Agnès Lewden
email: a.m.lewden@leeds.ac.uk

Batshéva Bonnet

Andreas Nord

The metabolic cost of subcutaneous and abdominal rewarming in King Penguins after long-term immersion in cold water

Keywords: Subcutaneous temperature, Rewarming, Metabolism, Normothermia

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Introduction

Aquatic endotherms, such as diving birds, experience higher heat loss rate in water than in air of the same temperature, owing to the considerably higher thermal conductivity and specific heat capacity of water [START_REF] Bullard | Problems of body heat loss in water immersion[END_REF][START_REF] Gagge | Heat exchange between human skin surface and thermal environment[END_REF][START_REF] Dejours | Water and air physical characteristics and their physiological consequences[END_REF]. This explains why metabolic rate may be twice as high when the animal is submerged and there is no heat substitution from exercise [START_REF] Scholander | Experimental investigations on the respiratory function in diving mammals and birds[END_REF][START_REF] Kooyman | Heat loss in penguins during immersion and compression[END_REF][START_REF] Stahel | Temperature regulation in the little penguin, Eudyptula minor, in air and water[END_REF][START_REF] Ponganis | Diving physiology in marine mammals and seabirds[END_REF]. Heat loss rate in water is reduced by body insulation provided by subcutaneous fat and a coat that is largely impenetrable water [START_REF] Kooyman | Heat loss in penguins during immersion and compression[END_REF], as well as by counter-current heat exchange in the appendages and vasoconstriction in the trunk that diminishes the thermal gradient between the body and the environment (Bulbard and Rapp, 1970).

The king penguin (Aptenodytes patagonicus) has a resting metabolic rate (RMR) that is 83% higher in water than in air [START_REF] Froget | Heart rate and energetics of free-ranging king penguins (Aptenodytes patagonicus)[END_REF]. RMR is also higher in fasting compared to fed king penguins [START_REF] Fahlman | Metabolism and thermoregulation during fasting in king penguins, Aptenodytes patagonicus, in air and water[END_REF]. This difference could be explained by lower thermal conductance in fed individuals if they have a thicker layer of subcutaneous fat than fasted birds [START_REF] Fahlman | Metabolism and thermoregulation during fasting in king penguins, Aptenodytes patagonicus, in air and water[END_REF]. However, RMR in fasting king penguins is higher also than in refed (i.e. fed once after several days of fasting) birds, where there is no difference in thickness of the subcutaneous fat layer between the two groups [START_REF] Halsey | Recovery from swimming-induced hypothermia in king penguins: effects of nutritional condition[END_REF]. It is, thus, possible that differences in RMR between birds in different nutritional conditions could be due to adjustments of thermal conductance other than that following changes in body composition. For example, re-fed birds (that need to digest food) may divert most blood flow to the splanchnic region and limit circulation to peripheral tissue, which would result in lower heat loss rate. Moreover, the flexibility of subcutaneous blood perfusion, which is reflected by changes in subcutaneous temperature, is well known in this species. In water, fed adult king penguins maintained lower subcutaneous temperature than fasted individuals (Lewden et al., 2017b), and showed an increase of metabolic rate in parallel to increasing flank temperature (Lewden et al., 2017b). Recently, variation in subcutaneous blood perfusion has also been demonstrated in moulting immature king penguins ashore [START_REF] Enstipp | The dive performance of immature king penguins following their annual molt suggests physiological constraints[END_REF]. However, there has been no measurement of how peripheral blood perfusion changes when king penguins recover normothermia on land after long periods in cold water. This could inform on whether local heterothermy is involved in the lower RMR observed in fed compared to fasted and re-fed king penguins.

The aim of this study was to measure subcutaneous and abdominal tissue temperatures during rewarming to normothermia (≥ 37.5°C) on land in fasted and fed king penguins, and to determine the energy cost of rewarming in these different nutritional states. To this end, we equipped wild king penguins with four temperatures loggers; one in the abdominal cavity (i.e. deep tissue) and three in subcutaneous adipose tissues (flank, back and brood-patch areas). The birds were then maintained in a cold sea water tank for several days without, or with food, where they showed soaking-induced hypothermia (Lewden et al., 2017a). We then removed the birds from the sea water tank and recorded the deep and subcutaneous temperature patterns during recovery to normothermia when they dried on land. We hypothesized that, compared to fasted birds, fed individuals should have a slower recovery of subcutaneous temperatures and, as a result, possibly also a faster recovery of abdominal temperature if warm blood has to be directed to the viscera to aid digestion. Because slower recovery of subcutaneous temperatures would contribute to reduced heat loss rate, a retarded response at the body surface might also convey energy savings during internal recovery. Thus, secondly, we also measured metabolic rate during the recovery phase (MRR), to test the hypothesis that MRR is lower before subcutaneous temperatures have recovered, possibly owing to reduced heat loss rate, and that this reduction would be even larger in fed birds.

