vs. Placebo), the Sex of the parent, $T_a$ and egg volume as fixed effects. Eggs were always measured when incubated by the implanted parents (i.e. 3 days after implantation of CORT or Placebo implants), while chicks at day 20 could be brooded by the implanted parent or its non-implanted partner. Thus, we investigated variation chick $T_{eye}$ and $T_{beak}$ by entering parental Treatment (CORT vs. Placebo), the category of the brooding parent at the time of measurement (implanted individual vs. non-implanted partner), Handling stress (before vs. after), and $T_a$ as fixed effects, and chick identity as a random intercept. Parental sex was not included in the models for chick $T_{eye}$ and $T_{beak}$ due to limited sample size available for chicks. Interactions were treated as described above for adults. All statistical analyses were conducted using R (version 3.5.3) and the packages *lme4* (Bates et al., 2015), *emmeans* (Lenth et al., 2018) and *pbkrtest* (Halekoh and Højsgaard, 2014). Results are reported as least-square means ± SE.

# 3. Results

## 3.1. Adult surface temperatures

Treatment (CORT vs. Placebo), either alone or in interaction with Handling stress, had no significant effect on eye ($T_{eye}$) or beak ($T_{beak}$) surface temperature in incubating adults (Table 2, Fig. 3A and B). Handling stress significantly affected $T_{eye}$ and $T_{beak}$, in opposite directions (Table 2). Over an 8-min handling stress, $T_{eye}$ increased by 0.8 ± 0.3 °C, on average (Fig. 3A), while $T_{beak}$ decreased by -1.5 ± 0.4 °C, on average (Fig. 3B). The increase in $T_{eye}$ appeared to be sex-specific (marginally significant interaction Handling * Sex: $p = 0.060$), with only females showing a significant increase in $T_{eye}$ in response to Handling stress (Fig. 3C; males: $t = 0.63, p = 0.53$; females: $t = 3.08, p = 0.003$). As expected, $T_{eye}$ and $T_{beak}$ were significantly and positively associated with $T_a$ (Table 2).

<caption>Table 2</caption>

Summary of linear mixed models (LMMs) investigating the determinants of (A) maximum eye temperature ($T_{eye}$) and (B) minimum beak temperature ($T_{beak}$) in adult king penguins.

<table><thead><tr><th colspan="6">A. T<sub>eye</sub> (n = 138<br>observations; N = 49<br>individuals)</th></tr><tr><th colspan="2">Random effects:</th><th colspan="4">Variance</th></tr><tr><th></th><th></th><th>SE</th><th>t</th><th>df</th><th>p</th></tr></thead><tbody><tr><td>Bird ID</td><td>Intercept</td><td>5.04</td><td></td><td></td><td></td></tr><tr><td>Residual</td><td></td><td>3.21</td><td></td><td></td><td></td></tr><tr><th colspan="2">Fixed effects:</th><th>SE</th><th>t</th><th>df</th><th>p</th></tr><tr><td>Intercept</td><td>24.94</td><td>1.71</td><td>14.54</td><td>47.9</td><td>&lt;0.001</td></tr><tr><td>Treatment (CORT)</td><td>-0.10</td><td>0.72</td><td>-0.15</td><td>45.8</td><td>0.89</td></tr><tr><td>Handling (after)</td><td>1.45</td><td>0.47</td><td>3.04</td><td>89.3</td><td>0.003</td></tr><tr><td>Sex (Male)</td><td>0.76</td><td>0.78</td><td>0.97</td><td>63.7</td><td>0.34</td></tr><tr><td>T<sub>a</sub></td><td>0.29</td><td>0.13</td><td>2.33</td><td>45.0</td><td>0.024</td></tr><tr><td>Handling*Sex<br>(Treatment x Handling)</td><td>-1.19</td><td>0.63</td><td>-1.90</td><td>89.7</td><td>0.060<br>(0.44)</td></tr><tr><th colspan="6">B. T<sub>beak</sub> (n = 138<br>observations; N = 49<br>individuals)</th></tr><tr><th colspan="2">Random effects:</th><th colspan="4">Variance</th></tr><tr><th></th><th></th><th>SE</th><th>t</th><th>df</th><th>p</th></tr><tr><td>Bird ID</td><td>Intercept</td><td>15.91</td><td></td><td></td><td></td></tr><tr><td>Residual</td><td></td><td>6.40</td><td></td><td></td><td></td></tr><tr><th colspan="2">Fixed effects:</th><th>SE</th><th>t</th><th>df</th><th>p</th></tr><tr><td>Intercept</td><td>13.84</td><td>2.92</td><td>4.74</td><td>45.0</td><td>&lt;0.001</td></tr><tr><td>Treatment (CORT)</td><td>-1.13</td><td>1.24</td><td>-0.91</td><td>44.5</td><td>0.37</td></tr><tr><td>Handling (after)</td><td>-1.47</td><td>0.45</td><td>-3.29</td><td>84.6</td><td>0.001</td></tr><tr><td>Sex (Male)</td><td>0.90</td><td>1.23</td><td>0.73</td><td>44.6</td><td>0.47</td></tr><tr><td>T<sub>a</sub></td><td>0.54</td><td>0.22</td><td>2.45</td><td>43.8</td><td>0.018<br>(0.61)</td></tr><tr><td>(Treatment x Handling)</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

## 3.2. Egg surface temperature

Neither parental Treatment ($F_{1,45} = 1.38, p = 0.25$; CORT: 34.5 ± 0.2 °C vs. Placebo: 34.9 ± 0.3 °C; N = 46), Sex ($F_{1,45} = 0.47, p = 0.49$), ambient temperature ($F_{1,45} = 0.45, p = 0.50$), nor egg volume ($F_{1,45} = 0.01, p = 0.93$) significantly affected $T_{egg}$.

## 3.3. Chick surface temperatures

The interaction between parental Treatment (CORT vs. Placebo) and the category of the parent brooding the chick (implanted vs. non-implanted partner) at the time of measurement significantly affected chick $T_{eye}$ ($F_{1,11} = 6.37, p = 0.028$, Table 3) and chick $T_{beak}$ ($F_{1,11} = 5.49, p = 0.039$, Table 3). Chicks brooded by the partner of CORT-implanted individuals had higher overall $T_{eye}$ and $T_{beak}$ than chicks brooded by CORT parents, or by Placebo and associated partners (Fig. 4A and B). While we found no significant interaction between parental Treatment and Handling stress (Table 3), both chick $T_{eye}$ (Fig. 4C) and chick $T_{beak}$ (Fig. 4D) significantly decreased after handling (by -0.9 ± 0.3 °C and -3.3 ± 0.6 °C respectively, Table 3). As expected, chick $T_{eye}$ and $T_{beak}$
<figure><img src="image_6.png" /><figcaption>Fig. 2. Infrared images of (A) breeding adult, (B) incubated egg and (C) 20 days-old chick.</figcaption></figure>

## 2.4. Ethical note

All the procedures were approved by the French Ethical Committee (APAFIS#16465-2018080111195526 v4) and the Terres Australes et Antartiques Françaises (Arrêté TAAF A-2018-118).

## 2.5. Statistical analysis

We ran separate analyses for adults, eggs and chicks. In adults, we investigated variations in $T_{eye}$ and $T_{beak}$ using two separated linear mixed models (LMMs) where Treatment (CORT vs. Placebo), Handling stress (before vs. after), Sex and $T_a$ were specified as fixed effects. Relative humidity was initially tested but removed from final models since it was never significant. All two-ways interactions were also initially included but removed from the final model if $p > 0.10$ using a backward stepwise procedure. The p-values (just before removal in the backward stepwise procedure) for the focal interaction between Treatment and Handling stress are reported in tables. Bird identity was included as a random intercept to control for repeated measures. We investigated variation in $T_{egg}$ by entering parental CORT Treatment (CORT vs. Placebo), the Sex of the parent, $T_a$ and egg volume as fixed effects. Eggs were always measured when incubated by the implanted parents (i.e. 3 days after implantation of CORT or Placebo implants), while chicks at day 20 could be brooded by the implanted parent or its non-implanted partner. Thus, we investigated variation chick $T_{eye}$ and $T_{beak}$ by entering parental Treatment (CORT vs. Placebo), the category of the brooding parent at the time of measurement (implanted individual vs. non-implanted partner), Handling stress (before vs. after), and $T_a$ as fixed effects, and chick identity as a random intercept. Parental sex was not included in the models for chick $T_{eye}$ and $T_{beak}$ due to limited sample size available for chicks. Interactions were treated as described above for adults. All statistical analyses were conducted using R (version 3.5.3) and the packages *lme4* (Bates et al., 2015), *emmeans* (Lenth et al., 2018) and *pbkrtest* (Halekoh and Højsgaard, 2014). Results are reported as least-square means ± SE.

# 3. Results

## 3.1. Adult surface temperatures

Treatment (CORT vs. Placebo), either alone or in interaction with Handling stress, had no significant effect on eye ($T_{eye}$) or beak ($T_{beak}$) surface temperature in incubating adults (Table 2, Fig. 3A and B). Handling stress significantly affected $T_{eye}$ and $T_{beak}$, in opposite directions (Table 2). Over an 8-min handling stress, $T_{eye}$ increased by 0.8 ± 0.3 °C, on average (Fig. 3A), while $T_{beak}$ decreased by -1.5 ± 0.4 °C, on average (Fig. 3B). The increase in $T_{eye}$ appeared to be sex-specific (marginally significant interaction Handling * Sex: $p = 0.060$), with only females showing a significant increase in $T_{eye}$ in response to Handling stress (Fig. 3C; males: $t = 0.63, p = 0.53$; females: $t = 3.08, p = 0.003$). As expected, $T_{eye}$ and $T_{beak}$ were significantly and positively associated with $T_a$ (Table 2).

<caption>Table 2</caption>

Summary of linear mixed models (LMMs) investigating the determinants of (A) maximum eye temperature ($T_{eye}$) and (B) minimum beak temperature ($T_{beak}$) in adult king penguins.

