Chloé Chabaud
email: chloe.chabaud@normalesup.org

George A Brusch

Anouk Pellerin

Olivier Lourdais

Jean-François Le Galliard

Prey consumption does not restore hydration state but mitigates the energetic costs of water deprivation in an insectivorous lizard

Keywords:

To cope with limited availability of drinking water in their environment, terrestrial animals have developed numerous behavioral and physiological strategies including maintaining an optimal hydration state through dietary water intake. Recent studies performed in snakes, which are carnivorous reptiles with a specialized diet, suggest that benefits of dietary water intake are negated by hydric costs of digestion. Most lizards are generalist insectivores that can shift their prey types, but firm experimental demonstration of dietary water intake is currently missing in these organisms. Here, we performed an experimental study in the common lizard Zootoca vivipara, a keystone mesopredator from temperate climates exhibiting a great diversity of prey in its mesic habitats, in order to investigate the effects of food consumption and prey type on physiological responses to water deprivation. Our results indicate that common lizards cannot improve their hydration state through prey consumption, irrespective of prey type, suggesting that they are primarily dependent upon drinking water.

Yet, high-quality prey consumption reduced the energetic costs of water deprivation, potentially helping lizards to conserve a better body condition during periods of limited water availability. These findings have important implications for understanding the physiological responses of ectotherms to water stress, and highlight the complex interactions between hydration status, energy metabolism, and feeding behavior in insectivorous lizards.

Introduction

As climatic conditions shift towards longer and more frequent warm and dry spells in many regions (IPCC 2019), it is critical to understand and quantify the contributions of different water sources in wild animals. Optimal hydration state can be maintained by drinking freestanding water [START_REF] Karasov | Physiological Ecology: How Animals Process Energy, Nutrients, and Toxins[END_REF], by food consumption (dietary water intake, [START_REF] Nagy | Energy, Water, and Food Use by Springbok Antelope (Antidorcas marsupialis) in the Kalahari Desert[END_REF] or cutaneous drinking (e.g., [START_REF] Jørgensen | Water economy in a terrestrial toad (Bufo bufo), with special reference to cutaneous drinking and urinary bladder function[END_REF], or by internal metabolic production (metabolic water, [START_REF] Bailey | Sources of Water Supply for Desert Animals[END_REF][START_REF] Jindra | Linkage between diet humidity, metabolic water production and heat dissipation in the larvae of Galleria mellonella[END_REF]. Free-standing water is the most widespread, primary source of water intake in animals, but it is not continuously available in many terrestrial ecosystems and most organisms have developed strategies to cope with limited water availability. For example, water requirements can be met through dietary shifts [START_REF] Leinbach | Predator water balance alters intraguild predation in a streamside food web[END_REF], utilizing internal water reservoirs (e.g., urinary bladder, [START_REF] Davis | The urinary bladder as a physiological reservoir that moderates dehydration in a large desert lizard, the Gila monster Heloderma suspectum[END_REF], or even harvesting water from fog [START_REF] Seely | Irregular fog as a water source for desert dune beetles[END_REF].

Conversely, water loss can be minimized through behavioral changes in activity patterns [START_REF] Bissonette | The Influence of Extremes of Temperature on Activity Patterns of Peccaries[END_REF] or microhabitat use [START_REF] Dupoué | Physiological state influences evaporative water loss and microclimate preference in the snake Vipera aspis[END_REF][START_REF] Dezetter | Behavioural hydroregulation protects against acute effects of drought in a dry-skinned ectotherm[END_REF], but also through internal osmoregulatory strategies (e.g., nitrogen excretion via increased uric acid production, [START_REF] Braun | Integration of organ systems in avian osmoregulation[END_REF]. Finally, some species can tolerate hyperosmolality and deviate from homeostatic set-points to cope with water constraints [START_REF] Peterson | Anhomeostasis: Seasonal Water and Solute Relations in Two Populations of the Desert Tortoise (Gopherus agassizii) during Chronic Drought[END_REF][START_REF] Bradshaw | Thermal Homeostasis[END_REF]. This diversity of strategies has important implications for the capacity to survive in arid environments [START_REF] Cloudsley-Thompson | Adaptations of Arthropoda to Arid Environments[END_REF], but has been little investigated in non-arid adapted species.