Material and methods

King penguins were caught during the courtship phase of their breeding cycle at the colony 'La Baie du Marin', on Possession Island, Crozet Archipelago, in the Southern Indian Ocean (46°4' S, 51°8' E). The experiments were conducted during three Austral summers (November to March in 2013/2014;2014[START_REF] Ponganis | Diving physiology in marine mammals and seabirds[END_REF][START_REF] Ponganis | Diving physiology in marine mammals and seabirds[END_REF]/2016). Experimental procedures were approved by the French Ethical Committee (APAFIS; permit no. 02015041411414001) and the French Polar Environmental Committee (permit no. 2013-76, 2014-121).

On the day of capture, individuals were weighed and equipped with four temperature loggers (iButton MXMDS1922L-F5; AVNET-MEMEC; resolution: ± 0.0625°C; range: 0-50°C; accuracy ± 0.1°C) implanted into subcutaneous tissues in the flank, back, and brood patch, and into the abdominal cavity (behind the brood patch), following the surgical procedures described in Lewden et al. (2017a). The abdominal temperature recorded this way is similar to the 'lower abdominal temperature' measured in previous studies on the same species [START_REF] Fahlman | Metabolism and thermoregulation during fasting in king penguins, Aptenodytes patagonicus, in air and water[END_REF][START_REF] Halsey | Recovery from swimming-induced hypothermia in king penguins: effects of nutritional condition[END_REF]. Even if this measurement may not be as close to true core as stomach temperature (cf. [START_REF] Handrich | Hypothermia in foraging king penguins[END_REF]) (see also below), lower abdominal temperature still responds to foraging and digestive activity in a qualitatively similar manner (cf. Lewden et al. 2017a, b). This makes it an appropriate metric when studying thermal and metabolic responses to manipulation of nutritional condition (see 2.1, below). The loggers recorded temperature every 11 min. Following surgery, birds were kept together in pairs in a wooden enclosure on land (3 × 3 m, without roof) without disturbance, apart from daily feeding, during 6.0 ± 0.1 (s.d.) days. This recovery period was longer than what is required for king penguins to revert to normal behavior after surgery (i.e. 2 days; [START_REF] Froget | Heart rate and energetics of free-ranging king penguins (Aptenodytes patagonicus)[END_REF]. As a result, our birds had ample time to acclimate to the feeding manipulation. After the recovery period, the penguins were immersed together in a sea water tank (2.5 m length × 1.3 m width × 1.2 m height; with a water volume of 2.5 m 3 ) with a constant flow of clean sea water pumped from an adjacent bay. This ensured a stable water temperature of 7.7 ± 0.6°C (s.e.) during the three years of experiment.

Nutritional state

During the three years, a total of 18 individuals with a mean body mass at capture of 13.6 ± 0.2 kg (s.e.) were maintained fasting in the sea water tank for 58:44 ± 04:18 hours (s.e.; range: 20:32 to 111:13 h) after having completely recover from surgery. During this time, they lost 210 ± 0.02 g (s.e.) body mass daily. After the fasting period, the birds were allowed to recover from soaking-induced hypothermia in the wooden enclosure, ashore at thermoneutrality (following [START_REF] Froget | The heart rate/oxygen consumption relationship during cold exposure of the king penguin: a comparison with that during exercise[END_REF]. Mean body mass after fasting was 11.4 ± 0.1 kg. During the second and third years of the experiment, we also studied body temperature recovery in 12 individuals that were re-fed once the fasting measurements were completed.

Just as when measured in the fasting condition, the fed birds were maintained day and night in the sea water tank, but were briefly (≤ 10 min) removed from the tank to be fed with a fish meal of 0.3 ± 0.1 kg (s.e.) four times daily (i.e. 1.2 kg fish per bird and day) distributed evenly throughout the photophase (Lewden et al., 2017a). After each meal, the individuals were immediately put back into the sea water tank. In the second year of the study, the fed birds then fasted for one night (fasting duration of 15:14 ± 00:03 h) before being removed from the water and allowed to recover once on land. In the third year, fed penguins were measured more frequently (1 to 6 times per bird) after a shorter mean fasting duration of 06:04 ± 01:08. The birds gained 279 ± 0.05 g (s.e.) daily during the feeding treatment. Mean body mass during the whole measurement period in the fed birds (11.9 ± 0.2 kg) did not differ significantly from that during measurements in the fasting condition (P=0.9). When averaging the full data set, the fasted birds had not eaten for 58:44 ± 04:18 h (range between 20:32 and 111:13), and the fed birds were measured 08:13 ± 01:07 h (range: 04:01 to 15:25 h) after the last meal.

Body temperatures during recovery

During the first year, we measured recovery temperatures once, after the penguins had spent 39:10 ± 00:10 h in the sea water tank. In the second year of the study, the penguins were measured twice; once in a fasted condition after 94:18± 00:07 h spent in the sea water tank, and once in a fed condition when the birds had been in the sea water tank for 116:38 ± 00:19 h. In the third year, the individuals were removed more frequently (means of 4 times in fasted and 3 times fed condition per bird) to be measured (see above), starting when they had spent 03:17 h in the sea water tank, and extending until 168:35 h into the experiment.