<table><thead><tr><th colspan="6">A. T<sub>eye</sub> (n = 138<br>observations; N = 49<br>individuals)</th></tr><tr><th colspan="2">Random effects:</th><th colspan="4">Variance</th></tr><tr><th></th><th></th><th>SE</th><th>t</th><th>df</th><th>p</th></tr></thead><tbody><tr><td>Bird ID</td><td>Intercept</td><td>5.04</td><td></td><td></td><td></td></tr><tr><td>Residual</td><td></td><td>3.21</td><td></td><td></td><td></td></tr><tr><th colspan="2">Fixed effects:</th><th>SE</th><th>t</th><th>df</th><th>p</th></tr><tr><td>Intercept</td><td>24.94</td><td>1.71</td><td>14.54</td><td>47.9</td><td>&lt;0.001</td></tr><tr><td>Treatment (CORT)</td><td>-0.10</td><td>0.72</td><td>-0.15</td><td>45.8</td><td>0.89</td></tr><tr><td>Handling (after)</td><td>1.45</td><td>0.47</td><td>3.04</td><td>89.3</td><td>0.003</td></tr><tr><td>Sex (Male)</td><td>0.76</td><td>0.78</td><td>0.97</td><td>63.7</td><td>0.34</td></tr><tr><td>T<sub>a</sub></td><td>0.29</td><td>0.13</td><td>2.33</td><td>45.0</td><td>0.024</td></tr><tr><td>Handling*Sex<br>(Treatment x Handling)</td><td>-1.19</td><td>0.63</td><td>-1.90</td><td>89.7</td><td>0.060<br>(0.44)</td></tr></tbody><tbody><tr><th colspan="6">B. T<sub>beak</sub> (n = 138<br>observations; N = 49<br>individuals)</th></tr><tr><th colspan="2">Random effects:</th><th colspan="4">Variance</th></tr><tr><th></th><th></th><th>SE</th><th>t</th><th>df</th><th>p</th></tr><tr><td>Bird ID</td><td>Intercept</td><td>15.91</td><td></td><td></td><td></td></tr><tr><td>Residual</td><td></td><td>6.40</td><td></td><td></td><td></td></tr><tr><th colspan="2">Fixed effects:</th><th>SE</th><th>t</th><th>df</th><th>p</th></tr><tr><td>Intercept</td><td>13.84</td><td>2.92</td><td>4.74</td><td>45.0</td><td>&lt;0.001</td></tr><tr><td>Treatment (CORT)</td><td>-1.13</td><td>1.24</td><td>-0.91</td><td>44.5</td><td>0.37</td></tr><tr><td>Handling (after)</td><td>-1.47</td><td>0.45</td><td>-3.29</td><td>84.6</td><td>0.001</td></tr><tr><td>Sex (Male)</td><td>0.90</td><td>1.23</td><td>0.73</td><td>44.6</td><td>0.47</td></tr><tr><td>T<sub>a</sub></td><td>0.54</td><td>0.22</td><td>2.45</td><td>43.8</td><td>0.018<br>(0.61)</td></tr><tr><td>(Treatment x Handling)</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

## 3.2. Egg surface temperature

Neither parental Treatment ($F_{1,45} = 1.38, p = 0.25$; CORT: 34.5 ± 0.2 °C vs. Placebo: 34.9 ± 0.3 °C; N = 46), Sex ($F_{1,45} = 0.47, p = 0.49$), ambient temperature ($F_{1,45} = 0.45, p = 0.50$), nor egg volume ($F_{1,45} = 0.01, p = 0.93$) significantly affected $T_{egg}$.

## 3.3. Chick surface temperatures

The interaction between parental Treatment (CORT vs. Placebo) and the category of the parent brooding the chick (implanted vs. non-implanted partner) at the time of measurement significantly affected chick $T_{eye}$ ($F_{1,11} = 6.37, p = 0.028$, Table 3) and chick $T_{beak}$ ($F_{1,11} = 5.49, p = 0.039$, Table 3). Chicks brooded by the partner of CORT-implanted individuals had higher overall $T_{eye}$ and $T_{beak}$ than chicks brooded by CORT parents, or by Placebo and associated partners (Fig. 4A and B). While we found no significant interaction between parental Treatment and Handling stress (Table 3), both chick $T_{eye}$ (Fig. 4C) and chick $T_{beak}$ (Fig. 4D) significantly decreased after handling (by -0.9 ± 0.3 °C and -3.3 ± 0.6 °C respectively, Table 3). As expected, chick $T_{eye}$ and $T_{beak}$
<figure><img src="image_7.png" /><figcaption>Fig. 3. Adult king penguin surface temperature responses to an experimental manipulation of glucocorticoid levels (CORT vs. Placebo subcutaneous implants) and handling stress: (A) Maximum eye temperature response to handling stress, (B) Minimum beak temperature response to handling stress, and (C) Sex-specific response of eye temperature to handling stress. In panels A and B, CORT (red) vs. Placebo (blue) individual responses (raw data) are presented. Since CORT Treatment had no significant effect either alone or in interaction with Handling stress (see Table 2 for details), the overall mean ± SE is shown in black. Least-square means ± SE from final statistical models (Table 2; n = 138 observations; N = 49 individuals) are presented. Different letters indicate significant differences in panels A and B, and ** in panel C represent the significant increase of T<sub>eye</sub> observed only in females.</figcaption></figure>

<table><caption>**Table 3**
Summary of linear mixed models (LMMs) investigating the determinants of (A) maximum eye temperature (T<sub>eye</sub>) and (B) minimum beak temperature (T<sub>beak</sub>) in king penguin chicks. Only one parent was implanted with CORT or Placebo implant, thus we considered both parental treatment (CORT vs. Placebo), the category of the parent brooding the chick at the time of measurement (implanted parent vs. non-implanted partner) and their interaction in our analyses.</caption><thead><tr><th colspan="6">A. T<sub>eye</sub> (n = 63<br>observations; N = 16<br>chicks)</th></tr><tr><th colspan="2">Random effects:</th><th colspan="4">Variance</th></tr><tr><th>Bird ID</th><th>Intercept</th><th colspan="4">0.81</th></tr><tr><th>Residual</th><th>1.18</th><th colspan="4"></th></tr><tr><th colspan="2">Fixed effects:</th><th>SE</th><th>t</th><th>df</th><th>p</th></tr></thead><tbody><tr><th>Intercept</th><td>29.47</td><td>1.39</td><td>21.26</td><td>11.1</td><td>&lt;0.001</td></tr><tr><th>Treatment (CORT)</th><td>-0.88</td><td>0.68</td><td>-1.29</td><td>11.3</td><td>0.22</td></tr><tr><th>Parent (implanted)</th><td>-2.84</td><td>0.82</td><td>3.48</td><td>11.1</td><td>0.005</td></tr><tr><th>Handling (after)</th><td>-0.85</td><td>0.28</td><td>-3.08</td><td>47.2</td><td>0.003</td></tr><tr><th>T<sub>a</sub></th><td>0.21</td><td>0.09</td><td>2.41</td><td>10.9</td><td>0.035</td></tr><tr><th>Treatment*Parent<br>(Treatment x Handling)</th><td>2.78</td><td>1.10</td><td>2.53</td><td>11.0</td><td>0.028<br>(0.79)</td></tr></tbody><tbody><tr><th colspan="6">B. T<sub>beak</sub> (n = 63<br>observations; N = 16<br>chicks)</th></tr><tr><th colspan="2">Random effects:</th><th colspan="4">Variance</th></tr><tr><th>Bird ID</th><th>Intercept</th><th colspan="4">5.53</th></tr><tr><th>Residual</th><th>4.89</th><th colspan="4"></th></tr><tr><th colspan="2">Fixed effects:</th><th>SE</th><th>t</th><th>df</th><th>p</th></tr><tr><th>Intercept</th><td>13.55</td><td>3.42</td><td>3.96</td><td>11.1</td><td>0.002</td></tr><tr><th>Treatment (CORT)</th><td>1.15</td><td>1.68</td><td>0.69</td><td>11.3</td><td>0.51</td></tr><tr><th>Parent (implanted)</th><td>-6.82</td><td>2.02</td><td>-3.38</td><td>11.1</td><td>0.006</td></tr><tr><th>Handling (after)</th><td>-3.26</td><td>0.56</td><td>-5.77</td><td>46.9</td><td>&lt;0.001</td></tr><tr><th>T<sub>a</sub></th><td>0.57</td><td>0.21</td><td>2.65</td><td>11.0</td><td>0.023</td></tr><tr><th>Treatment*Parent<br>(Treatment x Handling)</th><td>6.37</td><td>2.72</td><td>2.34</td><td>11.0</td><td>0.039<br>(0.94)</td></tr></tbody></table>

were significantly positively associated with T<sub>a</sub> (Table 3).

## 4. Discussion

By experimentally manipulating CORT levels in adult breeding king penguins, we show that corticosterone is unlikely to play a major role in determining changes in surface temperatures (both before or in response to handling stress) in king penguins. Indeed, we found no significant difference in baseline surface temperature between CORT and Placebo implanted individuals. However, whatever the parental CORT treatment, our handling stress protocol led to an increase in T<sub>eye</sub> and a decrease in T<sub>beak</sub> in response to capture and handling. The absence of effect of the CORT treatment on baseline surface temperature and its dynamics during acute handling stress suggest that changes in surface temperature are probably driven primarily by the activity of the SAM axis in king penguins (but see below for a potential causal involvement of CORT in house sparrow *Passer domesticus* (Ouyang et al., 2021)). Contrary to our prediction, parental treatment had no significant effect on egg temperature. Yet, we found an unexpected pattern for chick surface temperatures: chicks brooded by the non-implanted partner of CORT individuals had higher surface temperatures (both T<sub>eye</sub> and T<sub>beak</sub>) than chicks brooded by Placebo (or associated non-implanted partner) or CORT individuals. Finally, in the thermally non-emancipated chicks, both T<sub>eye</sub> and T<sub>beak</sub> decreased with handling, but irrespectively of the parental treatment.

### 4.1. CORT and adult surface temperatures

Previous correlative studies have considered baseline and stress-
<figure><img src="image_7.png" /><figcaption>Fig. 3. Adult king penguin surface temperature responses to an experimental manipulation of glucocorticoid levels (CORT vs. Placebo subcutaneous implants) and handling stress: (A) Maximum eye temperature response to handling stress, (B) Minimum beak temperature response to handling stress, and (C) Sex-specific response of eye temperature to handling stress. In panels A and B, CORT (red) vs. Placebo (blue) individual responses (raw data) are presented. Since CORT Treatment had no significant effect either alone or in interaction with Handling stress (see Table 2 for details), the overall mean ± SE is shown in black. Least-square means ± SE from final statistical models (Table 2; n = 138 observations; N = 49 individuals) are presented. Different letters indicate significant differences in panels A and B, and ** in panel C represent the significant increase of T<sub>eye</sub> observed only in females.</figcaption></figure>

<caption>Table 3</caption>

Summary of linear mixed models (LMMs) investigating the determinants of (A) maximum eye temperature (T<sub>eye</sub>) and (B) minimum beak temperature (T<sub>beak</sub>) in king penguin chicks. Only one parent was implanted with CORT or Placebo implant, thus we considered both parental treatment (CORT vs. Placebo), the category of the parent brooding the chick at the time of measurement (implanted parent vs. non-implanted partner) and their interaction in our analyses.

