# Microhabitat humidity rather than food availability drives thermo-hydroregulation responses to drought in a lizard

Théo Bodineau, Chloé Chabaud, Beatriz Decencière, Simon Agostini, Olivier Lourdais, Sandrine Meylan, Jean-françois Le Galliard

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Théo Bodineau, Chloé Chabaud, Beatriz Decencière, Simon Agostini, Olivier Lourdais, et al., Micro-habitat humidity rather than food availability drives thermo-hydroregulation responses to drought in a lizard. Oikos, 2024, 2024 (6), 10.1111/oik.10535. hal-04738801

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https://cnrs.hal.science/hal-04738801v1

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Distributed under a Creative Commons Attribution 4.0 International License
23 The authors thank Damien Lopez Santin and Anouk Pellerin who contributed to behavioural

24 and physiological data acquisition. This work was supported by an Agence Nationale de la

25 Recherche grant (ANR-17-CE020013, 'AQUATHERM') to J.-F. Le Galliard. Théo

26 Bodineau' PhD grant is funded by the Ministère de l'Enseignement Supérieur et de la

27 Recherche. This work has benefited from human and technical resources provided by the

28 CEREEP-Ecotron IleDeFrance (CNRS/ENS UMS 3194).

29

30 Authors' contributions:

31 Théo Bodineau, Jean-François Le Galliard, Chloé Chabaud and Olivier Lourdais conceived

32 the ideas and designed methodology; Théo Bodineau, Chloé Chabaud, Simon Agostini,

33 Beatriz Decencière, Sandrine Meylan and Jean-François Le Galliard participated to lizard

34 captures and breeding; Théo Bodineau and Jean-François Le Galliard collected and analysed

35 the data; Théo Bodineau led the writing of the manuscript with Jean-François Le Galliard. All

36 authors contributed critically to the drafts and gave final approval for publication.

37

38

39 Conflict of Interest:

40 The authors declare no competing or financial interests.

41

42 Data accessibility:

43 Data will be deposited in Zenodo after acceptance.

44

45
46

**Abstract**

47

1. The regulation of energy, water and thermal balance involves integrated processes that

48

should drive ecological responses of ectotherms to climate change. Functional trade-offs

49

between thermoregulation and hydroregulation are exacerbated during hot or dry spells, but

50

how microhabitat hydric properties and trophic resource availability influence these trade-offs

51

remains unknown.

52

2. Here, we investigated the effects of microhabitat humidity and food availability on thermo-

53

hydroregulation strategies in the ground-dwelling common lizard (Zootoca vivipara) during a

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simulated hot and dry spell event. We exposed lizards to a five-day long acute water

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restriction in hot conditions in the laboratory and manipulated hydric quality of the retreat site

56

(wet or dry shelter) as well as food availability (ad libitum food or food deprivation).

57

3. Water restriction and food deprivation caused physiological responses such as muscle

58

catabolism and mobilization of caudal energy reserves. Lizards also developed behavioural

59

strategies to conserve water or energy via decreased thermoregulation effort, higher shelter

60

use and increased eye closure behaviours through time.

61

4. These physiological and behavioural changes were importantly buffered by the presence of

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a wet shelter but not by food availability. A wet retreat site reduced the behavioural conflicts

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between thermoregulation and hydroregulation, allowed lizards to maintain a better condition

64

and reduced physiological dehydration. Instead, food intake did not play a major role in the

65

regulation of hydration state and increased behavioural conflicts between thermoregulation

66

and hydroregulation.

67

5. A better consideration of thermo-hydroregulation behaviours and microhabitat hydric

68

quality is required to address ectotherm responses to future climate change.

69

Keywords: behaviour, food, microhabitat, shelter, thermo-hydroregulation, trade-off, water

70

**Introduction**
71 Behavioural thermoregulation can mitigate the impact of climate warming in ectotherms
72 (Gunderson & Leal, 2015; Kearney, 2013). Recent empirical studies and mechanistic models
73 indicate that the behavioural capacity of ectotherms to buffer consequences of climate
74 warming will be constrained by water availability in the environment, which may further
75 exacerbate the deleterious effects of heat stress (Kearney et al., 2018; Rozen-Rechels et al.,
76 2020; Sannolo & Carretero, 2019). A proximate conflict exists between regulation of water
77 balance and thermoregulatory behaviours, since basking and maintenance of high body
78 temperatures typically increase water loss rates and eventually lead to dehydration (Anderson
79 & Andrade, 2017; Dmi'El, 2001). Conversely, dehydration risks can lead to water
80 conservation strategies such as reduced activity, thermal depression, diurnal or nocturnal
81 selection of cool and wet microhabitats or minimisation of time spent with the eyes open,
82 which can compromise energy acquisition, body condition and survival (e.g. Davis &
83 DeNardo, 2009; Le Galliard et al., 2021; Rozen-Rechels et al., 2020; Sannolo & Carretero,
84 2019). Despite the relevance of this functional trade-off for behavioural patterns of activity or
85 microhabitat selection, we have limited knowledge of environmental factors that influence
86 conflicts between thermoregulation and hydroregulation (Dezetter et al., 2023).