The start of the recovery period was defined as the time when the individual was removed from the sea water tank (time = 0). During the first two years of the study, we analyzed flank (Tflk), back (Tback), brood patch (Tbrood) and abdominal cavity (Tabd) temperatures during 106 ± 01 min (s.e.) after the start of recovery (range: 60 to 121 min). Data were collected from N = 13 birds in n = 26 session (i.e., two measurements per bird; Fig. 1). In the third year, measurements (N =8; n = 40) were collected during 71 ± 03 min (s.e.) from the start of recovery (range: 33 to 121 min). Normothermia was defined as a tissue temperature ≥ 37.5°C, which is within ± 1 °C of normothermic tissue temperatures for the four body parts previously measured in dry king penguins resting on land (Lewden et al., 2017a, b).

Recovery metabolic rate (MRR)

In the third year of the study, we measured oxygen consumption during the recovery phase (recovery metabolic rate, MRR) ashore at an air temperature of 8.11 ± 0.28°C (s.e.).

Measurements were performed on 8 individuals in both nutritional conditions (1 to 12 measurements per individual; n = 20 in each of the fasted and fed groups, respectively) during 60 ± 5 min (s.e.) in the beginning of the recovery period. The first MRR data were recorded within 2 min of removing the bird from the sea water tank. On average, the fasted birds here had not eaten for 59:50 ± 05:27 h (range: 15:21 to 112:09 h) during MRR recordings, whereas the fed birds were measured 06:59 ± 00:42 h after last meal (range: 01:32 to 13:34 h). In this sub-sample, fasted individuals (11.5 ± 0.1 kg) had lower body mass than the fed ones (12.0 ± 0.2 kg; paired t-test: P = 0.0143). Six of these 8 penguins were also measured once when they were dry, fed and normothermic (i.e. all tissues ≥ 37.5°C), to collect steady-state data on resting metabolic rate (RMR) for comparison to MRR.

We used an open flow respirometry system ('Turbofox'; Sable Systems, Henderson, NV, USA) composed of a cylindrical 60 l metabolic chamber put over the bird like a dome. Ambient air was pulled through the metabolic chamber at rate of 40 l min -1 . Flow rate was measured and maintained by the mass flow meter and controller unit in the Turbofox. A sub-sample of 200 ml min -1 sample air was measured for oxygen and carbon dioxide concentration. These data were recorded every 4 s using the software Expedata (Sable Systems). Baseline air was recorded before and after each measurement session. We calibrated the instruments before each trial as detailed in Lewden et al., 2017b. Briefly, the O2 analysers was span calibrated to 20.95 % O2 using outside air scrubbed of water vapour, and the CO2 analyser zero-calibrated using a CO2 scrubber (Ascarite and soda lime; all chemicals from Sigma-Aldrich Chemie, Lyon, France). Then, the CO2 analyser was spanned with 0.49% CO2 (Alphagaz; Air Liquide, Paris, France). We calculated oxygen consumption (ml.O2.min -1 ) using eq. 11.2 and CO2 production using eq. 11.4 in [START_REF] Lighton | Measuring metabolic rates -a manual for scientists[END_REF], and converted these data to metabolic rates (W) using an energy equivalence of 20 J.ml O2 -1 [START_REF] Kleiber | The Fire of Life. An Introduction to Animal Energetics[END_REF]. We used these data to calculated the respiratory quotient (RQ) (i.e. VCO2 / VO2). There was no significant difference in RQ in the fasted and fed birds (both 0.74 ± 0.05; for 20 fasted and 20 fed measurements) (paired ttest: P = 0.9).

Data analyses

Statistical analyses were conducted in R 3.4.4 (R Core Team 2018) and JMP® v. 13 (SAS Institute Inc., Cary, North Carolina, USA). For each measurement session, we determined individual breakpoint temperatures and times for each tissue using the 'lm.br' function in lm.br package [START_REF] Adams | br: Linear Model with Breakpoint[END_REF], to identify the inflection point that marked the end of rewarming. All tissue temperatures were stable after the estimated breakpoint (change 0.00 ± 0.00 °C min -1 in all cases). When tissue temperature was stable (± 0.5 °C relative to initial temperature) from the start of the trial onwards, we set breakpoint temperature to equal initial temperature and breakpoint time to 0. To describe body temperature during the rewarming period (i.e. before the breakpoint), we separately modelled the initial temperature (Tinitial), the rewarming rate (°C.min -1 gained before the breakpoint when tissue temperatures increased linearly with time), the time to reach the breakpoint and the temperature at recovery (Trecovery) using general linear mixed models (GLMM) including nutritional state (i.e. fasted and fed), tissue, the interaction "nutritional state × tissue" and year as fixed factors. Bird ID as a random intercept. Recovery patterns were not affected by respirometry measurement (all P > 0.1). We used the Tflk and Tabd data to estimate the metabolic rates during rewarming (i.e., MRR) of peripheral and deep tissues, alone and in combination. This was achieved by first defining five categories of body temperature recovery, viz. (i) rewarming of both subcutaneous (i.e. Tflk) and deep (i.e. Tabd) temperatures (henceforth 'global rewarming'); (ii) rewarming of deep temperature when subcutaneous temperature had recovered (henceforth 'deep rewarming'); or (iii) rewarming of subcutaneous temperature when deep temperature had recovered (henceforth 'subcutaneous rewarming'); (iv) recovered, with both tissues ≥ 37.5 °C and (v) stable normothermia throughout the experiment. We then aligned the body temperature and MR data sets by time, and used the breakpoint times defined for tissue temperatures to calculate MRR as the mean MR during each of the rewarming events.