<table><thead><tr><th colspan="6">A. T<sub>eye</sub> (n = 63<br>observations; N = 16<br>chicks)</th></tr><tr><th colspan="2">Random effects:</th><th colspan="4">Variance</th></tr><tr><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>Bird ID</td><td>Intercept</td><td>0.81</td><td></td><td></td><td></td></tr><tr><td>Residual</td><td></td><td>1.18</td><td></td><td></td><td></td></tr><tr><th colspan="2">Fixed effects:</th><th>SE</th><th>t</th><th>df</th><th>p</th></tr><tr><td>Intercept</td><td>29.47</td><td>1.39</td><td>21.26</td><td>11.1</td><td>&lt;0.001</td></tr><tr><td>Treatment (CORT)</td><td>-0.88</td><td>0.68</td><td>-1.29</td><td>11.3</td><td>0.22</td></tr><tr><td>Parent (implanted)</td><td>-2.84</td><td>0.82</td><td>3.48</td><td>11.1</td><td>0.005</td></tr><tr><td>Handling (after)</td><td>-0.85</td><td>0.28</td><td>-3.08</td><td>47.2</td><td>0.003</td></tr><tr><td>T<sub>a</sub></td><td>0.21</td><td>0.09</td><td>2.41</td><td>10.9</td><td>0.035</td></tr><tr><td>Treatment*Parent<br>(Treatment x Handling)</td><td>2.78</td><td>1.10</td><td>2.53</td><td>11.0</td><td>0.028<br>(0.79)</td></tr></tbody><thead><tr><th colspan="6">B. T<sub>beak</sub> (n = 63<br>observations; N = 16<br>chicks)</th></tr><tr><th colspan="2">Random effects:</th><th colspan="4">Variance</th></tr><tr><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>Bird ID</td><td>Intercept</td><td>5.53</td><td></td><td></td><td></td></tr><tr><td>Residual</td><td></td><td>4.89</td><td></td><td></td><td></td></tr><tr><th colspan="2">Fixed effects:</th><th>SE</th><th>t</th><th>df</th><th>p</th></tr><tr><td>Intercept</td><td>13.55</td><td>3.42</td><td>3.96</td><td>11.1</td><td>0.002</td></tr><tr><td>Treatment (CORT)</td><td>1.15</td><td>1.68</td><td>0.69</td><td>11.3</td><td>0.51</td></tr><tr><td>Parent (implanted)</td><td>-6.82</td><td>2.02</td><td>-3.38</td><td>11.1</td><td>0.006</td></tr><tr><td>Handling (after)</td><td>-3.26</td><td>0.56</td><td>-5.77</td><td>46.9</td><td>&lt;0.001</td></tr><tr><td>T<sub>a</sub></td><td>0.57</td><td>0.21</td><td>2.65</td><td>11.0</td><td>0.023</td></tr><tr><td>Treatment*Parent<br>(Treatment x Handling)</td><td>6.37</td><td>2.72</td><td>2.34</td><td>11.0</td><td>0.039<br>(0.94)</td></tr></tbody></table>

were significantly positively associated with T<sub>a</sub> (Table 3).

## 4. Discussion

By experimentally manipulating CORT levels in adult breeding king penguins, we show that corticosterone is unlikely to play a major role in determining changes in surface temperatures (both before or in response to handling stress) in king penguins. Indeed, we found no significant difference in baseline surface temperature between CORT and Placebo implanted individuals. However, whatever the parental CORT treatment, our handling stress protocol led to an increase in T<sub>eye</sub> and a decrease in T<sub>beak</sub> in response to capture and handling. The absence of effect of the CORT treatment on baseline surface temperature and its dynamics during acute handling stress suggest that changes in surface temperature are probably driven primarily by the activity of the SAM axis in king penguins (but see below for a potential causal involvement of CORT in house sparrow *Passer domesticus* (Ouyang et al., 2021)). Contrary to our prediction, parental treatment had no significant effect on egg temperature. Yet, we found an unexpected pattern for chick surface temperatures: chicks brooded by the non-implanted partner of CORT individuals had higher surface temperatures (both T<sub>eye</sub> and T<sub>beak</sub>) than chicks brooded by Placebo (or associated non-implanted partner) or CORT individuals. Finally, in the thermally non-emancipated chicks, both T<sub>eye</sub> and T<sub>beak</sub> decreased with handling, but irrespectively of the parental treatment.

### 4.1. CORT and adult surface temperatures

Previous correlative studies have considered baseline and stress-
<figure><img src="image_8.png" /><figcaption>Fig. 4. King penguin chick surface temperature in response to parental glucocorticoid manipulation and handling: (A) Maximum eye temperature according to parental treatment and category of the parent brooding the chick (i.e. partners are not implanted), (B) Minimum beak temperature according to parental treatment and category of the parent brooding the chick, (C) Maximum eye temperature response to handling, and (D) Minimum eye temperature response to handling. In panels C and D, CORT (red) vs. Placebo (blue) chick's individual responses (raw data) are presented, but only the global mean ± SE are presented since there was no significant interaction between parental CORT Treatment and Handling (see Table 3 for details). Least-square means ± SE from final statistical models (Table 3) are presented (n = 63 observations, N = 16 chicks). Different letters indicate significant differences.</figcaption></figure>

induced CORT levels to investigate the relationship between GC sig-
nalling and changes in body surface temperature in birds (blue tit,
*Cyanistes caerulescens*, (Jerem et al., 2018); domestic chicken, *Gallus*
*domesticus* (Giloh et al., 2012; Weimer et al., 2020),). To go deeper in the
involved mechanisms, we experimentally manipulated CORT in the king
penguin to help to causally distinguish the contribution of the HPA and
SAM axes to changes in body surface temperature. Since we did not
detect any significant effect of CORT manipulation on baseline or
stress-induced surface temperatures, our results provide little support
for a causal involvement of the HPA axis in mediating stress-related
changes in surface temperature.

Our results contrast with a recent study showing that an acute
stimulation of the HPA axis with adrenocorticotropic hormone (ACTH)
induced a decrease in Teye within 5 min in captive-held house sparrows
(Ouyang et al., 2021), which suggests a causal involvement of the HPA
axis in mediating stress-related changes in surface temperature. Yet, a
recent study in the same species found a significant correlation between
the acute change in surface temperature and heart rate variability, a
measure of SAM axis activation (Jerem and Romero, 2023). This sug-
gests that both the HPA and SAM axes could be involved in mediating
stress-related changes in surface temperature in house sparrow. While
we used an experimental approach and the lack of significant effect of
baseline CORT on body surface temperatures appears to be robust, we
cannot fully exclude that our handling stress protocol for thermal im-
aging was too short to detect a potential role of stress-induced CORT in
influencing changes in surface temperatures. Indeed, in 8 min of
handling, CORT levels are expected to double in king penguin, while
maximum CORT levels are only attained after ca. 80 min of handling in
our study species (Viblanc et al., 2018). Additionally, we may have

missed some early changes in surface temperatures occurring in
response to the stress of our approach prior to bird capture and handling.
Indeed, changes in Teye and Tbeak within 25 s before capture have been
observed in house sparrow (Passer domesticus) (Jerem and Romero,
2023) and our pre-handling thermal pictures were taken on average 38 s
after the first sign of alarm behaviour. Yet, our results suggest that
changes in surface temperature are probably more likely mediated by
the SAM axis rather than the HPA axis in adult king penguins. Future
studies testing for a direct acute stimulation of the HPA axis (using
ACTH) or SAM axis (using catecholamines) in the king penguin and
other bird species could be useful to gain greater insight on the contri-
bution of the HPA and SAM axes in mediating changes in body surface
temperature (Jerem and Romero, 2023; Ouyang et al., 2021).

4.2. *Stress-induced changes in surface temperatures*

In breeding adults, we observed a mild sex-specific significant in-
crease of Teye and a significant decrease of Tbeak in response to acute
capture-handling stress. Differences in temperature change between
body regions in response to acute stress are not rare (see Table 1). For
instance, (Moe et al., 2017) measured a decrease of Teye contrasting with
an increase of Tfeet in chicken, whereas in captive budgerigars (Melop-
sittacus undulatus) Teye increased transiently after exposure to a stressor
while Tfeet did not change (Ikkatai and Watanabe, 2015, Table 1).
Anatomical and functional reasons can likely explain the difference of
stress-induced changes in surface temperature between body regions in
penguins, with for instance a potential interest in maintaining or
increasing eye blood flow to maintain or enhance visual acuity when
acutely threatened by an environmental challenge.
<figure><img src="image_8.png" /><figcaption>Fig. 4. King penguin chick surface temperature in response to parental glucocorticoid manipulation and handling: (A) Maximum eye temperature according to parental treatment and category of the parent brooding the chick (i.e. partners are not implanted), (B) Minimum beak temperature according to parental treatment and category of the parent brooding the chick, (C) Maximum eye temperature response to handling, and (D) Minimum eye temperature response to handling. In panels C and D, CORT (red) vs. Placebo (blue) chick's individual responses (raw data) are presented, but only the global mean ± SE are presented since there was no significant interaction between parental CORT Treatment and Handling (see Table 3 for details). Least-square means ± SE from final statistical models (Table 3) are presented (n = 63 observations, N = 16 chicks). Different letters indicate significant differences.</figcaption></figure>

induced CORT levels to investigate the relationship between GC sig-
nalling and changes in body surface temperature in birds (blue tit,
*Cyanistes caerulescens*, (Jerem et al., 2018); domestic chicken, *Gallus*
*domesticus* (Giloh et al., 2012; Weimer et al., 2020),). To go deeper in the
involved mechanisms, we experimentally manipulated CORT in the king
penguin to help to causally distinguish the contribution of the HPA and
SAM axes to changes in body surface temperature. Since we did not
detect any significant effect of CORT manipulation on baseline or
stress-induced surface temperatures, our results provide little support
for a causal involvement of the HPA axis in mediating stress-related
changes in surface temperature.

Our results contrast with a recent study showing that an acute
stimulation of the HPA axis with adrenocorticotropic hormone (ACTH)
induced a decrease in Teye within 5 min in captive-held house sparrows
(Ouyang et al., 2021), which suggests a causal involvement of the HPA
axis in mediating stress-related changes in surface temperature. Yet, a
recent study in the same species found a significant correlation between
the acute change in surface temperature and heart rate variability, a
measure of SAM axis activation (Jerem and Romero, 2023). This sug-
gests that both the HPA and SAM axes could be involved in mediating
stress-related changes in surface temperature in house sparrow. While
we used an experimental approach and the lack of significant effect of
baseline CORT on body surface temperatures appears to be robust, we
cannot fully exclude that our handling stress protocol for thermal im-
aging was too short to detect a potential role of stress-induced CORT in
influencing changes in surface temperatures. Indeed, in 8 min of
handling, CORT levels are expected to double in king penguin, while
maximum CORT levels are only attained after ca. 80 min of handling in
our study species (Viblanc et al., 2018). Additionally, we may have

missed some early changes in surface temperatures occurring in
response to the stress of our approach prior to bird capture and handling.
Indeed, changes in Teye and Tbeak within 25 s before capture have been
observed in house sparrow (Passer domesticus) (Jerem and Romero,
2023) and our pre-handling thermal pictures were taken on average 38 s
after the first sign of alarm behaviour. Yet, our results suggest that
changes in surface temperature are probably more likely mediated by
the SAM axis rather than the HPA axis in adult king penguins. Future
studies testing for a direct acute stimulation of the HPA axis (using
ACTH) or SAM axis (using catecholamines) in the king penguin and
other bird species could be useful to gain greater insight on the contri-
bution of the HPA and SAM axes in mediating changes in body surface
temperature (Jerem and Romero, 2023; Ouyang et al., 2021).