A strong dependence of water budgets on dietary water intake has been shown in some animals living in semi-arid or arid environments characterized by low annual rainfall, high ambient temperatures, and recurrent drought episodes [START_REF] Nagy | Water economy of free-living desert animals[END_REF]. For example, many herbivorous desert vertebrates feed on plant tissues or seeds to sustain their standard activity and water balance even when water is not available in the environment (in mammals, [START_REF] Nagy | Seasonal Water and Energy Metabolism of the Desert-Dwelling Kangaroo Rat (Dipodomys merriami)[END_REF]in birds, Tieleman 2002; in turtles, [START_REF] Nagy | Physiological Ecology of Desert Tortoises in Southern Nevada[END_REF]; in lizards, [START_REF] Minnich | Diet, Behavior and Water Turnover in the Desert Iguana, Dipsosaurus dorsalis[END_REF]. Such dietary water intake is thought to provide a considerable advantage in these environments and some species display adaptive shifts in food preferences toward resources with higher water content during the dry season [START_REF] Karasov | Water Flux and Water Requirement in Free-living Antelope Ground Squirrels Ammospermophilus leucurus[END_REF]. However, recent studies in carnivorous squamate reptiles (i.e., lizards, snakes, and amphisbaenians), suggest that they do not rely on dietary water intake due to the lower water content of their prey compared to plant material, and the important protein content that increases the hydric cost of digestion and leads to more nitrogenous waste [START_REF] Wright | Meal consumption is ineffective at maintaining or correcting water balance in a desert lizard, Heloderma suspectum[END_REF][START_REF] Lillywhite | Feeding begets drinking: insights from intermittent feeding in snakes[END_REF]. Even in arid environments, these species do not receive net hydric benefits from their diet [START_REF] Murphy | Rattlesnakes Must Drink: Meal Consumption Does Not Improve Hydration State[END_REF]. Indeed, fluid secretion is required during digestion to aid in transport and absorption of nutrients, and this hydric cost can be particularly important when carnivorous reptiles are already dehydrated [START_REF] Murphy | Rattlesnakes Must Drink: Meal Consumption Does Not Improve Hydration State[END_REF]. Feeding may also involve behavioral changes that increase water loss rates, such as locomotor activity during foraging and basking during digestion, and trade-offs can occur between food acquisition and water loss [START_REF] Gregory | Conflicts and interactions among reproduction, thermoregulation and feeding in viviparous reptiles: are gravid snakes anorexic?[END_REF]. In addition, carnivorous reptiles often feed intermittently and it has been suggested that this may increase the hydric cost of digestion and reduce opportunities for dietary shifts toward prey with higher water content [START_REF] Lillywhite | Feeding begets drinking: insights from intermittent feeding in snakes[END_REF].

Currently, there is limited knowledge about dietary water intake and hydric costs of digestion in insectivorous reptiles, which include the vast majority of lizards (see Table 1; [START_REF] Meiri | Traits of lizards of the world: Variation around a successful evolutionary design[END_REF]. In addition, it remains unknown if inferences drawn from studies of arid adapted species apply to species from moister tropical or temperate zones, where free-standing water is more regularly available. Most insectivorous lizards are generalist mesopredators with a non-intermittent diet dominated by invertebrates such as insects, spiders, or other arthropods [START_REF] Meiri | Traits of lizards of the world: Variation around a successful evolutionary design[END_REF]. The invertebrate prey of these lizards are highly variable in size, digestibility, and macro-and micro-nutrient content including water content [START_REF] Mccluney | The effects of hydration on growth of the house cricket, Acheta domesticus[END_REF].

Generalist insectivorous lizards can thus shift their diet to meet different demands depending on prey availability and resource expenditures across the activity season or between different life stages [START_REF] Bestion | Altered trophic interactions in warming climates: consequences for predator diet breadth and fitness[END_REF]. Previous work has focused on the energetic quality of food and its adequacy to optimal diet models based on energetic constraints [START_REF] Stamps | The Relationship between Selectivity and Food Abundance in a Juvenile Lizard[END_REF][START_REF] Dearing | Testing Models of Optimal Diet Assembly by the Generalist Herbivorous Lizard Cnemidophorus Murinus[END_REF], but very few studies have investigated dietary shifts due to water constraints in reptiles (but see [START_REF] Mautz | Ontogenetic Changes in Diet, Field Metabolic Rate, and Water Flux in the Herbivorous Lizard Dipsosaurus dorsalis[END_REF]). Yet, when prey have different water and protein content or different digestibility, this may influence the dietary water intake, leading to dietary shifts caused by water balance instead of energy balance. For example, if free-standing water is limited in the environment, insectivorous lizards might shift their diet towards high-water content prey to maintain water balance, which can impact the population dynamics and trophic ecology of these prey species [START_REF] Bestion | Non-consumptive effects of a toppredator decrease the strength of the trophic cascade in a four-level terrestrial food web[END_REF]. Thus, an understanding of the net dietary water intake and hydric costs of digestion of insectivorous lizards should include the possibility of different prey types that are representative of their standard diet. To our knowledge, no study has examined if different prey types are more or less effective at maintaining water balance in these organisms.