We, firstly, tested how MRR (W) and mass-specific MRR (i.e. W.kg -1 ) was affected by nutritional state using a GLMM with thermal state, nutritional state ('fasted' or 'fed'), and the interactions "thermal state × nutritional state" as covariates and bird ID as a random factor. We also fitted two separate linear mixed effects model (LMEs) to test how massspecific MRR (averaged of rewarming categories) was affected by ∆Tflk (i.e. recovery Tflkinitial Tflk) and ∆Tabd (i.e. recovery Tabd -initial Tabd), using bird ID as random factor. We could not include ∆Tflk and ∆Tabd in the same model because of collinearity between these variables (VIF > 4).

The final models were derived using backward elimination of non-significant (P > 0.05) terms, starting with the interactions. We used a Tukey's HSD test to assess differences between groups/tissues for significant model terms. All results are reported as predicted means ± s.e.

Results

Body temperatures during recovery

We recorded a total of 268 rewarming events in the four tissues (67 events per tissue; Table 1). Tback showed the highest temperatures (Fig. 1), and was already at normothermia at the first temperature measurement in 49 % of cases. The corresponding values for Tflk, Tabd and Tbrood was 19, 10 and 3 %, respectively (Table 1). These events (55 of 268) were not included in the models describing rewarming patterns. Moreover, 12 % of Tbrood recording and 15% of Tabd recordings never reached normothermia, compared to 1 % of events in Tback and Tflk (Table 1). These data (20 events) were included only when analyzing Tinitial. On average, rewarming of Tback and Tflk was the fastest, followed by Tbrood and Tabd. There was no effect of year of study on any measurement (Table 2).

The effect of nutritional state on Tinitial differed between the tissues ("nutritional state × tissue" P<0.0001; Table 2). Specifically, neither initial Tback nor initial Tflk differed between fasted and fed birds (global means: 36.82 ± 0.97°C and 33.98 ± 0.86°C, respectively; Table 2), but both initial Tbrood and Tabd was lower in fed than in fasted birds (Table 2).

Tflk rewarmed faster (0.29 ± 0.02°C min -1 ) than Tbrood (0.21 ± 0.02°C min -1 ), which was still faster than Tback and Tabd (0.11 ± 0.03°C min -1 and 0.10 ± 0.02°C min -1 respectively)(Table 2; Fig. 1). However, rewarming rate was not affected by nutritional state (Table 2 and Fig. 2).

Nor did nutritional state affect the time needed for recovery (Table 2). However, the different tissues required different times to reach stable temperature (P<0.0001; Table 2).

Tflk and Tback recovered first, with a similar recovery time (21.36 ± 2.76 min and 24.15 ± 3.18 min, respectively; Table 2). This was followed by the recovery of Tbrood some 7 min later (31.22 ± 2.65 min, with no differences between these tissues; Table 2). Tabd recovered significantly later than all of the others tissues, after an additional 16 min (46.82 ± 3.00 min;

Table 2). It should be noted that while both Tabdo and Tbrood reached stable temperature during the study, neither tissue reached the normothermic values (≥ 37.5°C) that are characteristic of fully recovered birds that have been in the colony for several hours (Lewden et al. 2017a, b).

When the birds had regained recovered and/or normothermia, temperature differed between tissues depending on nutritional state ("nutritional state × tissue" Table 2). More precisely, recovery Tback and Tflk were similar and independent of nutritional state (global means: 38.57 ± 0.63°C and 38.65 ± 0.55°C, respectively; Table 2). In the fasted birds, recovery Tbrood and Tabd (37.98 ± 0.49°C and 38.03 ± 0.55°C, respectively) were similar to recovery Tback and Tflk (Table 2). In the fed birds, recovery Tabd was 1.5°C lower (36.46 ± 0.64°C) than in the fasted birds. This was also true for recovery Tbrood, which was 2.1°C lower in the fed (35.87 ± 0.59°C) compared to the fasted birds (Table 2).