87 The availability and overall quality of microhabitats should importantly influence
88 behavioural trade-offs between thermoregulation and hydroregulation. In terrestrial
89 ectotherms, wet and cold microhabitats provide thermal shelters to escape extreme surface
90 temperatures (Beck & Jennings, 2003; Moore et al., 2018). They also reduce evaporative
91 water loss, dehydration stress and physiological alterations caused by dehydration such as
92 muscle wasting (Dezetter et al., 2023). Yet, only few studies have quantified the effects of
93 microhabitat quality on thermo-hydroregulation strategies during hot and dry weather event
94 (Dezetter et al., 2023). Food resources should also influence thermo-hydroregulation
95 strategies because investment in these behaviours should be constrained by energy intake
96 according to the cost-benefit model of thermoregulation (Angilletta Jr., 2009; Huey & Slatkin,
1976). Food intake could influence hydroregulation behaviours if it allows to recoup some of the energetic costs of hydroregulation and because food can provide dietary water, a scenario which we called the "food for water hypothesis" (Chabaud et al., 2023). This hypothesis has been little tested so far in terrestrial ectotherms and some studies indicate that they are more dependent on free water rather than dietary water to regulate their water balance (Chabaud et al., 2023; Lillywhite, 2017; Perez et al., 2021; Wright et al., 2013).

Here, we designed an experiment to quantify the effects of food availability and microhabitat humidity on thermo-hydroregulation strategies, as well as body condition and whole-organism energetics, during a hot and dry weather event simulated in the laboratory in the common lizard Zootoca vivipara (Lichenstein, 1823). This lacertid is found in cool and moist environments and is highly sensitive to water restriction (Herczeg et al., 2003; Rozen-Rechels et al., 2020). We simulated a summer drought with water restriction and manipulated food availability (*ad libitum* food vs. no food) as well as the humidity of a surface shelter (access to a wet shelter or to a dry shelter). In the same time, we quantified the behavioural adjustments of the lizards as well as their body condition, plasma osmolality and energy-related plasma metabolites. We examined the following four hypotheses and predictions.
First, water and food restriction should challenge homeostasis and compromise the energy and water balance of lizards. We predict a decrease in body condition in response to water restriction and food deprivation, an increase in plasma osmolality in response to water restriction and a reduction in circulating glucose and triglycerides caused by food deprivation.
Second, compensatory physiological mechanisms should occur under conditions of water and food restriction. We predict muscle catabolism and mobilisation of caudal lipid reserves to release amino acids, lipids, and bound water and recoup some of the missing inputs of food and water (Brusch et al., 2018, 2020, 2022). Third, behavioural responses should mitigate water and energy constraints. We predict a decrease in selected body temperatures and an increase in shelter use in response to water and food restrictions. Frequency of water
conservation behaviour such as eye closure behaviours should also increase in response to
dehydration. If wet shelter reduces water losses, lizards provided with wet shelter should use
it more and dehydration should be negatively correlated with the frequency of shelter use
(Dezetter et al., 2023). Fourth, deleterious effects of water restriction and the behavioural
trade-off between thermoregulation and hydroregulation should be weaker when shelters are
optimal (i.e., wet microclimatic conditions) and when food is available if food provides a net
dietary water intake.

130 **Material and methods**

131 **Study species and sampling**

132 Between April 21st and May 24th 2021, we captured 60 adult male European common lizards
133 (*Zootoca vivipara*) inside semi-natural populations maintained in outdoor enclosures at
134 CEREEP-Ecotron IleDeFrance. Lizards were captured in three sequential capture sessions to
135 generate three sequential batches of 20 lizards. Prior to experiments, we acclimated lizards for
136 a minimum of five days in standard laboratory conditions (see Rozen-Rechels et al., 2020) in
137 order to standardise habituation to laboratory conditions and food satiety.

138 **Experimental conditions and study design**

139 The experiment was performed in 20 neutral arenas lit automatically between 8:30 am and 6
140 pm by a UVB-enhanced neon tube and halogen heating bulbs in order to generate a thermal
141 gradient (operative temperatures from hot to cold spot: 35.8 °C ± 7.5 SD to 22.1 °C ± 2.5
142 SD). Each arena was filled with 3 cm of peat and water-filled Petri dishes to provide lizards
143 with free-standing water. In the cold side of the arena, we installed a “shelter” that was
144 thermally sub-optimal for basking but had optimal hydric conditions or not depending on
145 treatment conditions in our study (see Supplementary figures S1 and S2). We manipulated
146 both food availability (*ad libitum* feeding with 200 ± 20 mg of crickets per day for the control
147 treatment or no feeding) and humidity of the shelter (access to a wet shelter for the control
148 treatment or access to a dry shelter) in a balanced full-factorial experimental design. Each
batch of 20 lizards was divided into four experimental groups formed by the factorial combination of the two treatments. The study was repeated three times independently with our three batches of lizards. During the course of the experiment (see Supplementary figure S3), lizards spent seven successive days alone in the same arena including one day of acclimation (called “day -1”) when lizards were maintained under control conditions with water, *ad libitum* food, wet shelter, and three water sprays per day. During “day 0”, we observed lizards under the same control conditions as during the acclimation day. At the end of this first day, we removed water cups from all arenas, removed crickets for the food-deprived arenas, and replaced the wet shelter with a dry shelter for dry shelter treatment group. From day 1 to day 5, a limited amount of water was provided to the lizards at 8:30 am by spraying arenas with water for five seconds. This manipulation was done to generate a chronic, sub-lethal water restriction similar to exposure to several days of warm and dry weather in natural conditions (Dupoué et al., 2020). After the last behavioural observation on day 5, all individuals were provided with water and food *ad libitum*.