Water deprivation induces multiple physiological responses to restore hydration state, including lipolysis, reduced hepatic acid synthesis, or protein catabolism to release bound water, as previously demonstrated in birds, lizards, and snakes [START_REF] Gerson | House sparrows (Passer domesticus) increase protein catabolism in response to water restriction[END_REF][START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF][START_REF] Brusch | Additive effects of temperature and water availability on pregnancy in a viviparous lizard[END_REF]. These physiological responses are known to induce energy and structural costs with reduced body reserves and muscle condition [START_REF] Dezetter | Additive effects of developmental acclimation and physiological syndromes on lifetime metabolic and water loss rates of a dry-skinned ectotherm[END_REF][START_REF] Brusch | 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[END_REF]. Therefore, besides the direct effect of diet on the water budget, we need to consider the whole-organism metabolic response according to different prey types, since food might be important to recoup the energetic and structural costs of water deprivation.

Here, we designed a laboratory experiment to test if consumption of different prey types can compensate for water deprivation. We investigated these responses in the common lizard (Zootoca vivipara, Lichtenstein 1823), a generalist mesopredator with a widespread distribution across Eurasia, which is dependent on cool and moist environments inside humid grasslands and bogs. In these natural habitats, the common lizard is a keystone mesopredator due to its great diversity of invertebrate prey and its abundance (densities as high as 1,000 lizards per ha, [START_REF] Avery | Estimates of Food Consumption by the Lizard Lacerta vivipara Jacquin[END_REF][START_REF] Bestion | Non-consumptive effects of a toppredator decrease the strength of the trophic cascade in a four-level terrestrial food web[END_REF]. Previous field and laboratory studies have suggested that the species is dependent on free-standing water to maintain water balance [START_REF] Grenot | Water Budget in Some Populations of the European Common Lizard, Lacerta vivipara Jacquin[END_REF][START_REF] Lorenzon | Effect of water constraint on growth rate, activity and body temperature of yearling common lizard (Lacerta vivipara)[END_REF][START_REF] Rozen-Rechels | Water restriction induces behavioral fight but impairs thermoregulation in a dry-skinned ectotherm[END_REF]. Additionally, water deprivation causes a range of systemic responses associated with a reduction of body mass, proximal tail width loss (where lipids are stored, [START_REF] Chapple | Distribution of energy reserves in a viviparous skink: Does tail autotomy involve the loss of lipid stores?[END_REF], and limb thickness reduction (due to muscle wasting, [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF]. However, no previous study has investigated the link between diet, water balance, and body condition.

We tested for the occurrence of dietary water intake by comparing changes in plasma osmolality of mildly dehydrated lizards following the consumption of three distinct types of invertebrate prey with contrasted nutrient and water contents (crickets, spiders, and woodlice).

These prey represent the trophic diversity of prey in natural communities (see [START_REF] Bestion | Non-consumptive effects of a toppredator decrease the strength of the trophic cascade in a four-level terrestrial food web[END_REF]. These lizards were fed but had no access to free-standing water during the 6 days of the experimental treatment in a temperature-controlled room. They were compared to two groups of unfed lizards: one "hydrated control" group with no food but ad libitum access to free-standing water and one "dehydrated control" group without food or water. Using this experimental design, we tested the following three hypotheses:

1) If lizards are able to extract dietary water resulting in a net water gain, we predict that lizards held with food will have responses closer to hydrated versus dehydrated control lizards.

2) Alternatively, if eating imposes a net hydric cost, lizards held with food would exhibit responses closer to those of dehydrated rather than hydrated control lizards.

3) Third, we hypothesized that prey characteristics should have an impact on the response.