4.2. *Stress-induced changes in surface temperatures*

In breeding adults, we observed a mild sex-specific significant in-
crease of Teye and a significant decrease of Tbeak in response to acute
capture-handling stress. Differences in temperature change between
body regions in response to acute stress are not rare (see Table 1). For
instance, (Moe et al., 2017) measured a decrease of Teye contrasting with
an increase of Tfeet in chicken, whereas in captive budgerigars (Melop-
sittacus undulatus) Teye increased transiently after exposure to a stressor
while Tfeet did not change (Ikkatai and Watanabe, 2015, Table 1).
Anatomical and functional reasons can likely explain the difference of
stress-induced changes in surface temperature between body regions in
penguins, with for instance a potential interest in maintaining or
increasing eye blood flow to maintain or enhance visual acuity when
acutely threatened by an environmental challenge.
$T_{eye}$ has also been shown to positively correlate with internal (cloacal) temperature in various avian species (budgerigars: Ikkatai and Watanabe, 2015; chicken: Cândido et al., 2020). Consequently, it is possible that the increase in $T_{eye}$ we observe in adults could reflect an increase in internal body temperature (Oka, 2018). Consistently, in closely related Emperor penguins (*Aptenodytes forsteri*), acute stress leads to an approximate 1.5 °C increase in internal (stomach) temperature (Regel and Pütz, 1997). Such an increase in $T_{eye}$ in response to handling stress has been reported two times in avian species to the best of our knowledge (Ikkatai and Watanabe, 2015; Jakubas et al., 2022), while most previous studies have shown either a significant decrease (4 studies) or no significant change (4 studies; Table 1). Those contrasted findings might be explained by at least three factors: 1. variation in body size, since smaller individuals/species are expected to favour internal heat conservation by reducing more markedly peripheral blood flow and surface temperature compared to large ones (*thermoprotective hypothesis*; Robertson et al., 2020a); 2. differences in the thermal environment, since it has been shown that during acute stress response, heat conservation is favoured below the thermoneutral zone while heat dissipation is favoured above its upper limit (Robertson et al., 2020a); 3. The various delays at which $T_{eye}$ was measured in response to acute stress, since for instance the increase in $T_{eye}$ found by Ikkatai and Watanabe (2015) was short-lived (visible 5 min after the stressor, but not later on during the 30 min measurement period). It is important to note that $T_{eye}$ and $T_{beak}$ can show very rapid and non-linear changes in response to acute stress, that are likely to be missed by most studies not using continuous thermal image recording (Jerem et al., 2019; Jerem and Romero, 2023; Zuluaga and Danner, 2023). For instance, Zuluaga et al. (2023) found an initial drop in $T_{beak}$ following exposure to stressor, with a recovery to baseline levels after only 2 min, while Jerem et al. (2019) found an initial drop in $T_{eye}$ lasting less than 15s, followed by an increase above baseline from 15 to 75 s.

Although the interaction between handling stress and sex was only marginally significant ($p$ = 0.060), it appeared that only females exhibited a noticeable increase in $T_{eye}$ in response to handling stress in our study. Differences in body size or mass between sexes are very modest (ca. 5%) in king penguin (Kriesell et al., 2018), which thus seems unlikely to fully explain the sex-difference observed here. There are known sex-differences in stress physiology (Handa and McGivern, 2017), but sex differences in surface temperature changes induced by acute stress have been rarely investigated (but see Robertson et al. (2020a) for a result opposite to ours between sexes). We previously observed no sex effect in HPA axis responsiveness between males and females king penguins (Viblanc et al., 2016), but as mentioned above, the SAM axis is the likely driver of acute changes in surface temperatures. Consequently, this suggests that sexes might differ in the stress-sensitivity of their SAM axis, although this would need to be confirmed by direct measurements of SAM axis activity.

The decrease we observe in $T_{beak}$ in response to acute stress in adults appears like the typical peripheral vasoconstriction response previously reported for instance by Tabh et al. (2021). Such peripheral vasoconstriction enables the redistribution of the peripheral blood circulation to internal organs and tissues, such as the brain or muscles, favouring their oxygenation and nutrition to sustain the fight-or-flight response. King penguins breed in a highly dense and aggressive colonial environment (up to 500 aggressive interactions per hour; Côté, 2000). Aggressive interactions, frequently leading to injuries, are known to result in increased heart rates (Viblanc et al., 2012). A decrease of $T_{beak}$ during acute stress response likely reflects peripheral vasoconstriction, that is likely widespread to other peripheral body parts, which according to the *haemoprotective hypothesis* could help to reduce blood loss in case of injury (Robertson et al., 2020a).

The changes observed in surface temperatures in chicks differ from those found in adults. Indeed, we observed a significant decrease in chick's $T_{eye}$, whereas $T_{eye}$ increased in adult females and stayed stable in adult males. While $T_{beak}$ significantly decreased as observed in adults,

the decrease was more pronounced in chicks (−3.3 ± 0.6 °C vs. −1.5 ± 0.4 °C, in chicks and adults, respectively). This is likely explained by greater peripheral thermal losses in chicks suddenly exposed to an ambient temperature drop (ca. 35 °C under the brood pouch of the adults vs. 15 °C when taken out for measurement and handling), despite their ability to maintain their internal temperature at this stage at ambient temperature (Duchamp et al., 2002). The more pronounced drop in surface temperature we observed in chicks during acute handling could reflect their greater need to conserve heat at a relatively mild $T_a$ compared to adults.

### 4.3. Impact of parental CORT on egg and chick surface temperature

Contrary to our prediction, we observed no significant impact of parental CORT treatment on egg temperature, suggesting little alteration of incubation behaviour by increased CORT levels, contrary to a previous study in Adelie penguin reporting a reduction of 1.3 ± 0.2 °C in $T_{egg}$ (Thierry et al., 2013a). It is possible that incubating two eggs in the colder environment of Adélie penguins is more challenging (and thus more likely to be impacted by high CORT levels) than incubating a single egg under the milder climate experienced by king penguins. Alternatively, it is also possible that king penguins are more resilient to increased CORT levels than Adelie penguins, which is supported by the good reproductive success of king penguins implanted with CORT (X et al. unpublished), contrary to what has been observed in Adelie penguins (Thierry et al., 2013b). Measurements of incubation quality using a dummy egg with temperature and rotation sensors (Thierry et al., 2013a) may provide more accurate data on this question, but it is important to note that the $T_{egg}$ measured in this study (Placebo: 34.9 ± 0.3 °C) was close to the $T_{egg}$ measured in the same penguin colony using internal sensors in dummy eggs (35.7 ± 0.4 °C (Groscolas et al., 2000)). Our results therefore suggest that further studies may benefit from the use of minimally invasive thermal imaging to measure incubation quality, for instance in the context of parental behaviour and climate change (Cook et al., 2020).

The measure of chick surface temperatures revealed some surprising patterns. While we expected CORT chicks to have lower surface temperatures due to poorer parental care (less efficient brooding), we found some opposite result: chicks had higher surface temperatures when brooded by the non-implanted partner of the CORT parent. This result should be considered with caution considering the limited sample size available for chick surface temperatures (N = 16). One possibility is that partners from CORT-implanted individuals somehow perceived the 'stress' levels of their partner (as shown between siblings in yellow legged gulls (*Larus michahellis*); Noguera et al., 2017) and somehow compensated for parental care through more efficient brooding. This hypothesis requires further study to determine whether the concept of 'family-transmitted stress' (Noguera et al., 2017) applies to the king penguin. In this species, both parents must rely on each other over more than one year to successfully fledge their single chick, which itself is entirely dependent on its parents for its food supply throughout its growth. Hence, the king penguin could be promising system to investigate the 'family-transmitted stress' hypothesis.

### 4.4. Conclusion

Evaluating stress levels and responses of free-living animals in a non-invasive manner using thermal imaging is a burgeoning field of research (Jerem et al., 2015; Tabh et al., 2022). However, little is still known about the underlying pathways of stress physiology that influence the changes in surface temperature. Our experimental study in king penguin points toward a likely preponderant role of the SAM axis. Additionally, by summarizing the current evidence for acute changes in surface temperatures during stress exposure (Table 1), we highlight the relative complexity and inconsistency between the effects observed by different studies. Further experimental studies related to SAM and HPA axes
$T_{eye}$ has also been shown to positively correlate with internal (cloacal) temperature in various avian species (budgerigars: Ikkatai and Watanabe, 2015; chicken: Cândido et al., 2020). Consequently, it is possible that the increase in $T_{eye}$ we observe in adults could reflect an increase in internal body temperature (Oka, 2018). Consistently, in closely related Emperor penguins (*Aptenodytes forsteri*), acute stress leads to an approximate 1.5 °C increase in internal (stomach) temperature (Regel and Pütz, 1997). Such an increase in $T_{eye}$ in response to handling stress has been reported two times in avian species to the best of our knowledge (Ikkatai and Watanabe, 2015; Jakubas et al., 2022), while most previous studies have shown either a significant decrease (4 studies) or no significant change (4 studies; Table 1). Those contrasted findings might be explained by at least three factors: 1. variation in body size, since smaller individuals/species are expected to favour internal heat conservation by reducing more markedly peripheral blood flow and surface temperature compared to large ones (*thermoprotective hypothesis*; Robertson et al., 2020a); 2. differences in the thermal environment, since it has been shown that during acute stress response, heat conservation is favoured below the thermoneutral zone while heat dissipation is favoured above its upper limit (Robertson et al., 2020a); 3. The various delays at which $T_{eye}$ was measured in response to acute stress, since for instance the increase in $T_{eye}$ found by Ikkatai and Watanabe (2015) was short-lived (visible 5 min after the stressor, but not later on during the 30 min measurement period). It is important to note that $T_{eye}$ and $T_{beak}$ can show very rapid and non-linear changes in response to acute stress, that are likely to be missed by most studies not using continuous thermal image recording (Jerem et al., 2019; Jerem and Romero, 2023; Zuluaga and Danner, 2023). For instance, Zuluaga et al. (2023) found an initial drop in $T_{beak}$ following exposure to stressor, with a recovery to baseline levels after only 2 min, while Jerem et al. (2019) found an initial drop in $T_{eye}$ lasting less than 15s, followed by an increase above baseline from 15 to 75 s.