Recovery metabolic rate (MRR)

We measured MRR of 77 thermal states (Fig. 3A). Of these, 28 (36%) were 'global rewarming', and 27 (35%) 'deep rewarming', i.e. rewarming of deep tissue temperature after the subcutaneous had recovered. There were few observations of 'subcutaneous rewarming', i.e. rewarming of the subcutaneous once deep temperature had recovered (one each in 'fasted' and 'fed' conditions; 3% of total). For this reason, these data were not used in the model, but are shown in Fig. 3B for comparison. Finally, we recorded 13 cases (17%) where the birds were categorized as 'recovered', and in 6 cases (9%) birds were normothermic ('stable normothermia' category) throughout the metabolic measurements.

On average, MRR and mass-specific MRR during 'global rewarming' (52.46 ± 4.55 W and 4.56 ± 0.42 W.kg -1 , respectively) were twice as high as RMR during 'stable normothermia' (25.94 ± 6.60 W and 2.16 ± 0.59 W.kg -1 , respectively).

MRR and mass-specific MRR were not affected by nutritional state, neither alone nor in combination with thermal state (Table 3). However, MRR and mass-specific MRR during 'global rewarming' were significantly higher than all other categories (Table 3; Fig. 3B). In fact, MRR and mass-specific MRR during 'deep rewarming' was similar to MRR in the 'recovered' thermal state, and also not significantly higher than MRR at 'normothermia' (Table 3; Fig. 3B). Finally, we measured a significant positive relationship between MRR and ∆Tflk (F1, 70=12.53 P=0.0007; GLMM R 2 =0.28 n=72; Fig. 3C), but ∆Tabd did not affect MRR (P=0.3641; GLMM R 2 =0.09 n=72).

Discussion

Subcutaneous tissues (back and flank) recovered in half the time (24 ± 3 min and 22 ± 3 min, respectively) taken for internal tissues (abdomen; 47 ± 3 min) when king penguins rewarmed to normothermia after cold water immersion (Fig. 1). As previously measured in water (Lewden et al. 2017a, b), Tbrood did not follow the same pattern as Tback and Tflk despite both temperature loggers being implanted subcutaneously. Rather, temperature in this body part was constantly lower than in the three other tissues (Fig. 1), both on land (this study) and in water (Lewden et al. 2017a, b) independent of nutritional state. The subsequent recovery pattern, which was similar to that of Tabd, was probably indicative of a response to reduce heat loss from this bare, and richly vascularized, skin area until a more favourable (drier) thermal state had been restored. In contrast, the back region, which was constantly above the surface when the birds rested in the sea water tank (cf. Time -11 in Fig. 1), consistently showed the highest initial temperatures (see also Lewden et al. 2017a, b). MRR during global rewarming (4.56 ± 0.42 W.kg -1 ) was about two times higher than RMR at normothermia (2.16 ± 0.59 W.kg -1 ), which indicates that the birds actively produced heat to rewarm (or to counter increased heat loss when the plumage was still wet) during the initial period on land. Once subcutaneous temperature had recovered, (but the deep tissues were still rewarming), MRR was not significantly elevated above RMR (Fig. 3). All these changes were independent of nutritional state, despite fed birds having a steeper internal temperature gradient to recover. Hence, a delayed recovery of subcutaneous temperature probably does not explain lower MR observed in fed king penguins in other studies [START_REF] Fahlman | Metabolism and thermoregulation during fasting in king penguins, Aptenodytes patagonicus, in air and water[END_REF][START_REF] Halsey | Recovery from swimming-induced hypothermia in king penguins: effects of nutritional condition[END_REF].

The rapid increase of subcutaneous temperatures prior to deep tissue recovery is consistent with the increasing flipper temperature that has been measured in rewarming emperor (Aptenodytes forsteri) and king penguins after emergence from water in other studies [START_REF] Ponganis | Regional heterothermy and conservation of core temperature in emperor penguins diving under sea ice[END_REF][START_REF] Schmidt | Etude de la thermorégulation en mer chez le manchot royal : Mécanismes et conséquences énergétiques[END_REF]Lewden et al., submitted), and suggests that fed king penguins in this study did not use subcutaneous vasoconstriction to reduce heat loss rate during rewarming, as previously hypothesized [START_REF] Halsey | Recovery from swimming-induced hypothermia in king penguins: effects of nutritional condition[END_REF].

We measured lower initial Tabd in fed than in fasted birds (Table 2; Fig. 1). This was probably a consequence of daily ingestion of cold fish meals (Culik et al., 1996;Eichhorn et al., 2011;Lewden et al., 2017a), or a delay of digestive activity (see below). Even so, the rewarming rate and the recovery time was independent of nutritional state (Table 2; Fig. 1). However, both Tbrood and Tabd stabilized at lower temperature in the fed birds (Fig. 2). Thus, these birds would have required a longer time to reach the same high temperature as in the fasted individuals.