Notably digestively "costly" prey (low energy content and hard exoskeleton) such as woodlice should provide limited energy and water benefits, and could induce a shift from hypothesis one to two.

Material and methods

Study species, sampling, and acclimation

The common lizard Zootoca vivipara is a small lacertid lizard (Reptilia: Lacertidae) with a wide Eurosiberian distribution [START_REF] Surget-Groba | Multiple origins of viviparity, or reversal from viviparity to oviparity? The European common lizard (Zootoca vivipara, Lacertidae) and the evolution of parity[END_REF]. It inhabits cold and wet habitats, such as humid grasslands and bogs, and is highly sensitive to water deprivation due to high standard water loss rates [START_REF] Lorenzon | Effect of water constraint on growth rate, activity and body temperature of yearling common lizard (Lacerta vivipara)[END_REF][START_REF] Massot | Condition-dependent dispersal and ontogeny of the dispersal behaviour: an experimental approach[END_REF][START_REF] Dupoué | Water availability and environmental temperature correlate with geographic variation in water balance in common lizards[END_REF]. This lizard is a mesopredator in its food web with a diet of various invertebrates (hexapods, spiders, isopods, annelids) ranging in size from 1 to 9 mm (Avery, 1996), which includes decomposers, herbivorous, and predatory species [START_REF] Bestion | Altered trophic interactions in warming climates: consequences for predator diet breadth and fitness[END_REF]. Common lizards used in this study were captured in semi-natural populations maintained in 25 outdoor enclosures in CEREEP-Ecotron IleDeFrance in Saint Pierre lès Nemours, France (48°17'N, 2°41'E).

Experiments were carried out in accordance with institutional guidelines, under license from the French Ministry of the Environment (permit APAFIS#25281-2020042915424095).

We captured 128 lizards by hand in June 2020 and brought them back to a laboratory located nearby. We selected only sexually mature males based on external morphology, body size, and known age from previous population monitoring. Upon capture, one person measured snout-vent length (SVL, mean=59.3 ± 6.5 mm SD) and total length (TL) to the nearest millimeter with a plastic ruler as well as body mass (BM) to the nearest milligram using an electronic balance (mean=4.19 ± 1.62 g SD). Animals were then kept separated in individual terraria (18×11×12 cm) with sterilized peat substrate, a basking rock, a shelter buried in the substrate and a water cup with ad libitum access to water. During captivity, we provided all lizards with the same thermal conditions in a temperature-controlled room (23°C for 9h per day 09:00-18:00, and 16°C at night) in addition to heating lamp and UV above their terrarium during daytime (33°C below heating lamp). Lizards were fed with live crickets (Acheta domesticus) every two days and had water sprayed on the sides of their cages three times a day during acclimation.

After one week of acclimation, we collected morphology data of hydrated and fed lizards (referred to as T0 measurements). During the same day, they were weighed with an electronic balance and photographed with a digital scanner (ventral view, CanoScan LiDE 700F, Canon) to measure tail width, a trait linked to fat and muscle storage of the tail. Tail width was measured at the 7 th and 10 th subcaudal scales by processing the digital images with the software ImageJ (Schneider, Rasband & Eliceiri, 2012). We also assessed the transverse muscle thickness of the right hind leg at the mid region of the upper limb with digital calipers (ID-C1012BS, Mitutoyo, Japan, ±0.01 mm). These three morphological variables (body mass, tail width, and hindlimb width) were used as indicators of dehydration and well-being during the whole study. We also took a blood sample from the post-orbital sinus using 1 to 2 microcapillary tubes (ca. 20-40 µl whole blood). In the laboratory, samples were centrifuged at 11,000 rpm for 5 min to separate plasma from red blood cells. Plasma samples (approx. 10 µl)

were immediately frozen at -28°C until osmolality assays (see section 2.3).

Experimental design

In order to investigate the effects of food consumption on water and energy balance in mildly dehydrated lizards, we first exposed all lizards to a moderate water restriction for 10 days (Fig 1A). To do so, we removed the water cup from terraria and reduced water spray to once every two days while feeding lizards every two days. Water spray only provided lizards a few milliliters of water which they can ingest by licking, but that dried in less than 30 minutes (no water at all during 6 days is fatal for adults in this species). At the end of the 10 th day, we weighed lizards and measured tail and muscle width as indicated above (referred to as T1 measurements). We collected blood samples using 2 to 3 micro-capillary tubes (ca. 30-40 µl whole blood) and stored plasma samples like indicated above until osmolality and metabolite assays (see section 2.3).