Although the interaction between handling stress and sex was only marginally significant ($p$ = 0.060), it appeared that only females exhibited a noticeable increase in $T_{eye}$ in response to handling stress in our study. Differences in body size or mass between sexes are very modest (ca. 5%) in king penguin (Kriesell et al., 2018), which thus seems unlikely to fully explain the sex-difference observed here. There are known sex-differences in stress physiology (Handa and McGivern, 2017), but sex differences in surface temperature changes induced by acute stress have been rarely investigated (but see Robertson et al. (2020a) for a result opposite to ours between sexes). We previously observed no sex effect in HPA axis responsiveness between males and females king penguins (Viblanc et al., 2016), but as mentioned above, the SAM axis is the likely driver of acute changes in surface temperatures. Consequently, this suggests that sexes might differ in the stress-sensitivity of their SAM axis, although this would need to be confirmed by direct measurements of SAM axis activity.

The decrease we observe in $T_{beak}$ in response to acute stress in adults appears like the typical peripheral vasoconstriction response previously reported for instance by Tabh et al. (2021). Such peripheral vasoconstriction enables the redistribution of the peripheral blood circulation to internal organs and tissues, such as the brain or muscles, favouring their oxygenation and nutrition to sustain the fight-or-flight response. King penguins breed in a highly dense and aggressive colonial environment (up to 500 aggressive interactions per hour; CôTé, 2000). Aggressive interactions, frequently leading to injuries, are known to result in increased heart rates (Viblanc et al., 2012). A decrease of $T_{beak}$ during acute stress response likely reflects peripheral vasoconstriction, that is likely widespread to other peripheral body parts, which according to the *haemoprotective hypothesis* could help to reduce blood loss in case of injury (Robertson et al., 2020a).

The changes observed in surface temperatures in chicks differ from those found in adults. Indeed, we observed a significant decrease in chick's $T_{eye}$, whereas $T_{eye}$ increased in adult females and stayed stable in adult males. While $T_{beak}$ significantly decreased as observed in adults,

the decrease was more pronounced in chicks (−3.3 ± 0.6 °C vs. −1.5 ± 0.4 °C, in chicks and adults, respectively). This is likely explained by greater peripheral thermal losses in chicks suddenly exposed to an ambient temperature drop (ca. 35 °C under the brood pouch of the adults vs. 15 °C when taken out for measurement and handling), despite their ability to maintain their internal temperature at this stage at ambient temperature (Duchamp et al., 2002). The more pronounced drop in surface temperature we observed in chicks during acute handling could reflect their greater need to conserve heat at a relatively mild $T_a$ compared to adults.

### 4.3. Impact of parental CORT on egg and chick surface temperature

Contrary to our prediction, we observed no significant impact of parental CORT treatment on egg temperature, suggesting little alteration of incubation behaviour by increased CORT levels, contrary to a previous study in Adelie penguin reporting a reduction of 1.3 ± 0.2 °C in $T_{egg}$ (Thierry et al., 2013a). It is possible that incubating two eggs in the colder environment of Adélie penguins is more challenging (and thus more likely to be impacted by high CORT levels) than incubating a single egg under the milder climate experienced by king penguins. Alternatively, it is also possible that king penguins are more resilient to increased CORT levels than Adelie penguins, which is supported by the good reproductive success of king penguins implanted with CORT (X et al. unpublished), contrary to what has been observed in Adelie penguins (Thierry et al., 2013b). Measurements of incubation quality using a dummy egg with temperature and rotation sensors (Thierry et al., 2013a) may provide more accurate data on this question, but it is important to note that the $T_{egg}$ measured in this study (Placebo: 34.9 ± 0.3 °C) was close to the $T_{egg}$ measured in the same penguin colony using internal sensors in dummy eggs (35.7 ± 0.4 °C (Groscolas et al., 2000)). Our results therefore suggest that further studies may benefit from the use of minimally invasive thermal imaging to measure incubation quality, for instance in the context of parental behaviour and climate change (Cook et al., 2020).

The measure of chick surface temperatures revealed some surprising patterns. While we expected CORT chicks to have lower surface temperatures due to poorer parental care (less efficient brooding), we found some opposite result: chicks had higher surface temperatures when brooded by the non-implanted partner of the CORT parent. This result should be considered with caution considering the limited sample size available for chick surface temperatures (N = 16). One possibility is that partners from CORT-implanted individuals somehow perceived the 'stress' levels of their partner (as shown between siblings in yellow legged gulls (*Larus michahellis*); Noguera et al., 2017) and somehow compensated for parental care through more efficient brooding. This hypothesis requires further study to determine whether the concept of 'family-transmitted stress' (Noguera et al., 2017) applies to the king penguin. In this species, both parents must rely on each other over more than one year to successfully fledge their single chick, which itself is entirely dependent on its parents for its food supply throughout its growth. Hence, the king penguin could be promising system to investigate the 'family-transmitted stress' hypothesis.

### 4.4. Conclusion

Evaluating stress levels and responses of free-living animals in a non-invasive manner using thermal imaging is a burgeoning field of research (Jerem et al., 2015; Tabh et al., 2022). However, little is still known about the underlying pathways of stress physiology that influence the changes in surface temperature. Our experimental study in king penguin points toward a likely preponderant role of the SAM axis. Additionally, by summarizing the current evidence for acute changes in surface temperatures during stress exposure (Table 1), we highlight the relative complexity and inconsistency between the effects observed by different studies. Further experimental studies related to SAM and HPA axes
involvement are thus required to clarify what stress component(s) are
measured through non-invasive thermal imaging.

Data availability

The datasets used in this manuscript are available on FigShare
(10.6084/m9 figshare.20134181).

CRediT authorship contribution statement

**Agnès Lewden:** Writing – review & editing, Writing – original draft, Data curation, Conceptualization. **Chelsea Ward:** Investigation. **Aude Noiret:** Writing – review & editing, Data curation. **Sandra Avril:** Writing – review & editing, Investigation. **Lucie Abolivier:** Writing – review & editing, Investigation. **Caroline Gérard:** Writing – review & editing, Investigation. **Tracey L. Hammer:** Investigation, Writing – review & editing. **Émilie Raymond:** Investigation. **Jean-Patrice Robin:** Funding acquisition, Project administration, Writing – review & editing. **Vincent A. Viblanc:** Writing – review & editing, Funding acquisition, Conceptualization. **Pierre Bize:** Writing – review & editing, Supervision, Formal analysis, Conceptualization. **Antoine Stier:** Writing – original draft, Visualization, Validation, Supervision, Project administration, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

Antoine Stier was an Associate Editor of J Thermal Biol at the time of
submitting the manuscript but took no part in the peer review and
decision-making processes for this research article.

Acknowledgements

We are grateful to the French Polar Institute (IPEV) and the Terres
Australes et Antarctiques Françaises for providing financial and logis-
tical support for this study through the polar program #119 (ECO-
NERGY). This study is part of the long-term Studies in Ecology and
Evolution (SEE-Life) program of the CNRS. We wish to thank the Zone
Atelier Antarctique et Terres Australes (ZATA) from the CNRS for
financial support, and the members of the Alfred Faure field station for
their help and support in the field. AL was supported by ISblue project,
Interdisciplinary graduate school for the blue planet (ANR-17-EURE-
0015) and co-funded by a grant from the French government under the
program "Investissements d'Avenir" embedded in France (2030). AS was
supported by a 'Turku Collegium for Science and Medicine' Fellowship,
a Marie Sklodowska-Curie Postdoctoral Fellowships (#894963) and the
IdEx Université de Strasbourg (HotPenguin). SA and CG were funded by
the French Polar Institute.

References

Bates, D., Mächler, M., Bolker, B., Walker, S., 2015. Fitting linear mixed-effects models using lme4. J Stat Softw 67. https://doi.org/10.18637/jss.v067.i01.

Brandl, H.B., Pruessner, J.C., Farine, D.R., 2022. The social transmission of stress in animal collectives. Proc Royal Soc B 289, 20212158. https://doi.org/10.1098/rspb.2021.2158.

Cabanac, A.J., Guillemette, M., 2001. Temperature and heart rate as stress indicators of handled common eider. Physiol. Behav. 74, 475-479. https://doi.org/10.1016/
s0031-9384(01)00586-8.

Cándido, M.G.L., Tinóco, I.F.F., Albino, L.F.T., Freitas, L.C.S.R., Santos, T.C., Cecon, P.R., Gates, R.S., 2020. Effects of heat stress on bullet cloacal and body temperature. Poultry Sci. 99, 2469-2477. https://doi.org/10.1016/j.psy.2019.11.062.

Carere, C., Oers, K. van, 2004. Shy and bold great tits (Parus major): body temperature and breath rate in response to handling stress. Physiol. Behav. 82, 905–912. https://doi.org/10.1016/j.physbeh.2004.07.009.

Cook, T.R., Martin, R., Roberts, J., Häkkinen, H., Botha, P., Meyer, C., Sparks, E., Underhill, L.G., Ryan, P.G., Sherley, R.B., 2020. Parenting in a warming world: thermoregulatory responses to heat stress in an endangered seabird. Conservation Physiology 8, 2283. https://doi.org/10.1093/conphys/coz109, 13.

CoTé, S.D., 2000. Aggressiveness in king penguins in relation to reproductive status and territory location. Anim. Behav. 59, 813-821. https://doi.org/10.1006/anbe.1999.1384.

Deavers, D.R., Musacchia, X.J., 1979. The function of glucocorticoids in thermogenesis. Fed. Proc. 38, 2177-2181.

Duchamp, C., Rouanet, J.-L., Barre, H., 2002. Ontogeny of thermoregulatory mechanisms in king penguin chicks (Aptenodytes patagonicus). Comparative Biochemistry and Physiology. Part. Accel. 131, 765-773. https://doi.org/10.1016/s1095-6433(02) 00014-4.

Edgar, J.L., Lowe, J.C., Paul, E.S., Nicol, C.J., 2011. Avian maternal response to chick distress. Proc. Biol. Sci. 278, 3129-3134. https://doi.org/10.1098/rspb.2010.2701.

Edgar, J.L., Nicol, C.J., 2018. Socially-mediated arousal and contagion within domestic chick broods. Sci Rep-uk 8, 10509. https://doi.org/10.1038/s41598-018-28923-8.

Edgar, J.L., Nicol, C.J., Pugh, C.A., Paul, E.S., 2013. Surface temperature changes in response to handling in domestic chickens. Physiol. Behav. 119, 195-200. https://doi.org/10.1016/j.physbeh.2013.06.020.

Ellenberg, U., Setiawan, A.N., Cree, A., Houston, D.M., Seddon, P.J., 2007. Elevated hormonal stress response and reduced reproductive output in Yellow-eyed penguins exposed to unregulated tourism. Gen Comp Endoc 152, 54-63. https://doi.org/10.1016/j.ygcen.2007.02.022.