It should be noted that we measured a lower internal temperature than in previous studies [START_REF] Halsey | Recovery from swimming-induced hypothermia in king penguins: effects of nutritional condition[END_REF][START_REF] Fahlman | Metabolism and thermoregulation during fasting in king penguins, Aptenodytes patagonicus, in air and water[END_REF]. This could be an effect of logger placement, because it is known that king penguins may show several temperature differences of several degrees in a small area (e.g. [START_REF] Handrich | Hypothermia in foraging king penguins[END_REF][START_REF] Fahlman | Metabolism and thermoregulation during fasting in king penguins, Aptenodytes patagonicus, in air and water[END_REF]. Our abdominal logger was positioned closer to the 'lower' abdomen measured by [START_REF] Halsey | Recovery from swimming-induced hypothermia in king penguins: effects of nutritional condition[END_REF]. These authors also measured 'upper' and 'middle abdominal' temperatures, which are likely closer to true core temperature. The differences between the studies could also be explained by variation in the duration in water preceding the recovery measurements. This time was 120 and 160 min in the studies by [START_REF] Fahlman | Metabolism and thermoregulation during fasting in king penguins, Aptenodytes patagonicus, in air and water[END_REF] and [START_REF] Halsey | Recovery from swimming-induced hypothermia in king penguins: effects of nutritional condition[END_REF], but on average 5024 min (83 h) in our study. Moreover, we measured that it takes around 5 h of constant immersion before all subcutaneous and deep tissues have reach a low stable temperature (Lewden, A., et al. unpublished data). Thus, the shorter water immersion period of 2-2.5 h in previous studies could explain why a higher 'lower abdominal' than in this study has been reported elsewhere. Even if we assume that Tabd as it was measured by us is closer to a 'peripheral' tissue than in the previous studies, our conclusion that king penguins to do not delay rewarming of the subcutaneous tissues to save energy during recovery remains the same.

Both physiological and physical factors could explain why we observed a faster recovery of subcutaneous compared to deep temperature (Fig. 2). On the one hand, the rapid increase of subcutaneous temperatures could indicate increased circulation required for fuel mobilisation (Lewden et al., 2017a) to cover energy costs of the rewarming phase. On the other hand, faster subcutaneous rewarming could facilitate evaporation of water within the plumage. Because a dry plumage confers better insulation than a wet one (de Vries and van Eerden, 1995), increased peripheral temperature might actually restore body insulation and, hence, reduce heat loss rate during subsequent rewarming of deep tissues. This is consistent with the drop in MRR that occurred as soon as the periphery had recovered, from which point onwards it was not different from RMR in a normothermic bird (Fig. 3). Finally, we cannot completely exclude the possibility that there was also a slight effect of heat gain from long-wave and solar radiation in the subcutaneous trunk area when the bird was on land.

The contribution of radiative gain to the overall energy budget of rewarming penguins should be quantified in future studies.

The absence of an effect of nutritional state on MRR contrasts previous studies [START_REF] Fahlman | Metabolism and thermoregulation during fasting in king penguins, Aptenodytes patagonicus, in air and water[END_REF][START_REF] Halsey | Recovery from swimming-induced hypothermia in king penguins: effects of nutritional condition[END_REF], despite overall similarly in metabolic rates measured here and elsewhere (Table 4). Thus, both MRR (global mean across states: 2.52 W.kg -1 ) and RMR during normothermia (2.09 W.kg -1 ) was similar to previously measured MRR (2.76 W.kg -1 ; Fahlman et al., 2006) and RMR values (2.16 to 2.72 W.kg -1 ; [START_REF] Fahlman | Metabolism and thermoregulation during fasting in king penguins, Aptenodytes patagonicus, in air and water[END_REF]Fahlman et al. , 2014) ) (Table 4). This might be explained by methodological differences between the studies. Our fed group is most similar to 're-fed' birds in [START_REF] Halsey | Recovery from swimming-induced hypothermia in king penguins: effects of nutritional condition[END_REF], who force-fed one meal 24 h before the measurements. In contrast, our fed birds ate daily over a period of 47:42 ± 05:53 h and had ingested their most recent meal 04:01 to 15:25 h before metabolic rate measurements. Even so, MRR during global rewarming was very similar in the fed birds in our studies and those in the study by [START_REF] Halsey | Recovery from swimming-induced hypothermia in king penguins: effects of nutritional condition[END_REF] (4.15 W.kg -1 and 4.22 W.kg -1 , respectively) (Table 4). [START_REF] Fahlman | Metabolism and thermoregulation during fasting in king penguins, Aptenodytes patagonicus, in air and water[END_REF] hypothesized that differences in MRR between fasted and fed penguins was caused by differences in body mass. While, on average, body mass differed between nutritional states in the subset where we measured metabolic responses, this difference was only 500 g. This might not have been large enough detect any effects on the metabolic cost of rewarming. However, one individual lost body mass twice as fast as the others (i.e. 628 ± 0.19 g.day -1 , compared to 335 ± 0.08 g.day -1 for the other penguins) possibly even entering fasting phase III which is characterized by protein catabolism (LeMaho et al., 1988[START_REF] Cherel | Diving behaviour of female northern rockhopper penguins, Eudyptes chrysocome moseleyi, during the brooding period at Amsterdam Island (Southern Indian Ocean)[END_REF][START_REF] Groscolas | Long-term fasting and refeeding in penguins[END_REF]. This bird showed a clear pattern of energy conservation, with lower peripheral tissues in the sea water tank (Supp. Fig. 1A), but its rewarming pattern on land did not differ from that in other individuals (Supp. Fig. 1). It would be interesting to repeat this study using a more extensive fasting protocol that more strongly significantly affected nutritional state of the birds to investigate the body mass hypothesis more broadly.