We selected three types of prey known to be part of this species diet and with distinct trophic roles. First, we provided some lizards with crickets Acheta domesticus, an herbivorous insect that can be purchased at reptile breeding shops. Crickets were kept with dry food and with or without water in a climatic chamber at 25°C based on earlier studies showing that such water restriction conditions can trigger dehydration [START_REF] Mccluney | The effects of hydration on growth of the house cricket, Acheta domesticus[END_REF]. We also wanted to compare a same type of prey but with different water content, in order to test the quality of prey in addition to prey type. To do so, we provided lizards either with "hydrated" crickets (held with access to water) or with "dehydrated" crickets (held for at least a week without access to water). Second, we provided other lizards with woodlice (several species including Armadillum vulgare, Porcellio scaber, Philloscia muscorum and Oniscus asellus),.

Woodlice are detritivorous isopods and were collected in the same outdoor enclosures as the lizards. They were kept in a dark, humid box with leaf and compost until being provided to the lizards. Third, we provided some lizards with spiders (several species of Lycosidae or wolf spiders), which are carnivorous invertebrates. All arachnids were collected by hand in the nearby forest plot as well as in outdoor enclosures the day before feeding the lizard and were kept alone with humid cotton and no food prior to being used. The three species of prey are different in their macronutrient components (proteins, carbohydrates and lipids, see [START_REF] Reeves | Arthropod prey vary among orders in their nutrient and exoskeleton content[END_REF] and differences in those ratios (spiders having higher protein: lipid ratio than crickets and woodlice) can inform us about the hydric cost of digestion depending on prey type.

To measure prey water content, invertebrates were first weighed alive individually with a high precision scale (±0.01 mg). Then, they were put in the freezer at -20°C for a few hours to be killed, before being dried in a laboratory oven at 60°C for 24 hours and weighed again to determine dry mass. Water content was calculated as the difference between wet mass and dry mass. Despite the absence of a statistically significant difference in cricket water amounts (Table 2), we chose to maintain the two groups as distinct treatments. In addition, energy content was determined with dry aliquots (0.5-1.1g) by using an isoperibol bomb calorimeter (Parr 6200), with benzoic acid as a standard. Woodlice differed significantly in water content and energetic content, being of poorer quality, while the other prey types were similar (Table 2).

We separated lizards into six different treatments groups (G1 to G6) for six days (Fig 1A) including a "hydrated control" where lizards had access to ad libitum water but no food (G1), four groups of lizards that did not have access to ad libitum water but were provided 200 mg per day of either hydrated crickets (G2), dehydrated crickets (G3), woodlice (G4), or spiders (G5) and a "dehydrated control" group where lizards did not have access to ad libitum water or food (G6). We provided the same quantity of each prey type which corresponded to similar energy intake for crickets and spiders but not for woodlice (Table 2).

Parameters considered and plasma assays:

We assessed plasma osmolality before and after the period of food consumption to get a direct indicator of the response: a decrease in plasma osmolality indicates that lizards can compensate for a lack of drinking water by consuming food, whereas an increase in plasma osmolality indicates that meal consumption (including secretion, digestion, absorption, and associated behavioral changes) has a non-negligible hydric cost [START_REF] Wright | Meal consumption is ineffective at maintaining or correcting water balance in a desert lizard, Heloderma suspectum[END_REF]. We also tracked plasma circulating metabolites associated with catabolism and fasting in reptiles. We considered glucose which is liberated in the liver during fasting from stored glycogen, triglycerides stored in adipose tissue or neoglucogenesis, and also ketone bodies produced by the breakdown of fatty acids in the liver during periods of food restriction (McCue, [START_REF] Mccue | Physiological Responses to Starvation in Snakes: Low Energy Specialists[END_REF][START_REF] Price | Serum triglycerides and ßhydroxybutyrate predict feeding status in green turtles (Chelonia mydas): Evaluating a single blood sample method for assessing feeding/fasting in reptiles[END_REF]. In particular, the brain, unlike other organs, can only use glucose and ketone bodies to function, which makes them particularly essential during food restriction [START_REF] Price | The physiology of lipid storage and use in reptiles[END_REF]. We also monitored morphology over time (tail and leg width) as indicators of structural mobilization. We measured the body mass of lizards every two days, and we weighed any remaining food in the terrarium to assess how much was eaten by each individual (Table 2). On the 7 th day, we measured body mass, leg and muscle width (T2 measurement), and collected a blood sample to measure plasma osmolality and metabolites. All lizards were then removed from the experiment, fed and provided water ad libitum for 2-3 days, and released into the outdoor enclosures.