Gauchet, L., Grémillet, D., 2022. Using facial infrared thermography to infer avian body temperatures in the wild. Marine Biology.

Giloh, M., Shinder, D., Yahav, S., 2012. Skin surface temperature of broiler chickens is correlated to body core temperature and is indicative of their thermoregulatory status. Poultry Sci. 91, 175-188. https://doi.org/10.3382/ps.2011-01497.

Groscolas, R., Decrock, F., Thil, M.-A., Fayolle, C., Boissery, C., Robin, J.-P., 2000. Refeeding signal in fasting-incubating king penguins: changes in behavior and egg temperature. Am J Physiology-regulatory Integr Comp Physiol 279, R2104-R2112. https://doi.org/10.1152/ajpregu.2000.279.6.r2104.

Halekoh, U., Højsgaard, S., 2014. A Kenward-Roger approximation and Parametric Bootstrap methods for tests in linear mixed models - the R package pbkrtest. J Stat Softw 59. https://doi.org/10.18637/jss.v059.i09.

Handa, R.J., McGivern, R.F., 2017. Reference Module in Neuroscience and Biobehavioral Psychology. https://doi.org/10.1016/b978-0-12-809324-5.02865-0.

Handrich, Y., Bevan, R.M., Charrassin, J.-B., Butler, P.J., Ptz, K., Woakes, A.J., Lage, J., Maho, Y.L., 1997. Hypothermia in foraging king penguins. Nature 388, 64-67. https://doi.org/10.1038/40392.

Herborn, K.A., Graves, J.L., Jerem, P., Evans, N.P., Nager, R., McCafferty, D.J., McKeegan, D.E.F., 2015. Skin temperature reveals the intensity of acute stress. Physiol. Behav. 152, 225-230. https://doi.org/10.1016/j.physbeh.2015.09.032.

Herborn, K.A., Jerem, P., Nager, R.G., McKeegan, D.E.F., McCafferty, D.J., 2018. Surface temperature elevated by chronic and intermittent stress. Physiol. Behav. 191, 47-55. https://doi.org/10.1016/j.physbeh.2018.04.004.

Ikkatai, Y., Watanabe, S., 2015. Eye surface temperature detects stress response in budgerigars (Melopsittacus undulatus). Neuroreport 26, 642-646. https://doi.org/10.1097/wnr.0000000000000403.

Jakubas, D., Wojczulaniak-Jakubas, K., Grissot, A., Devogel, M., Cendrowska, M., Chastel, O., 2022. Eye region surface temperature and corticosterone response to acute stress in a high-Arctic seabird, the little Auk. Animals Open Access J Mdpi 12, 499. https://doi.org/10.3390/ani12040499.

Jerem, P., Herborn, K., McCafferty, D., McKeegan, D., Nager, R., 2015. Thermal imaging to study stress non-invasively in Unrestrained birds. J. Vis. Exp. 53184 https://doi.org/10.3791/53184.

Jerem, P., Jenni-Eiermann, S., Herborn, K., McKeegan, D., McCafferty, D.J., Nager, R.G., 2018. Eye region surface temperature reflects both energy reserves and circulating glucocorticoids in a wild bird. Sci Rep-uk 8, 1907. https://doi.org/10.1038/s41598-018-20204-4.

Jerem, P., Jenni-Eiermann, S., McKeegan, D., McCafferty, D.J., Nager, R.G., 2019. Eye region surface temperature dynamics during acute stress relate to baseline glucocorticoids independently of environmental conditions. Physiol. Behav. 11267 https://doi.org/10.1016/j.physbeh.2019.11267.

Jerem, P., Romero, L.M., 2023. It's cool to be stressed: body surface temperatures track sympathetic nervous system activation during acute stress. J. Exp. Biol. 226, jet246552. https://doi.org/10.1242/jeb.246552.

Knoch, S., Whiteside, M.A., Madden, J.R., Rose, P.E., Fawcett, T.W., 2022. Hot-headed peckers: thermographic changes during aggression among juvenile pheasants (Phasianus colchicus). Philosophical Transactions Royal Soc B 377, 20200442. https://doi.org/10.1098/rstb.2020.0442.

Kriesell, H.J., Aubin, T., Planas-Bielsa, V., Benoiste, M., Bonadonna, F., Gachot-Neveu, H., Maho, Y.L., Schull, Q., Vallas, B., Zahn, S., Bohec, C.L., 2018. Sex identification in King Penguins Aptenodytes patagonicus through morphological and acoustic cues. Ibis 160, 755-768. https://doi.org/10.1111/ibi.12577.

Lenth, R., Singmann, H., Love, J., Buerkner, P., Herve, M., 2018. Emmeans: estimated marginal means, aka least-squares means. R package version. https://doi.org/10.1080/00031305.1980.10483031.

Lewden, A., Bonnet, B., Nord, A., 2020. The metabolic cost of subcutaneous and abdominal rewarming in king penguins after long-term immersion in cold water. J. Therm. Biol. 91, 102638 https://doi.org/10.1016/j.jtherbio.2020.102638.

Lewden, Agnes, Enstipp, M.R., Bonnet, B., Boost, C., Georges, J.-Y., Handrich, Y., 2017a. Thermal strategies of king penguins during prolonged fasting in water. J. Exp. Biol. 220, 4600-4611. https://doi.org/10.1242/jeb.168807.

Lewden, Agnes, Enstipp, M.R., Picard, B., Walsum, T. van, Handrich, Y., 2017b. High peripheral temperatures in king penguins while resting at sea: thermoregulation versus fat deposition. J. Exp. Biol. 220, 3084-3094. https://doi.org/10.1242/jeb.158980.
involvement are thus required to clarify what stress component(s) are
measured through non-invasive thermal imaging.

Data availability

The datasets used in this manuscript are available on FigShare
(10.6084/m9 figshare.20134181).

CRediT authorship contribution statement

Agnès Lewden: Writing – review & editing, Writing – original draft, Data curation, Conceptualization. Chelsea Ward: Investigation. Aude Noiret: Writing – review & editing, Data curation. Sandra Avril: Writing – review & editing, Investigation. Lucie Abolivier: Writing – review & editing, Investigation. Caroline Gérard: Writing – review & editing, Investigation. Tracey L. Hammer: Investigation, Writing – review & editing. Émilie Raymond: Investigation. Jean-Patrice Robin: Funding acquisition, Project administration, Writing – review & editing. Vincent A. Viblanc: Writing – review & editing, Funding acquisition, Conceptualization. Pierre Bize: Writing – review & editing, Supervision, Formal analysis, Conceptualization. Antoine Stier: Writing – original draft, Visualization, Validation, Supervision, Project administration, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

Antoine Stier was an Associate Editor of J Thermal Biol at the time of
submitting the manuscript but took no part in the peer review and
decision-making processes for this research article.

Acknowledgements

We are grateful to the French Polar Institute (IPEV) and the Terres
Australes et Antarctiques Françaises for providing financial and logis-
tical support for this study through the polar program #119 (ECO-
NERGY). This study is part of the long-term Studies in Ecology and
Evolution (SEE-Life) program of the CNRS. We wish to thank the Zone
Atelier Antarctique et Terres Australes (ZATA) from the CNRS for
financial support, and the members of the Alfred Faure field station for
their help and support in the field. AL was supported by ISblue project,
Interdisciplinary graduate school for the blue planet (ANR-17-EURE-
0015) and co-funded by a grant from the French government under the
program "Investissements d'Avenir" embedded in France (2030). AS was
supported by a 'Turku Collegium for Science and Medicine' Fellowship,
a Marie Sklodowska-Curie Postdoctoral Fellowships (#894963) and the
IdEx Université de Strasbourg (HotPenguin). SA and CG were funded by
the French Polar Institute.

References

Bates, D., Mächler, M., Bolker, B., Walker, S., 2015. Fitting linear mixed-effects models using lme4. J Stat Softw 67. https://doi.org/10.18637/jss.v067.i01.

Brandl, H.B., Pruessner, J.C., Farine, D.R., 2022. The social transmission of stress in animal collectives. Proc Royal Soc B 289, 20212158. https://doi.org/10.1098/rspb.2021.2158.

Cabanac, A.J., Guillemette, M., 2001. Temperature and heart rate as stress indicators of handled common eider. Physiol. Behav. 74, 475-479. https://doi.org/10.1016/
s0031-9384(01)00586-8.

Cándido, M.G.L., Tinóco, I.F.F., Albino, L.F.T., Freitas, L.C.S.R., Santos, T.C., Cecon, P.R., Gates, R.S., 2020. Effects of heat stress on bullet cloacal and body temperature. Poultry Sci. 99, 2469-2477. https://doi.org/10.1016/j.psy.2019.11.062.

Carere, C., Oers, K. van, 2004. Shy and bold great tits (Parus major): body temperature and breath rate in response to handling stress. Physiol. Behav. 82, 905-912. https://doi.org/10.1016/j.physbeh.2004.07.009.

Cook, T.R., Martin, R., Roberts, J., Häkkinen, H., Botha, P., Meyer, C., Sparks, E., Underhill, L.G., Ryan, P.G., Sherley, R.B., 2020. Parenting in a warming world: thermoregulatory responses to heat stress in an endangered seabird. Conservation Physiology 8, 2283. https://doi.org/10.1093/conphys/coz109, 13.

CoTé, S.D., 2000. Aggressiveness in king penguins in relation to reproductive status and territory location. Anim. Behav. 59, 813-821. https://doi.org/10.1006/anbe.1999.1384.

Deavers, D.R., Musacchia, X.J., 1979. The function of glucocorticoids in thermogenesis. Fed. Proc. 38, 2177-2181.

Duchamp, C., Rouanet, J.-L., Barre, H., 2002. Ontogeny of thermoregulatory mechanisms in king penguin chicks (Aptenodytes patagonicus). Comparative Biochemistry and Physiology. Part. Accel. 131, 765-773. https://doi.org/10.1016/s1095-6433(02) 00014-4.

Edgar, J.L., Lowe, J.C., Paul, E.S., Nicol, C.J., 2011. Avian maternal response to chick distress. Proc. Biol. Sci. 278, 3129-3134. https://doi.org/10.1098/rspb.2010.2701.

Edgar, J.L., Nicol, C.J., 2018. Socially-mediated arousal and contagion within domestic chick broods. Sci Rep-uk 8, 10509. https://doi.org/10.1038/s41598-018-28923-8.

Edgar, J.L., Nicol, C.J., Pugh, C.A., Paul, E.S., 2013. Surface temperature changes in response to handling in domestic chickens. Physiol. Behav. 119, 195-200. https://doi.org/10.1016/j.physbeh.2013.06.020.

Ellenberg, U., Setiawan, A.N., Cree, A., Houston, D.M., Seddon, P.J., 2007. Elevated hormonal stress response and reduced reproductive output in Yellow-eyed penguins exposed to unregulated tourism. Gen Comp Endoc 152, 54-63. https://doi.org/10.1016/j.ygcen.2007.02.022.