The similar MRR in fasted and fed birds could also be due to absence of digestive activity in fed individuals. We do not think that it is likely that the fed birds had already digested the fish meal by the time the measurements started, in which case we would have expected similar Tabd between treatments. The capacity to delay digestive activity is well known in this species [START_REF] Gauthier-Clerc | Penguin fathers preserve food for their chicks[END_REF][START_REF] Thouzeau | Evidence of stress in bacteria associated with long-term preservation of food in the stomach of incubating king penguins (Aptenodytes patagonicus)[END_REF][START_REF] Thouzeau | Adjustments of gastric pH, motility and temperature during long-term preservation of stomach contents in free-ranging incubating king penguins[END_REF]. If this is associated with changes to circulation of the abdominal region, delayed digestions could explain the lower Tabd observed in our fed birds. This is supported by the similar RQs in the two nutritional conditions (see Materials and Methods). Thus, it is possible that the fed group was not as homogenous with regards to digestive activity as we had intended. To evaluate this possibility, future studies need to measure recovery temperatures and MRR over a range of time periods from the feeding event onwards.

Finally, in line with our previous study (Lewden et al., 2017b), we found a positive relationship between MRR and ∆Tflk during recovery on land (Fig. 3C). However, according to the higher heat loss rate in water than in air, the effect of ∆Tflk on MRR ashore was more than three times lower (0.22 W.kg -1 .°C -1 ) than in water (0.73 W.kg 1 .°C -1 ) (Lewden et al., 2017b). In contrast, there was no relationship between MRR and ∆Tabd. These results indicate that while subcutaneous vasodilation increases heat loss rate (and, thus, impacts metabolic rate), temperature change inside the well-insulated trunk does not necessarily increase heat loss from the body. To this end, ∆Tflk may be viewed as the driver of the energetic cost needed to rewarm the subcutaneous layer. Distribution of rewarming events recorded in four tissues of king penguins during the three years. In total, 67 rewarming events were recorded. However, due to device failure ('without data' category) there are missing data for some tissues.

Rewarming events where a given tissue was already at normothermia before recovery (i.e. 'Tinitial ≥ 37.5°C' category) were not included in the statistical analyses. Nor were data where the tissue never reached stable temperature during rewarming the event ('Never reached stable state' category). Thus, we analysed 73% of the 67 rewarming events recorded in the flank, 48% of those in the back tissue, and 79% and 60% of those recorded in the brood patch and abdominal cavity, respectively.

T flk T back T brood T abd

Rewarming events -n (%) 49 ( 73

Figures:Figure 1 :

 1 Figures:Figure1: Mean ± s.e. tissue temperatures recorded every 11 min in subcutaneous tissue on the back (blue), flank (green), and brood patch (pink), and in the abdominal cavity (red) in fasted (A) and fed (B) king penguins. The first measurement (time = -11) is the last temperature recorded before the bird was removed from the cold sea water tank.

Figure 2 :

 2 Figure 2: Schematic representation of the rewarming rate in the four tissue from the point in timewhere the birds were removed from the sea water tank (i.e. Tinitial) to the end of the linear rewarming phase (i.e. Trecovery). Rewarming rate ± s.e. (°C.min -1 ) for each tissue is printed above the curve. There was no effect of nutritional state on Tflk and Tback, but fed birds (dotted line) had lower Tbrood and Tabd than fasted (dash-dotted line) birds throughout the rewarming phase.

Figure 3 :

 3 Figure 3: (A) Metabolic rate (MRR, W) ± s.e. and mass-specific MRR (W.kg -1 ) ± s.e in each thermal state during recovery to normothermia on land in king penguins after long-term immersion in cold. 'Subcutaneous rewarming' was not included in the statistical model on account of very few occurrences, as described in the main text. The number of segments representing each category is indicated on the bars, and different letters denote significant differences between categories. MRR is delimited by the horizontal line in each bar. Mass-specific MRR is represented by the upper range of the bars. (B) Temporal changes in mean ± s.e. Tflk (green), Tabd (red), and metabolic rate (black) in the sea water tank immediately before the recovery measurements started (Time = -11), and during subsequent recovery to normothermia on land (starting at Time =0). Metabolic rate in the sea water tank before recovery started (hexagon) was measured in different individuals but during the same experiment (N = 4; n = 13; Lewden et al. 2017b). MRR recovery (downward facing triangle) was calculated as every 10 min means during all thermal states (cf. panel A) (N = 8; n = 40). RMR (upward facing triangle) was measured in dry birds at normothermia (N = 6; n = 6). (C) MRR in relation to the change in flank temperature (ΔTflk) during rewarming. Thermal states are indicated with different symbols for reference only.