First, plasma osmolality was determined using a vapor pressure osmometer (Vapro 5600, ELITechGroup) with the protocol described in [START_REF] Wright | Meal consumption is ineffective at maintaining or correcting water balance in a desert lizard, Heloderma suspectum[END_REF] adjusted to small plasma volumes [START_REF] Dupoué | Water availability and environmental temperature correlate with geographic variation in water balance in common lizards[END_REF]. Before analyses, plasma was diluted (1:3) in standard saline solution (280 mOsm kg -1 ) to obtain duplicates for each sample (CV<0.4%). Normosmic plasma osmolality values (280-320 mOsm kg -1 ) indicate that lizards are hydrated, whereas hyperosmotic values (> 350 mOsm kg -1 ) indicate that lizards are moderately to sub-clinically dehydrated [START_REF] Dupoué | Water restriction causes an intergenerational trade-off and delayed mother-offspring conflict in a viviparous lizard (J Grindstaff[END_REF]. Second, we measured plasma concentrations of ketone bodies β-Hydroxybutyrate (β-HB) with a colorimetric method (β-Hydroxybutyrate Ketone Body Colorimetric Assay Kit, ref n°700190, Cayman Chemical, USA). β-HB are carbohydrates that are produced in the liver as a byproduct of the catabolism of fatty acid βoxidation during fasting periods in a process known as ketogenesis. β-HB can be used as an energy source for the brain during periods of starvation and thus provide information on the nutritional status [START_REF] Mccue | Fatty acid analyses may provide insight into the progression of starvation among squamate reptiles[END_REF][START_REF] Mccue | Physiological Responses to Starvation in Snakes: Low Energy Specialists[END_REF]. Here, plasma samples were diluted to 1:8 (using 8 μl of plasma) in the dilution buffer of the assay kit. Absorbance was measured at 450 nm and then transformed to concentration values using a dilution range provided by the kit. We performed duplicates on a few samples to measure a coefficient of variation within plates (CVwithin=7%) and between plates (CVbetween=11%) Third, we measured levels of circulating triglycerides in plasma by a colorimetric assay (Triglyceride Colorimetric Assay kit, ref n°10010303, Cayman Chemical, USA) using 2.5 μl of plasma diluted at 1:4 in provided dilution buffer. This method quantifies the total triglycerides using a chain of three enzymatic reactions ending up in hydrogen peroxide production, which is converted into quinoneimine dye quantified by absorbance at 540 nm (CVwithin=12% ; CVbetween=13%).

Finally, we measured glucose concentration in the plasma with a colorimetric assay (Glucose Colorimetric Assay Kit, ref n°10009582, Cayman Chemical, USA). We used 1.5 μl of plasma diluted at 1:13 in the provided dilution buffer as the glucose concentrations were very high compared to kit sensibility. After three chemical reactions, the product of glucose is pink and quantified by absorbance at 500 nm (CVwithin=4% ; CVbetween > 30% so we only compared samples measured on the same kit).

Statistical analyses

All statistical analyses were performed using R statistical software (version 3.6.3; R Core Team 2020). First, we checked plasma osmolality values during the first phase of our manipulation to verify that lizards were mildly dehydrated and dehydration was similar among treatment groups. This test was performed with an analysis of variance of the intraindividual change in plasma osmolality from T0 to T1. There was no difference among groups (F5,120=0.89, p=0.49) since all lizards had similar increases in plasma osmolality (mean ± SE: 41.08 ± 3.86 mOsm kg -1 ). Then, to assess the effects of treatments on morphology, plasma osmolality, and metabolites over the course of the experiment, we performed independent linear models for each response variable. We calculated intra-individual changes as the difference between T1 and T2 (referred to as Δvariable) and included the fixed effects of treatment groups and initial value of the response variable in a linear regression model fitted with the lm procedure. Prior to statistical tests, we used diagnostic plots of residuals to verify the model assumptions with regard to the normality and homogeneous variance of residuals (Heumann, Schomaker, & Shalabh, 2016). Post-hoc tests of differences among groups were performed using the multcomp package [START_REF] Hothorn | Package 'multcomp'. Simultaneous inference in general parametric models[END_REF]. All results are reported as means ± SE unless otherwise stated. As we noticed a difference in the amount of food consumed among treatment groups with access to food (F3,83=10.22, p<0.0001), we conducted a second set of analyses with fed lizards using the total amount of food consumed during the manipulation period as a covariate.