## Pre- and post-experimental measurements

At the beginning and at the end of the experiment (day -1 and day 5), lizards were bled using a standard protocol (see Meylan et al., 2003) to measure plasma osmolality, circulating triglyceride and glucose levels in plasma in order to quantify metabolic changes during the experiment. Plasma osmolality (an indicator of hydration status in species lacking a salt gland which increases in dehydrated individuals) was measured using a Vapro® vapour pressure osmometer and circulating triglyceride and glucose levels were measured in plasma by colorimetric assays (Triglyceride Colorimetric Assay kit and Glucose Colorimetric Assay kit, ref. 10010303 and 10009582, Cayman Chemical, USA). At the beginning (day -2) and at the end of the experiment (day 5), lizards were weighed using a precision scale (PX 323 Pioneer, OHAUS Corp., USA) and scanned using a flatbed scanner (CanoScan LiDE 700F, Canon) to measure tail width at the 9th sub-caudal scale row using ImageJ software (Version 1.52v), as a
proxy for tail reserves. Hind limb thickness was measured to the nearest 0.01 mm on both legs using a spessimeter (ID-C1012BS, Mitutoyo, Japan).

## 177 Behavioural measurements

We recorded body temperature, basking behaviours (increased exposure to radiation from heat bulbs or not) and the position of lizard (inside or outside the shelter) for 6 days (day 0 to day 5) with an instantaneous sampling every 30 minutes from 9 am to 5 pm (i.e., 17 readings during the day). Surface body temperature was recorded using an infrared thermometer (Raytek, Raynger MX2). We then calculated body temperature $T_b$ using a calibration curve (see Supplementary figure S4). When the lizard was visible, we noted whether its eyes were open or closed. When the lizard was not visible, the roof of the shelter was slowly lifted to note the presence or not of the lizard in the shelter.

## 186 Statistical analyses

Analyses were performed with R software version 4.2.0 (R Core Team, 2022). We used mixed models to control for the fully randomised design of our experiment where we attributed different individuals from different treatments to the same arena three times (three experimental batches). We also controlled for inter-individual variability by including a random effect of individual identity. For each analysis, we built a full model, checked its assumptions and performed a goodness-of-fit test. Non-significant effects were then eliminated using a stepwise model selection procedure starting from the full model. First, we calculated intra-individual changes in body mass, in average limb thickness, in tail width, in plasma osmolality, in triglyceride and in glucose concentration by subtracting the final values measured after the experiment from the initial values measured before the experiment. To explain these morphological and physiological changes, we fitted linear mixed models (Pinheiro et al., 2012) with experimental batch, the initial centred value of the morphological or physiological trait and interactive effects of the two experimental treatments as fixed effects. For the analysis of the change in plasma osmolality, we added the individual-centred
daytime shelter use rate during the experiment (calculated as the number of observations where the lizard was in the shelter divided by the total number of daytime observations during the experiment) in interaction with the shelter and food treatments as fixed effects to test the relationship between shelter use and dehydration. To study thermo-hydroregulation behaviours, we counted the number of times each day out of 17 observations where lizards exhibited basking behaviour, when they closed their eyes, and when they were inside the shelter. We analysed basking, eye closure behaviour and shelter use as success-failure events with generalised linear mixed models assuming a binomial distribution and a logit link using the *glmer* function from the *lme4* package (Bates et al., 2014). T<sub>b</sub> was analysed using linear mixed models. We analysed these four behavioural traits with the experimental batch and the factorial, linear or quadratic effects of time (number of days since start of the experiment) in interaction with the shelter and food treatments as fixed effects.

## 213 Results

All lizards lost condition during the experiment, as manifested by a decrease of body mass, limb thickness and tail width (Table 1). Mass, limb thickness and tail width losses were additively influenced by shelter treatment and food treatment. Food restricted lizards and lizards provided with dry shelter lose more body mass, limb thickness and tail thickness than others. We also found an overall increase in plasma osmolality for all experimental groups indicating strong physiological dehydration (+57.09 mOsmol.kg⁻¹ ± 5.6 SE). Dehydration was influenced by the interaction between shelter use rate and shelter treatment but food treatment did not affect dehydration. On average, dehydration was greater in lizards provided with a dry shelter compared to a wet shelter. Surprisingly, dehydration was positively correlated with daily shelter use in lizards provided with a wet shelter, whereas it was not correlated in lizards provided with a dry shelter (see Figure 1B). In addition, fed lizards showed a significant increase in plasma glucose concentration relative to food-deprived lizards, and a greater increase in circulating plasma triglyceride concentration was observed in fed than food-
227 deprived lizards and also to some extent in lizards from the dry shelter group than from the
228 wet shelter group (Table 1).

229 During water restriction, daytime shelter use increased and was influenced by the
230 interaction between time, shelter treatment and food treatment (Figure 1A). In food-deprived
231 animals, daytime shelter use increased similarly in dry and wet shelter treatment groups,
232 whereas in fed lizards, daytime shelter use increased more strongly in lizards provided with a
233 dry shelter. Average daily T<sub>b</sub> and basking behaviours of lizards decreased through time as the
234 number of days of water restriction increase but these behavioural changes were only
235 influenced by the shelter treatment (Figure 2A, B, C, D). The diminution of T<sub>b</sub> and basking
236 behaviours was more pronounced in lizards provided with a dry shelter. Finally, we found
237 strong effects of shelter treatment in interaction with time on the ocular behaviour of lizards.
238 Eye closure rate increased remarkably more in lizards provided with a dry shelter compared to
239 lizards with a wet shelter (Figure 2E).