Gauchet, L., Grémillet, D., 2022. Using facial infrared thermography to infer avian body temperatures in the wild. Marine Biology.

Giloh, M., Shinder, D., Yahav, S., 2012. Skin surface temperature of broiler chickens is correlated to body core temperature and is indicative of their thermoregulatory status. Poultry Sci. 91, 175-188. https://doi.org/10.3382/ps.2011-01497.

Groscolas, R., Decrock, F., Thil, M.-A., Fayolle, C., Boissery, C., Robin, J.-P., 2000. Refeeding signal in fasting-incubating king penguins: changes in behavior and egg temperature. Am J Physiology-regulatory Integr Comp Physiol 279, R2104-R2112. https://doi.org/10.1152/ajpregu.2000.279.6.r2104.

Halekoh, U., Højsgaard, S., 2014. A Kenward-Roger approximation and Parametric Bootstrap methods for tests in linear mixed models - the R package pbkrtest. J Stat Softw 59. https://doi.org/10.18637/jss.v059.i09.

Handa, R.J., McGivern, R.F., 2017. Reference Module in Neuroscience and Biobehavioral Psychology. https://doi.org/10.1016/b978-0-12-809324-5.02865-0.

Handrich, Y., Bevan, R.M., Charrassin, J.-B., Butler, P.J., Ptz, K., Woakes, A.J., Lage, J., Maho, Y.L., 1997. Hypothermia in foraging king penguins. Nature 388, 64-67. https://doi.org/10.1038/40392.

Herborn, K.A., Graves, J.L., Jerem, P., Evans, N.P., Nager, R., McCafferty, D.J., McKeegan, D.E.F., 2015. Skin temperature reveals the intensity of acute stress. Physiol. Behav. 152, 225-230. https://doi.org/10.1016/j.physbeh.2015.09.032.

Herborn, K.A., Jerem, P., Nager, R.G., McKeegan, D.E.F., McCafferty, D.J., 2018. Surface temperature elevated by chronic and intermittent stress. Physiol. Behav. 191, 47-55. https://doi.org/10.1016/j.physbeh.2018.04.004.

Ikkatai, Y., Watanabe, S., 2015. Eye surface temperature detects stress response in budgerigars (Melopsittacus undulatus). Neuroreport 26, 642-646. https://doi.org/10.1097/wnr.0000000000000403.

Jakubas, D., Wojczulaniak-Jakubas, K., Grissot, A., Devogel, M., Cendrowska, M., Chastel, O., 2022. Eye region surface temperature and corticosterone response to acute stress in a high-Arctic seabird, the little Auk. Animals Open Access J Mdpi 12, 499. https://doi.org/10.3390/ani12040499.

Jerem, P., Herborn, K., McCafferty, D., McKeegan, D., Nager, R., 2015. Thermal imaging to study stress non-invasively in Unrestrained birds. J. Vis. Exp. 53184 https://doi.org/10.3791/53184.

Jerem, P., Jenni-Eiermann, S., Herborn, K., McKeegan, D., McCafferty, D.J., Nager, R.G., 2018. Eye region surface temperature reflects both energy reserves and circulating glucocorticoids in a wild bird. Sci Rep-uk 8, 1907. https://doi.org/10.1038/s41598-018-20204-4.

Jerem, P., Jenni-Eiermann, S., McKeegan, D., McCafferty, D.J., Nager, R.G., 2019. Eye region surface temperature dynamics during acute stress relate to baseline glucocorticoids independently of environmental conditions. Physiol. Behav. 11267 https://doi.org/10.1016/j.physbeh.2019.11267.

Jerem, P., Romero, L.M., 2023. It's cool to be stressed: body surface temperatures track sympathetic nervous system activation during acute stress. J. Exp. Biol. 226, jet246552. https://doi.org/10.1242/jeb.246552.

Knoch, S., Whiteside, M.A., Madden, J.R., Rose, P.E., Fawcett, T.W., 2022. Hot-headed peckers: thermographic changes during aggression among juvenile pheasants (Phasianus colchicus). Philosophical Transactions Royal Soc B 377, 20200442. https://doi.org/10.1098/rstb.2020.0442.

Kriesell, H.J., Aubin, T., Planas-Bielsa, V., Benoiste, M., Bonadonna, F., Gachot-Neveu, H., Maho, Y.L., Schull, Q., Vallas, B., Zahn, S., Bohec, C.L., 2018. Sex identification in King Penguins Aptenodytes patagonicus through morphological and acoustic cues. Ibis 160, 755-768. https://doi.org/10.1111/ibi.12577.

Lenth, R., Singmann, H., Love, J., Buerkner, P., Herve, M., 2018. Emmeans: estimated marginal means, aka least-squares means. R package version. https://doi.org/10.1080/00031305.1980.10483031.

Lewden, A., Bonnet, B., Nord, A., 2020. The metabolic cost of subcutaneous and abdominal rewarming in king penguins after long-term immersion in cold water. J. Therm. Biol. 91, 102638 https://doi.org/10.1016/j.jtherbio.2020.102638.

Lewden, Agnes, Enstipp, M.R., Bonnet, B., Boost, C., Georges, J.-Y., Handrich, Y., 2017a. Thermal strategies of king penguins during prolonged fasting in water. J. Exp. Biol. 220, 4600-4611. https://doi.org/10.1242/jeb.168807.

Lewden, Agnes, Enstipp, M.R., Picard, B., Walsum, T. van, Handrich, Y., 2017b. High peripheral temperatures in king penguins while resting at sea: thermoregulation versus fat deposition. J. Exp. Biol. 220, 3084-3094. https://doi.org/10.1242/jeb.158980.
MacDougall-Shackleton, S.A., Bonier, F., Romero, L.M., Moore, I.T., 2019. Glucocorticoids and “stress” are not Synonymous. Integr Org Biology 1, obz017. https://doi.org/10.1093/lob/obz017.

Moe, R.O., Bohlin, J., Flø, A., Vasdal, G., Stubsjøen, S.M., 2017. Hot chicks, cold feet. Physiol. Behav. 179, 42–48. https://doi.org/10.1016/j.physbeh.2017.05.025.

Narushin, V.G., 2005. Egg geometry calculation using the measurements of length and breadth. Poultry Sci. 84, 482–484. https://doi.org/10.1093/ps/84.3.482.

Noguera, J.C., Kim, S.-Y., Velando, A., 2017. Family-transmitted stress in a wild bird. Proc. Natl. Acad. Sci. USA 27, 201706164. https://doi.org/10.1086/680688.

Oka, T., 2018. Chapter 35 Stress-induced hyperthermia and hypothermia. Handb. Clin. Neurol. 157, 599–621. https://doi.org/10.1016/b978-0-444-64074-1.00035-5.

Ouyang, J.Q., Macaballug, P., Chen, H., Hodach, K., Tang, S., Francis, J.S., 2021. Infrared thermography is an effective, noninvasive measure of HPA activation. Ann Ny Acad Sci 24, 1–6. https://doi.org/10.1080/10253890.2020.1868431.

Pijpers, N., Heuvel, H. van den, Duncan, I.H., Yorzinski, J., Neethirajan, S., 2022. Understanding chicks' Emotions: are eye Blinks & facial temperatures reliable indicators? bioRxiv. https://doi.org/10.1101/2022.01.31.478468, 2022.01.31.478468.

Playà-Montmany, N., Tattersall, G.J., 2021. Spot size, distance and emissivity errors in field applications of infrared thermography. Methods Ecol. Evol. 12, 828–840. https://doi.org/10.1111/2041-210x.13563.

Regel, J., Pütz, K., 1997. Effect of human disturbance on body temperature and energy expenditure in penguins. Polar Biol. 18, 246–253. https://doi.org/10.1007/s003000050185.

Robertson, J.K., Mastromonaco, G., Burness, G., 2020a. Evidence that stress-induced changes in surface temperature serve a thermoregulatory function. J. Exp. Biol. 223, jeb213421. https://doi.org/10.1242/jeb.213421.

Robertson, J.K., Mastromonaco, G.F., Burness, G., 2020b. Social hierarchy reveals thermoregulatory trade-offs in response to repeated stressors. J. Exp. Biol. 223, jeb229047 https://doi.org/10.1242/jeb.229047.

Romero, L.M., 2004. Physiological stress in ecology: lessons from biomedical research. Trends Ecol. Evol. 19, 249–255. https://doi.org/10.1016/j.tree.2004.03.008.

Sapolsky, R.M., Romero, L.M., Munck, A.U., 2000. How do glucocorticoids influence stress responses? Integrating Permissive, Suppressive, stimulatory, and Preparative Actions. Endocr. Rev. 21, 55–89. https://doi.org/10.1210/edrv.21.1.0389.

Smulders, T.V., 2021. Telencephalic regulation of the HPA axis in birds. Neurobiol. Stress 15, 100351. https://doi.org/10.1016/j.ynstr.2021.100351.

Stier, A., Schull, Q., Bize, P., Lefol, E., Haussmann, M., Roussel, D., Robin, J.-P., Viblanc, V.A., 2019. Oxidative stress and mitochondrial responses to stress exposure suggest that king penguins are naturally equipped to resist stress. Sci. Rep. 9, 8545. https://doi.org/10.1038/s41598-019-44990-x.

Tabh, J.K.R., Burness, G., Wearing, O.H., Tattersall, G.J., Mastromonaco, G.F., 2021. Infrared thermography as a technique to measure physiological stress in birds: body region and image angle matter. Physiological Reports 9, e14865. https://doi.org/10.14814/phy2.14865.

Tabh, J.K.R., Mastromonaco, G.F., Burness, G., 2022. Stress-induced changes in body surface temperature are repeatable, but do not differ between urban and rural birds. Oecologia 1–15. https://doi.org/10.1007/s00442-022-05120-z.

Tattersall, G.J., Andrade, D.V., Abe, A.S., 2009. Heat Exchange from the Toucan bill reveals a controllable vascular thermal Radiator. Science 325, 468–470. https://doi.org/10.1126/science.1175553.

Thierry, A.-M., Massemin, S., Handrich, Y., Raclot, T., 2013a. Elevated corticosterone levels and severe weather conditions decrease parental investment of incubating Adélie penguins. Horm. Behav. 63, 475–483. https://doi.org/10.1016/j.ybeh.2012.12.011.

Thierry, A.-M., Ropert-Coudert, Y., Raclot, T., 2013b. Elevated corticosterone levels decrease reproductive output of chick-rearing Adélie penguins but do not affect chick mass at fledging. Conservation Physiology 1, cot007. https://doi.org/10.1093/conphys/cot007.

Thomas, D.B., Ksepka, D.T., Fordyce, R.E., 2011. Penguin heat-retention structures evolved in a greenhouse Earth. Biol. Lett. 7, 461–464. https://doi.org/10.1098/rsbl.2010.0993.