Results

Dehydration and water balance

Treatment groups had a significant effect on Δosmolality (F5,120=11.27, p<0.0001;

Plasma metabolites

There was a strong effect of food treatment on triglyceride concentration changes in plasma (F5,88=37.25, p<0.0001; Fig 3A), as well as an effect of initial concentration (F1,88=111.20, p<0.0001). Post-hoc analyses indicated three different treatment group categories. First, triglyceride concentrations on average decreased slightly in control lizards with no food irrespective of water availability. Second, triglyceride concentrations did not change on average in lizards provided with woodlice. Third, lizards fed with hydrated or dehydrated crickets and spiders had higher concentrations of plasma triglycerides (respectively, contrasts: β=420.8, β=413.4 and β=286.5, all p<0.0001; Fig 3A) compared to the other groups. Across all treatment groups with access to food, the amount of food consumed was positively correlated with the change in triglyceride concentrations (F1,53=7.83, p=0.007). At the same time, plasma β-HB concentrations slightly increased with a low variability among lizards and there was no significant difference among treatment groups (F5,118=1.44, p=0.22; initial concentration: F1,118=2.69, p<0.0001). Instead, intra-individual change of glucose concentration was highly variable and differed among treatment groups (F5,73=4.04, p=0.0027; F1,73=5.99, p=0.017; Fig 3B). In the dehydrated control treatment, lizard glucose concentrations increased (estimate=67.83 ± 18.94, p=0.0006) above the levels observed in the hydrated control treatment, whereas lizards provided with food had intermediate values. We found no effect of food consumption on glucose concentration changes (F1,45=0.01, p=0.92).

Discussion

In this study, we tested if prey consumption by a generalist mesopredator lizard could compensate for water deprivation via dietary water intake. Notably, our results revealed that eating does not restore water balance, irrespective of prey type. On the contrary, osmolality increased similarly in all water-restricted lizards, with or without access to prey, whereas access to free-standing water was associated with restoration of an optimal hydration status as expected. This result is remarkable given that non-fed animals lost about 0.8 g of body mass during the experiment, which corresponds to average water loss through excretion in addition to respiratory and cutaneous evaporative water losses (Le [START_REF] Galliard | A worldwide and annotated database of evaporative water loss rates in squamate reptiles[END_REF]. At the same time, we provided 1.2 g of food with a mean water content of 72%, which is the equivalent of 0.85 g potential dietary water intake (Table 2). Thus, if lizards could extract significant amount of water from their food, they would have been able to compensate fully or partly for the loss of drinking water, especially when fed with high quality prey types, but this was obviously not the case. In addition, our results suggest that the more the lizards eat, the more dehydrated they get, which would imply that food intake compromises water balance although this result must be interpreted with caution given that it is based on a correlation (Fig 1C).

Altogether, these results imply that food consumption and digestion have a significant water cost that counterbalance dietary water intake whatever the prey type and that common lizards are therefore dependent on free standing water intake (i.e., drinking) or air moisture to maintain their water balance [START_REF] Gettinger | Energy and Water Metabolism of Free-Ranging Pocket Gophers, Thomomys Bottae[END_REF].