240 Discussion

241 During the experiment, all lizards showed significant increase in plasma osmolality which
242 indicates physiological dehydration (Peterson, 2002; Rozen-Rechels et al., 2020) and the
243 food-deprived group had lower circulating triglyceride concentrations and blood glucose
244 levels suggesting fasting patterns consistent with previous studies on lipid or glucose
245 metabolism in reptiles (McCue, 2007; Moon et al., 1999; Price, 2017). Concurrently, water
246 and food restrictions led to reductions in body mass, hind limb width, and tail width. In snakes
247 and birds, muscle wasting from the tail and limbs is caused by protein catabolism and is
248 associated with a significant release of protein-bound water to improve the water status during
249 water restriction (Brusch et al., 2018; Gerson & Guglielmo, 2011). In lizards, the tail is also a
250 reserve organ consisting mostly of muscle and lipids stored as triglycerides (Avery, 1974;
251 Price, 2017). Thus, our data suggest that fasting and dehydration caused mobilisation of
caudal lipid reserves and muscle wasting to support water, energy, and amino acid requirements when water and dietary intakes are insufficient (Cherel et al., 1992).

At the same time, lizards developed behavioural strategies during the water restriction period including a lower basking effort, a lower body temperature and a higher eye closure rate, and those responses were more strongly expressed in the dry shelter treatment. Our study is the first experimental demonstration of an adjustment of eye closure behaviour in response to hydric stress. In dry-skinned ectotherms with an eyelid, eye closure behaviour should play an important role in organismal hydroregulation because the eye surface is poorly resistant to water loss and may represent a significant proportion of the body surface (Pirtle et al., 2019; Waldschmidt & Porter, 1987). In addition, eye opening is important for predators' detection and visual interactions with conspecifics so hydric stress may decrease vigilance and increase risks of predation. Altogether, these behavioural responses indicate a trade-off between heat and water balance regulation mediated by the hydric costs of thermoregulation (Rozen-Rechels et al., 2020). Hydric quality of the shelter rather than food availability was the main modulator of these thermo-hydroregulation strategies and an important buffer against the deleterious effects of water restriction. Presence of a wet shelter at day and night limited very strongly dehydration, muscle and lipid reserves mobilisation, thermal depression and the increase of eye closure rate, which can play an important role in organismal hydroregulation (Pirtle et al., 2019; Waldschmidt & Porter, 1987). These findings highlight the importance of shelter and wet microhabitat quality in the behavioural responses of ectotherms to future changes in climate conditions (Beck & Jennings, 2003; Moore et al., 2018).

Contrary to earlier studies (Dezetter et al., 2023; Pintor et al., 2016), lizards with access to a wet shelter did not use it more often than the ones with a dry shelter and we found a positive relationship between individual dehydration state and the daytime wet shelter use rate, whereas we predicted the opposite relationship since wet shelter should reduce evaporative water loss rates. These results suggest condition-dependent wet shelter use, for
example if lizards decided to use the wet shelter below a certain dehydration level, and
highlight the potential thermoregulatory costs of hydroregulation behaviours. Behavioural
selection of a wet microclimate inside the shelter allowed lizards to maintain a better
hydration state more efficiently but also involved the selection of a thermally suboptimal
microhabitat. Thus, wet shelter use had a strong opportunity and energetic cost because it was
detrimental to thermoregulatory activities essential for energy intake such as prey capture and
assimilation rates (Van Damme et al., 1991), which potentially explains why this behaviour
was less frequent in weakly dehydrated individual lizards (which privileged thermoregulation
activities) than in strongly dehydrated individuals (which privileged hydroregulation
activities). On the other hand, individual dehydration state was not correlated with the
daytime dry shelter use rate because a dry shelter does not effectively reduce water losses and
dehydration contrary to a wet shelter.

Food availability did not influence mean daily thermoregulation effort contrary to the
predictions of the cost-benefit model of thermoregulation and results of some earlier studies
(Brown & Griffin, 2005; Huey & Slatkin, 1976). Thus, common lizards have limited
behavioural acclimation responses to energy imbalance, maybe because fasting was too short
or because physiological responses to fasting were prioritized over behavioural responses.
Finally, in accordance to a recent study (Chabaud et al., 2023), none of our findings support
the “food for water hypothesis” because *ad libitum* food conditions did not reduce
dehydration nor the morphological and behavioural impacts of water restriction. On the
contrary, shelter use in the presence of dry shelter and *ad libitum* food was more frequent,
which suggests that food consumption and processing require a good hydration status and thus
food availability increases reliance on free standing water, as suggested for several snake
species and a carnivorous lizard (Lillywhite, 2017; Murphy & DeNardo, 2019; Wright et al.,
2013). Therefore, our experiment indicates that food acquisition can exacerbate the conflict
between thermoregulation and hydroregulation, most likely because behavioural optimisation
304 of food capture, processing and digestion implies behavioural selection of elevated body
305 temperatures or increased basking effort, which are water costly thermoregulation behaviours.
306 An alternative explanation is that feeding itself increases water requirements or that prey
307 capture and digestion requires a good hydration state as seen in some carnivorous snakes
308 (Lillywhite, 2017). Thus, food-deprived lizards use dry and wet shelters similarly, whereas in
309 the presence of food, lizards with a dry shelter must spend more time inside the shelter to
310 regulate their hydration state than lizards with a wet shelter.