Torres-Medina, F., Cabezas, S., Marchant, T.A., Wikelski, M., Romero, L.M., Hau, M., Carrete, M., Tella, J.L., Blas, J., 2018. Corticosterone implants make stress hyporesponsive birds. The Journal of Experimental Biology jeb 173864–43. https://doi.org/10.1242/jeb.173864.

Viblanc, V.A., Dobson, F.S., Stier, A., Schull, Q., Saraux, C., Gineste, B., Pardonnet, S., Kauffmann, M., Robin, J.-P., Bize, P., 2016. Mutually honest? Physiological ‘qualities’ signalled by colour ornaments in monomorphic king penguins. Biol. J. Linn. Soc. 118, 200–214. https://doi.org/10.1111/bij.12729.

Viblanc, V.A., Schull, Q., Cornioley, T., Stier, A., Ménard, J.-J., Groscolas, R., Robin, J.-P., 2018. An integrative appraisal of the hormonal and metabolic changes induced by acute stress using king penguins as a model. Gen Comp Endocr 269, 1–10. https://doi.org/10.1016/j.ygcen.2017.08.024.

Viblanc, V.A., Valette, V., Kauffmann, M., Malosse, N., Groscolas, R., 2012. Coping with social stress: heart rate responses to agonistic interactions in king penguins. Behav. Ecol. 23, 1178–1185. https://doi.org/10.1093/beheco/ars095.

Weimer, S.L., Wideman, R.F., Scanes, C.G., Mauromoustakos, A., Christensen, K.D., Vizzier-Thaxton, Y., 2020. Broiler stress responses to light intensity, flooring type, and leg weakness as assessed by heterophil-to-lymphocyte ratios, serum corticosterone, infrared thermography, and latency to lie. Poultry Sci. 99, 3301–3311. https://doi.org/10.1016/j.psj.2020.03.028.

Weimerskirch, H., Stahl, J.C., Jouventin, P., 1992. The breeding biology and population dynamics of king penguins Aptenodytes patagonica on the Crozet Islands. Ibis 134, 107–117.

Winder, L.A., White, S.A., Nord, A., Helm, B., McCafferty, D.J., 2020. Body surface temperature responses to food restriction in wild and captive great tits. J. Exp. Biol. 223, jeb220046 https://doi.org/10.1242/jeb.220046, 8.

Wingfield, J.C., Romero, L.M., 2015. In: Tempestes, Poxes, Predators, and People, pp. 149–196. https://doi.org/10.1093/acprof:oso/9780195366693.003.0005.

Zuluaga, J.D., Danner, R.M., 2023. Acute stress and restricted diet reduce bill-mediated heat dissipation in the song sparrow (Melospiza melodia): implications for optimal thermoregulation. J. Exp. Biol. 226 https://doi.org/10.1242/jeb.245316.
MacDougall-Shackleton, S.A., Bonier, F., Romero, L.M., Moore, I.T., 2019. Glucocorticoids and “stress” are not Synonymous. Integr Org Biology 1, obz017. https://doi.org/10.1093/lob/obz017.

Moe, R.O., Bohlin, J., Flø, A., Vasdal, G., Stubsjøen, S.M., 2017. Hot chicks, cold feet. Physiol. Behav. 179, 42–48. https://doi.org/10.1016/j.physbeh.2017.05.025.

Narushin, V.G., 2005. Egg geometry calculation using the measurements of length and breadth. Poultry Sci. 84, 482–484. https://doi.org/10.1093/ps/84.3.482.

Noguera, J.C., Kim, S.-Y., Velando, A., 2017. Family-transmitted stress in a wild bird. Proc. Natl. Acad. Sci. USA 27, 201706164. https://doi.org/10.1086/680688.

Oka, T., 2018. Chapter 35 Stress-induced hyperthermia and hypothermia. Handb. Clin. Neurol. 157, 599–621. https://doi.org/10.1016/b978-0-444-64074-1.00035-5.

Ouyang, J.Q., Macaballug, P., Chen, H., Hodach, K., Tang, S., Francis, J.S., 2021. Infrared thermography is an effective, noninvasive measure of HPA activation. Ann Ny Acad Sci 24, 1–6. https://doi.org/10.1080/10253890.2020.1868431.

Pijpers, N., Heuvel, H. van den, Duncan, I.H., Yorzinski, J., Neethirajan, S., 2022. Understanding chicks' Emotions: are eye Blinks & facial temperatures reliable indicators? bioRxiv. https://doi.org/10.1101/2022.01.31.478468, 2022.01.31.478468.

Playà-Montmany, N., Tattersall, G.J., 2021. Spot size, distance and emissivity errors in field applications of infrared thermography. Methods Ecol. Evol. 12, 828–840. https://doi.org/10.1111/2041-210x.13563.

Regel, J., Pütz, K., 1997. Effect of human disturbance on body temperature and energy expenditure in penguins. Polar Biol. 18, 246–253. https://doi.org/10.1007/s003000050185.

Robertson, J.K., Mastromonaco, G., Burness, G., 2020a. Evidence that stress-induced changes in surface temperature serve a thermoregulatory function. J. Exp. Biol. 223, jeb213421. https://doi.org/10.1242/jeb.213421.

Robertson, J.K., Mastromonaco, G.F., Burness, G., 2020b. Social hierarchy reveals thermoregulatory trade-offs in response to repeated stressors. J. Exp. Biol. 223, jeb229047 https://doi.org/10.1242/jeb.229047.

Romero, L.M., 2004. Physiological stress in ecology: lessons from biomedical research. Trends Ecol. Evol. 19, 249–255. https://doi.org/10.1016/j.tree.2004.03.008.

Sapolsky, R.M., Romero, L.M., Munck, A.U., 2000. How do glucocorticoids influence stress responses? Integrating Permissive, Suppressive, stimulatory, and Preparative Actions. Endocr. Rev. 21, 55–89. https://doi.org/10.1210/edrv.21.1.0389.

Smulders, T.V., 2021. Telencephalic regulation of the HPA axis in birds. Neurobiol. Stress 15, 100351. https://doi.org/10.1016/j.ynstr.2021.100351.

Stier, A., Schull, Q., Bize, P., Lefol, E., Haussmann, M., Roussel, D., Robin, J.-P., Viblanc, V.A., 2019. Oxidative stress and mitochondrial responses to stress exposure suggest that king penguins are naturally equipped to resist stress. Sci. Rep. 9, 8545. https://doi.org/10.1038/s41598-019-44990-x.

Tabh, J.K.R., Burness, G., Wearing, O.H., Tattersall, G.J., Mastromonaco, G.F., 2021. Infrared thermography as a technique to measure physiological stress in birds: body region and image angle matter. Physiological Reports 9, e14865. https://doi.org/10.14814/phy2.14865.

Tabh, J.K.R., Mastromonaco, G.F., Burness, G., 2022. Stress-induced changes in body surface temperature are repeatable, but do not differ between urban and rural birds. Oecologia 1–15. https://doi.org/10.1007/s00442-022-05120-z.

Tattersall, G.J., Andrade, D.V., Abe, A.S., 2009. Heat Exchange from the Toucan bill reveals a controllable vascular thermal Radiator. Science 325, 468–470. https://doi.org/10.1126/science.1175553.

Thierry, A.-M., Massemin, S., Handrich, Y., Raclot, T., 2013a. Elevated corticosterone levels and severe weather conditions decrease parental investment of incubating Adélie penguins. Horm. Behav. 63, 475–483. https://doi.org/10.1016/j.yhbeh.2012.12.011.

Thierry, A.-M., Ropert-Coudert, Y., Raclot, T., 2013b. Elevated corticosterone levels decrease reproductive output of chick-rearing Adélie penguins but do not affect chick mass at fledging. Conservation Physiology 1, cot007. https://doi.org/10.1093/conphys/cot007.

Thomas, D.B., Ksepka, D.T., Fordyce, R.E., 2011. Penguin heat-retention structures evolved in a greenhouse Earth. Biol. Lett. 7, 461–464. https://doi.org/10.1098/rsbl.2010.0993.

Torres-Medina, F., Cabezas, S., Marchant, T.A., Wikelski, M., Romero, L.M., Hau, M., Carrete, M., Tella, J.L., Blas, J., 2018. Corticosterone implants make stress hyporesponsive birds. The Journal of Experimental Biology jeb 173864–43. https://doi.org/10.1242/jeb.173864.

Viblanc, V.A., Dobson, F.S., Stier, A., Schull, Q., Saraux, C., Gineste, B., Pardonnet, S., Kauffmann, M., Robin, J.-P., Bize, P., 2016. Mutually honest? Physiological ‘qualities’ signalled by colour ornaments in monomorphic king penguins. Biol. J. Linn. Soc. 118, 200–214. https://doi.org/10.1111/bij.12729.

Viblanc, V.A., Schull, Q., Cornioley, T., Stier, A., Ménard, J.-J., Groscolas, R., Robin, J.-P., 2018. An integrative appraisal of the hormonal and metabolic changes induced by acute stress using king penguins as a model. Gen Comp Endocr 269, 1–10. https://doi.org/10.1016/j.ygcen.2017.08.024.

Viblanc, V.A., Valette, V., Kauffmann, M., Malosse, N., Groscolas, R., 2012. Coping with social stress: heart rate responses to agonistic interactions in king penguins. Behav. Ecol. 23, 1178–1185. https://doi.org/10.1093/beheco/ars095.

Weimer, S.L., Wideman, R.F., Scanes, C.G., Mauromoustakos, A., Christensen, K.D., Vizzier-Thaxton, Y., 2020. Broiler stress responses to light intensity, flooring type, and leg weakness as assessed by heterophil-to-lymphocyte ratios, serum corticosterone, infrared thermography, and latency to lie. Poultry Sci. 99, 3301–3311. https://doi.org/10.1016/j.psj.2020.03.028.

Weimerskirch, H., Stahl, J.C., Jouventin, P., 1992. The breeding biology and population dynamics of king penguins Aptenodytes patagonica on the Crozet Islands. Ibis 134, 107–117.

Winder, L.A., White, S.A., Nord, A., Helm, B., McCafferty, D.J., 2020. Body surface temperature responses to food restriction in wild and captive great tits. J. Exp. Biol. 223, jeb220046 https://doi.org/10.1242/jeb.220046, 8.

Wingfield, J.C., Romero, L.M., 2015. In: Tempestes, Poxes, Predators, and People, pp. 149–196. https://doi.org/10.1093/acprof:oso/9780195366693.003.0005.

Zuluaga, J.D., Danner, R.M., 2023. Acute stress and restricted diet reduce bill-mediated heat dissipation in the song sparrow (Melospiza melodia): implications for optimal thermoregulation. J. Exp. Biol. 226 https://doi.org/10.1242/jeb.245316.