Figure 1 :

 1 Figure 1:

  Figure 2:

Tissue temperature (°C)

	A -Fasted condition (N=18 individuals, n=36 rewarming events)
		40
		38
	Tissue temperature (°C)	30 32 34 36	Flank
		28	Back Brood patch
			Abdominal cavity
		26
		-11 0 11 22 33 44 55 66 77 88 99 110 121
		Recovery time (min)
		40
		38
		36
		34
		32
		30
			Flank
		28	Back Brood patch
			Abdominal cavity
		26

Table 1 :

 1

Table 2 :

 2 Effects of nutritional states, tissues, the interaction tissues x nutritional states and year on Tinitial, Trecovery, rewarming rate and recovery time in king penguins recovering normothermia on land after long immersion in cold water. Data were analyzed using general linear mixed effects models with bird ID as a random intercept, with separate models for each parameter Model estimates are not provided for main effects in the presence of significant interactions.

	Tinitial (°C)

Table 3 :

 3 Effects of thermal state and nutritional condition on recovery metabolic rate (MRR) and mass-specific MRR during the four thermal states in fed and fasted king penguins that recovered from soaking-induced hypothermia on land. Thermal states were assigned using inflection points in Tflk and Tabd recordings, as detailed in the main text.

				MRR (W)			Mass-specific MRR (W.kg -1 )
			R² = 0.35 N = 8	n = 72		R² = 0. 35 N = 8	n = 72
		df	F	Prob. > F	Prediction ± s.e.	Post-hoc test	df	F	Prob. > F	Prediction ± s.e.	Post-hoc test
	Nutritional state	-	-	0.2164				-	-	0.3449
	Thermal state	3.65 7.57 0.0002				3.64 7.64	0.0002
	Global rewarming				4.56 ± 0.42	a				52.46 ± 4.55	a
	Deep rewarming				3.42 ± 0.42	b				39.31 ± 4.54	b
	Recovered				2.92 ± 0.50	b				33.84 ± 5.49	b
	Stable normothermia				2.16 ± 0.59	b				25.94 ± 6.60	b

Table 4 :

 4 Summary table of field metabolic rate (FMR), field metabolic rate estimate (FMRest), resting metabolic rate (RMR) and recovery metabolic rate (MRR) in free-ranging or temporarily captive, wild king penguins in different nutritional conditions.Bold values correspond to results obtained in the current study.

	Condition	Methods	Metabolism Nutritional condition	RQ Body mass VO2 (ml.min -1 ) W.kg -1
	Captivity	Respirometry	FMR		-	-	-	2.80	LeMa
		Respirometry	FMR		-	-	-	3.00	Barré
	Free-ranging	Doubly labelled water	FMR		-	-	-	3.30	Kooy
		Heart rate	FMRest		-	-	122.1	3.15	Froge
					0.76	13.8	127.6	3.05
					0.76	12.6	107.2	2.81
					0.74	11.5	95.3	2.72	Fahlm
					0.75	10.7	79.1	2.44
	Captivity	Respirometry	RMR	Fasted	0.69	9.8	65.8	2.16
					-	11.1	86.9	2.55	Fahlm
					0.72	11.5	69.0	2.09	This
	Captivity	Respirometry	RMR	'Fed' (after foraging trip) Fed (manually fed)	-0.74	13.9 12.0	117.1 91.03	2.76 2.52	Fahlm This

Acknowledgements

We are grateful to Yves Handrich for his valuable help on this project and comments on a previous version of the manuscript. We also thank Jean-Yves Georges for his material support, Thibaut Hestin, Caroline Bost for their help and all the members of the 53 th mission in Crozet for their support in the field.

Funding sources

Logistic support in the field was provided by the Terres Australes et Antarctiques Françaises (TAAF). The research project No. 394 is funded by Institut Polaire Français Paul-Emile Victor and by the Centre National de la Recherche Scientifique (CNRS-INEE). A.L. was the recipient of a scholarship from the French Ministere de l'Education National, de la recherche et de la technologie. AN was supported by the Birgit and Hellmuth Hertz Foundation / The Royal Physiographic Society of Lund (grant no. 2017-39034).

Supplementary material

Fig. S1: Temperatures recorded subcutaneously in the flank, back, brood patch, and abdominal cavity, in king penguin A (A) that weighed 9.33 kg after having lost 628 ± 0.19 g.day -1 during fasting (and might have been metabolizing protein), compared to king penguin B (B) weighing 11.40 kg after having lost 335 ± 0.08 g.day -1 over the same fasting period.

Both individuals were maintained fasting together in a sea water tank for four days, after which they were allowed to recover to normothermia on land (starting at time = 0 min).