311 Conclusions

312 Our study challenges previous studies that have only focused on the effects of temperature or
313 water availability on thermoregulatory behaviours of ectotherms. Food intake did not play a
314 major role in regulating the hydration status of this insectivorous species but food intake
315 influenced water balance regulation by increasing a behavioural trade-off between
316 thermoregulatory and hydroregulatory activities. Our findings demonstrate the urgent need to
317 take into account the dual effect of warming and drought events, behavioural interactions
318 between thermoregulation and hydroregulation and the buffering role of microclimatic
319 conditions to understand the ecological and evolutionary responses of ectotherms to climate
320 change (Moore et al., 2018).

321 References

322 Anderson, R. C. O., & Andrade, D. V. (2017). Trading heat and hops for water : Dehydration
323 effects on locomotor performance, thermal limits, and thermoregulatory behavior of a
324 terrestrial toad. *Ecology and Evolution*, 7(21), 9066-9075.
325 https://doi.org/10.1002/ece3.3219

326 Avery, R. A. (1974). Storage lipids in the lizard Lacerta vivipara : A quantitative study.
327 *Journal of Zoology*, 173(3), 419-425. https://doi.org/10.1111/j.1469-3289.1974.x
329 Bates, D., Mächler, M., Bolker, B., & Walker, S. (2014). Fitting Linear Mixed-Effects Models Using lme4. *ArXiv e-prints*, arXiv:1406. https://doi.org/10.18637/jss.v067.i01
330
331 Beck, D. D., & Jennings, R. D. (2003). Habitat Use by Gila Monsters: The Importance of Shelters. *Herpetological Monographs*, 17, 111-129.
332
333 Brown, R. P., & Griffin, S. (2005). Lower selected body temperatures after food deprivation in the lizard Anolis carolinensis. *Journal of Thermal Biology*, 30(1), 79-83.
334
335 https://doi.org/10.1016/j.jtherbio.2004.07.005
336 Brusch, G. A., Gavira, R. S. B., Viton, R., Dupoué, A., Leroux-Coyau, M., Meylan, S., Galliard, J.-F. L., & Lourdais, O. (2020). Additive effects of temperature and water availability on pregnancy in a viviparous lizard. *Journal of Experimental Biology*, 223(19). https://doi.org/10.1242/jeb.228064
337
338
339
340 Brusch, G. A., Le Galliard, J., Viton, R., Gavira, R. S. B., Clobert, J., & Lourdais, O. (2022). Reproducing in a changing world: Combined effects of thermal conditions by day and night and of water constraints during pregnancy in a cold-adapted ectotherm. *Oikos*.
341
342
343 https://doi.org/10.1111/oik.09536
344 Brusch, G. A., Lourdais, O., Kaminsky, B., & DeNardo, D. F. (2018). Muscles provide an internal water reserve for reproduction. *Proceedings. Biological Sciences*, 285(1881).
345
346 https://doi.org/10.1098/rspb.2018.0752
347 Chabaud, C., Brusch, G., Pellerin, A., Lourdais, O., & Le Galliard, J.-F. (2023). Prey consumption does not restore hydration state but mitigates the energetic costs of water deprivation in an insectivorous lizard. *The Journal of experimental biology*, 226.
348
349
350 https://doi.org/10.1242/jeb.246129
351 Cherel, Y., Robin, J. P., Heitz, A., Calgari, C., & Le Maho, Y. (1992). Relationships between lipid availability and protein utilization during prolonged fasting. *Journal of Comparative Physiology. B, Biochemical, Systemic, and Environmental Physiology*, 162(4), 305-313. https://doi.org/10.1007/BF00260757
355 Davis, J. R., & DeNardo, D. F. (2009). Water supplementation affects the behavioral and physiological ecology of Gila monsters (Heloderma suspectum) in the Sonoran Desert.
356
357 *Physiological and Biochemical Zoology: PBZ*, 82(6), 739-748.
358 https://doi.org/10.1086/605933

359 Dezetter, M., Le Galliard, J.-F., & Lourdais, O. (2023). Behavioural hydroregulation protects against acute effects of drought in a dry-skinned ectotherm. *Oecologia*, 201(2), 355-367. https://doi.org/10.1007/s00442-022-05299-1

362 Dmi'El, R. (2001). SKIN RESISTANCE TO EVAPORATIVE WATER LOSS IN REPTILES: A PHYSIOLOGICAL ADAPTIVE MECHANISM TO ENVIRONMENTAL STRESS OR A PHYLETICALLY DICTATED TRAIT? *Israel Journal of Zoology*, 47(1), 56-67. https://doi.org/10.1560/ENQ9-KD7R-WFGW-KUQW

367 Dupoué, A., Blaimont, P., Rozen-Rechels, D., Richard, M., Meylan, S., Clobert, J., Miles, D. B., Martin, R., Decencière, B., Agostini, S., & Le Galliard, J.-F. (2020). Water availability and temperature induce changes in oxidative status during pregnancy in a viviparous lizard. *Functional Ecology*, 34(2), 475-485. https://doi.org/10.1111/1365-2435.13481

372 Gerson, A. R., & Guglielmo, C. G. (2011). House sparrows (Passer domesticus) increase protein catabolism in response to water restriction. *American Journal of Physiology-Regulatory, Integrative and Comparative Physiology*, 300(4), R925-R930.
375 https://doi.org/10.1152/ajpregu.00701.2010

376 Gunderson, A. R., & Leal, M. (2015). Patterns of thermal constraint on ectotherm activity.
377 *The American Naturalist*, 185(5), 653-664. https://doi.org/10.1086/680849

378 Herczeg, G., Kovács, T., Hettyey, A., & Merilä, J. (2003). To thermoconform or thermoregulate? An assessment of thermoregulation opportunities for the lizard
380 Zootoca vivipara in the subarctic. *Polar Biology*, 26, 486-490.

381 https://doi.org/10.1007/s00300-003-0507-y

382 Huey, R., & Slatkin, M. (1976). Cost and Benefits of Lizard Thermoregulation. *The Quarterly*

383 *review of biology*, 51, 363-384. https://doi.org/10.1086/409470

384 Kearney, M. R. (2013). Activity restriction and the mechanistic basis for extinctions under

385 climate warming. *Ecology Letters*, 16(12), 1470-1479.

386 https://doi.org/10.1111/ele.12192

387 Kearney, M. R., Munns, S. L., Moore, D., Malishev, M., & Bull, C. M. (2018). Field tests of a

388 general ectotherm niche model show how water can limit lizard activity and

389 distribution. *Ecological Monographs*. https://doi.org/10.1002/ecm.1326

390 Le Galliard, J. F., Rozen-Rechels, D., Lecomte, A., Demay, C., Dupoué, A., & Meylan, S.

391 (2021). Short-term changes in air humidity and water availability weakly constrain

392 thermoregulation in a dry-skinned ectotherm. *PLOS ONE*, 16(2), e0247514.

393 https://doi.org/10.1371/journal.pone.0247514

394 Lillywhite, H. B. (2017). Feeding begets drinking : Insights from intermittent feeding in

395 snakes. *The Journal of Experimental Biology*, 220(19), 3565-3570.

396 https://doi.org/10.1242/jeb.163725

397 Lorenzon, P., Clobert, J., Oppliger, A., & John-Alder, H. (1999). Effect of water constraint on

398 growth rate, activity and body temperature of yearling common lizard ( Lacerta

399 vivipara ). *Oecologia*, 118(4), 423-430. https://doi.org/10.1007/s004420050744

400 McCue, M. D. (2007). Western Diamondback Rattlesnakes Demonstrate Physiological and

401 Biochemical Strategies for Tolerating Prolonged Starvation. *Physiological and*

402 *Biochemical Zoology*, 80(1), 25-34. https://doi.org/10.1086/509057

403 Meylan, S., Dufty, A. M., & Clobert, J. (2003). The effect of transdermal corticosterone

404 application on plasma corticosterone levels in pregnant Lacerta vivipara. *Comparative*
405 *Biochemistry and Physiology Part A: Molecular & Integrative Physiology*, 134(3),

406 497-503. https://doi.org/10.1016/S1095-6433(02)00343-4

407 Moon, D.-Y., Owens, D. W., & MacKenzie, D. S. (1999). The Effects of Fasting and
408 Increased Feeding on Plasma Thyroid Hormones, Glucose, and Total Protein in Sea

409 Turtles. *Zoological Science*, 16(4), 579-586. https://doi.org/10.2108/zsj.16.579

410 Moore, D., Stow, A., & Kearney, M. R. (2018). Under the weather?—The direct effects of
411 climate warming on a threatened desert lizard are mediated by their activity phase and
412 burrow system. *Journal of Animal Ecology*, 87(3), 660-671.

413 https://doi.org/10.1111/1365-2656.12812

414 Murphy, M. S., & DeNardo, D. F. (2019). Rattlesnakes Must Drink : Meal Consumption Does
415 Not Improve Hydration State. *Physiological and Biochemical Zoology*, 92(4),

416 381-385. https://doi.org/10.1086/704081

417 Perez, D. J. P., Carvalho, J. E. de, & Navas, C. A. (2021). Effects of food intake and hydration
418 state on behavioral thermoregulation and locomotor activity in the tropidurid lizard
419 Tropidurus catalanensis. *Journal of Experimental Biology*, 224(6).

420 https://doi.org/10.1242/jeb.242199

421 Peterson, C. C. (2002). Temporal, population, and sexual variation in hematocrit of free-living
422 desert tortoises : Correlational tests of causal hypotheses. *Canadian Journal of*

423 *Zoology*. https://doi.org/10.1139/z02-021

424 Pinheiro, J. C., Bates, D. J., DebRoy, S., & Sakar, D. (2012). The Nlme Package : Linear and
425 Nonlinear Mixed Effects Models, R Version 3. In *R package version* (Vol. 6).

426 Pintor, A. F. V., Schwarzkopf, L., & Krockenberger, A. K. (2016). Hydroregulation in a
427 tropical dry-skinned ectotherm. *Oecologia*, 182(4), 925-931.

428 https://doi.org/10.1007/s00442-016-3687-1

429 Pirtle, E. I., Tracy, C. R., & Kearney, M. R. (2019). *Behavior of Lizards : Evolutionary and
430 Mechanistic Perspectives*. Routledge & CRC Press.
431 https://www.routledge.com/Behavior-of-Lizards-Evolutionary-and-Mechanistic-Perspectives/Bels-Russell/p/book/9780367655853

432 Perspectives/Bels-Russell/p/book/9780367655853

433 Price, E. R. (2017). The physiology of lipid storage and use in reptiles. *Biological Reviews*,

434 92(3), 1406-1426. https://doi.org/10.1111/brv.12288

435 R Core Team. (2022). *R: A language and environment for statistical computing*. R Foundation for Statistical Computing, Vienna, Austria.

436 Vienna, Austria. https://www.R-project.org/

437 Rozen-Rechels, D., Badiane, A., Agostini, S., Meylan, S., & Le Galliard, J.-F. (2020). Water

438 restriction induces behavioral fight but impairs thermoregulation in a dry-skinned

439 ectotherm. *Oikos*, 129(4), 572-584. https://doi.org/10.1111/oik.06910

440 Rozen-Rechels, D., Farigoule, P., Agostini, S., Badiane, A., Meylan, S., & Le Galliard, J.

441 (2020). Short-term change in water availability influences thermoregulation

442 behaviours in a dry-skinned ectotherm. *Journal of Animal Ecology*, 89(9), 2099-2110.

443 https://doi.org/10.1111/1365-2656.13279

444 Sannolo, M., & Carretero, M. A. (2019). Dehydration constrains thermoregulation and space

445 use in lizards. *PLOS ONE*, 14(7), e0220384.

446 https://doi.org/10.1371/journal.pone.0220384

447 Van Damme, R., Bauwens, D., & Verheyen, R. F. (1991). The Thermal Dependence of

448 Feeding Behaviour, Food Consumption and Gut-Passage Time in the Lizard Lacerta

449 vivipara Jacquin. *Functional Ecology*, 5(4), 507-517. https://doi.org/10.2307/2389633

450 Waldschmidt, S. R., & Porter, W. P. (1987). A Model and Experimental Test of the Effect of

451 Body Temperature and Wind Speed on Ocular Water Loss in the Lizard Uta

452 stansburiana. *Physiological Zoology*, 60(6), 678-686.

453 https://doi.org/10.1086/physzool.60.6.30159982

454 Wright, C. D., Jackson, M. L., & DeNardo, D. F. (2013). Meal consumption is ineffective at

455 maintaining or correcting water balance in a desert lizard, *Heloderma suspectum*.
456 *Journal of Experimental Biology*, 216(8), 1439-1447.

457 https://doi.org/10.1242/jeb.080895

458

459

460

461

462

463
**Table 1.** Summary statistics of morphological and physiological changes in 60 males *Zootoca vivipara* from the four treatment groups (n = 15 per group). Morphological and physiological changes were calculated by subtracting the initial value measured at the beginning of the experiment from the final value measured at the end of the experiment. Shelter and food treatments had an additive effect on change in body mass (F<sub>1,33</sub> = 82.95, p < 0.0001; F<sub>1,33</sub> = 69.41, p < 0.0001 respectively), in limb thickness (F<sub>1,37</sub> = 18.15, p = 0.0001; F<sub>1,37</sub> = 10.15, p = 0.003), in tail width (F<sub>1,36</sub> = 9.96, p = 0.003; F<sub>1,36</sub> = 6.39, p = 0.02) and in the concentration of circulating triglycerides (F<sub>1,33</sub> = 4.61, p = 0.04; F<sub>1,33</sub> = 152.5, p < 0.0001). Circulating glucose concentrations were influenced by food treatment (F<sub>1,33</sub> = 14.64, p = 0.0005) but not by shelter treatment (F<sub>1,33</sub> = 0.13, p = 0.71). Data are provided as mean (± SE) for each treatment group and different letters indicate significant differences between groups (p < 0.05; Tukey's HSD post hoc test).

476

<table><thead><tr><th rowspan="2">Treatment groups</th><th colspan="2">Wet shelter</th><th colspan="2">Dry shelter</th></tr><tr><th>Food</th><th>Food deprivation</th><th>Food</th><th>Food deprivation</th></tr></thead><tbody><tr><td><strong>Variables</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Δ body mass (g)</td><td>-0.22 ± 0.05 <sup>a</sup></td><td>-0.65 ± 0.05 <sup>b</sup></td><td>-0.67 ± 0.05 <sup>b</sup></td><td>-1.04 ± 0.05 <sup>c</sup></td></tr><tr><td>Δ limb thickness (mm)</td><td>-0.07 ± 0.02 <sup>a</sup></td><td>-0.14 ± 0.02 <sup>ab</sup></td><td>-0.15 ± 0.02 <sup>bc</sup></td><td>-0.21 ± 0.02 <sup>c</sup></td></tr><tr><td>Δ tail width (mm)</td><td>-0.14 ± 0.11 <sup>a</sup></td><td>-0.43 ± 0.11 <sup>ab</sup></td><td>-0.47 ± 0.11 <sup>ab</sup></td><td>-0.68 ± 0.11 <sup>b</sup></td></tr><tr><td>Δ [triglycerides] (mg.dL<sup>-1</sup>)</td><td>256.4 ± 28.9 <sup>a</sup></td><td>-57.2 ± 28.5 <sup>b</sup></td><td>378.3 ± 29.7 <sup>c</sup></td><td>-47.8 ± 29.6 <sup>b</sup></td></tr><tr><td>Δ [glucose] (mg.dL<sup>-1</sup>)</td><td>49.5 ± 11.0 <sup>ab</sup></td><td>24.1 ± 11.0 <sup>b</sup></td><td>71.8 ± 11.0 <sup>a</sup></td><td>8.22 ± 11.8 <sup>b</sup></td></tr></tbody></table>

477 Δ = intra-individual change

478

479
480 **Figure legends**

481 **Figure 1.** A. Interactive effects of shelter and food treatment on daytime shelter use as a
function of time (number of days since the start of the experiment). B. Plasma osmolality
482
483 change during the experiment as a function of daytime shelter use and shelter treatment.
484
485 Colours in panel A represent experimental groups formed by the factorial combination of the
two treatments (WS-FOOD (dark blue): wet shelter and *ad libitum* feeding, WS-FD (light
486
487 blue): wet shelter and food deprivation, DS-FOOD (orange): dry shelter and *ad libitum*
488 feeding, DS-FD (yellow): dry shelter and food deprivation). Points represent average daily
489 shelter use (± SE) and solid lines represent the average shelter use adjusted with a quadratic
time effect. We found a significant effect of the interaction between time, shelter treatment
490 and food treatment on shelter use ($\chi^2_1 = 8.68, p = 0.003$). Colours in (B) represent lizards with
491 access to wet shelter (WS (dark blue)) or dry shelter (DS (red)). Points represent individual
492 shelter use score during the experiment and a positive change in osmolality indicates
493 physiological dehydration. The change in plasma osmolality was influenced by the interaction
494 between shelter use rate and shelter treatment ($F_{1,30} = 4.44, p = 0.04$) independently of food
treatment ($F_{1,30} = 2.27, p = 0.14$). Dehydration was positively correlated with daytime shelter
495
496 use in lizards with access to wet shelter ($F_{1,10} = 7.13, p = 0.02$) but it was not correlated in
497 lizards with access to dry shelter ($F_{1,8} = 3.68, p = 0.09$). Daytime shelter use rates were
498 calculated as the number of observations where the lizard was in the shelter divided by the
499 total number of daytime observations of the day in (A) or of the experiment in (B). Water
500 restriction begins on day 1.

501

502

503
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>
**Figure 2.** Effects of experimental treatments on thermo-hydroregulation behaviours of male *Zootoca vivipara*. Mean body temperature (A, B), mean basking rate (C, D), and eye closure rate (E) of lizards as a function of day and experimental treatment (WS: wet shelter, DS: dry shelter, FOOD: food *ad libitum*, FD: food deprivation). Points and error bars represent temperature, basking rate, and average daily eye closure rate of lizards (± SE). Basking and eye closure rates was calculated as the number of observations where the lizard exhibited basking behaviour or had its eyes closed divided by the total number of observations of the day. Water restriction begins on day 1. Shelter treatment in interaction with time significantly influenced mean body temperature ($F_{1,2514} = 11.04, p < 0.0001$), mean basking rate ($\chi^2_5 = 45.35, p < 0.001$), and eye closure rate ($\chi^2_5 = 17.05, p < 0.001$) in contrast to food treatment (p > 0.05 for all).

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527
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

Treatment - WS - DS - FOOD - FD
530 Supplementary Information

531 Figure S1

532 Daily variation of operative temperature on the hot and cold side of the thermal gradient. Jittered 533 points correspond to raw operative temperature values. Smoothing with a generalized additive 534 model was applied to visualize the nonlinear patterns (solid blue and red line). Thermal 535 preferences (black dashed line) and upper critical thermal limit (orange solid line) of *Zootoca 536 vivipara* (Gvoždík & Castilla, 2001) are represented.

<figure><img src="image_9.png" /></figure>

537

538
Daily variation of temperature (A) and relative humidity (B) in shelters as a function of shelter treatment (WS: wet shelter, DS: dry shelter). Jittered points correspond to raw values, red and blue lines are predictions from a generalized additive model. Thermal preference of *Zootoca vivipara* (Gvoždík & Castilla, 2001) is represented by a black dashed line. Mean temperature in the shelter did not differ significantly between dry and wet treatments ($F_{1,16123} = 2.28, p = 0.95$).

546

<figure><img src="image_10.png" /></figure>

547

548

549

550
551 **Figure S3**

552 Chronology of the experiment for each experimental batch.

<figure><img src="image_11.png" /></figure>

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569
570 **Figure S4**

571 Relationship between internal body temperature measured with a K-type thermocouple

572 inserted in the cloaca and surface body temperature measured with an infrared thermometer.

573 Surface body temperatures and cloacal temperatures were measured consecutively for each
574 individual using an infrared thermometer (Raytek, Raynger MX2) and a K-type thermocouple

575 (HI 935005N, Hanna) after the last behavioural record of day 0. The linear regression (with
576 95% confidence interval) is plotted against raw data. Cloacal temperatures were strongly
577 correlated with surface temperatures (N=60, T<sub>b</sub> = 7.46 (± 0.90) + 0.70 (± 0.03) × T<sub>IR</sub>, R<sup>2</sup> =

578 0.91).

<figure><img src="image_12.png" /></figure>

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