Only a few previous studies have investigated dietary water intake in squamate reptiles using experimental approaches. Direct tests on herbivorous and insectivorous species have found similar results, with food consumption reducing body mass loss due to water deprivation (Table 1, [START_REF] Munsey | Water loss in five species of lizards[END_REF]. However, osmolality was not measured, so previous knowledge of the effect of food consumption on animal physiological hydration state is lacking. Studies focusing on carnivorous species explain the water costs of digestion through intermittent feeding and consumption of protein-rich prey, two traits associated with a stronger water use during digestion, metabolism, and excretion [START_REF] Wright | Meal consumption is ineffective at maintaining or correcting water balance in a desert lizard, Heloderma suspectum[END_REF][START_REF] Lillywhite | Feeding begets drinking: insights from intermittent feeding in snakes[END_REF]. Thus, food consumption and metabolism, as well as the elimination of metabolic wastes, can increase the requirement for water, potentially exacerbating dehydration during periods of drought in carnivorous reptiles, such as snakes [START_REF] Lillywhite | Feeding begets drinking: insights from intermittent feeding in snakes[END_REF]. In our study, lizards are insectivores that fed continuously, but increased dehydration due to feeding could be the result of an increased activity and body temperature due to the thermal and energetic requirements of foraging and digestion (Reilly, McBrayer, & Miles, 2007). These behavioral aspects may be of great importance in explaining the differences between non-fed and fed individuals, particularly in widely foraging and active lizards of small body size. Our results further suggest that lizards provided with low-quality diet such as woodlice favored their energetic balance rather than their water balance, but we cannot determine if it was due to a digestion constraint or the result of a behavioral choice from woodlice-fed lizards to thermoregulate more, as we had no data on locomotor activity and thermoregulation. If locomotion for foraging and thermoregulation to reach an optimal temperature for digestion are mostly responsible for increased water losses and negate the possible dietary water gains, we expect these costs to be even more significant in the field.

Indeed, hunting for prey should have a much higher energetic and hydric cost in natural environments than in captivity [START_REF] Anderson | Contrasts in energy intake and expenditure in sit-andwait and widely foraging lizards[END_REF]. Therefore, complementary studies are needed to quantify the hydric costs of digestion and the behavioral trade-offs between feeding and hydration.

Despite no evidence for the hypothesis that water can be extracted from food from our data on plasma osmolality, a relevant metric of whole-organism hydration status [START_REF] Capehart | No drought about it: Effects of supplemental hydration on the ecology, behavior, and physiology of free-ranging rattlesnakes[END_REF], we found that food consumption can mitigate the structural and energetic costs caused by chronic water deprivation, which included a general loss of body condition, muscle loss from the limb due to protein catabolism to release bound water (as shown in reptiles [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF] and lipid catabolism from the reserves stored in the tail [START_REF] Price | The physiology of lipid storage and use in reptiles[END_REF]. We chose three prey types corresponding to the natural diet of Zootoca vivipara [START_REF] Vacheva | Diet of the Viviparous lizard Zootoca vivipara (Lichtenstein, 1823) (Reptilia: Lacertidae) from its southern range[END_REF] and representing different trophic roles ranging from decomposers to insectivores [START_REF] Bestion | Altered trophic interactions in warming climates: consequences for predator diet breadth and fitness[END_REF] with distinct mobility strategies, morphology, as well as energy and nutrient content [START_REF] Reeves | Arthropod prey vary among orders in their nutrient and exoskeleton content[END_REF]. In natural populations, common lizards forage actively on a wide diversity of invertebrate species including larvae and adult spiders, insects, isopods or gastropods [START_REF] Vacheva | Diet of the Viviparous lizard Zootoca vivipara (Lichtenstein, 1823) (Reptilia: Lacertidae) from its southern range[END_REF]. For example, [START_REF] Avery | Food and feeding habits of the Common lizard (Lacerta vivipara) in the west of England[END_REF] found mostly spiders and homopters in stomach content of common lizards at two localities but also rarer prey such as ants and woodlice. Here, lizards provided with woodlice ate less readily during the experiment (on average 0.8 g instead of 1 g for the other prey types), possibly because these prey are less water and energy-rich (Table 2) and are less easy to handle and digest, making them distasteful for lizards (see [START_REF] Pekár | Suitability of woodlice prey for generalist and specialist spider predators: a comparative study[END_REF]. The results on metabolites indicate that the concentration of plasma triglycerides was aligned with the amount of food consumed per treatment, suggesting that food intake was associated with nutrient uptake and transport. Triglycerides are the transport form of lipids and can be hydrolyzed to produce fatty acids that enter the ATP production pathways. The low levels of plasma triglycerides in non-fed lizards and those fed with woodlice indicate little food intake and little lipid transport and storage, which is consistent with previous results seen in other reptiles [START_REF] Price | Serum triglycerides and ßhydroxybutyrate predict feeding status in green turtles (Chelonia mydas): Evaluating a single blood sample method for assessing feeding/fasting in reptiles[END_REF]. As a consequence, the general body condition of woodlice-fed and water deprived lizards was much more impacted than in lizards fed with higher quality food such as crickets and spiders: these lizards lost more body mass, more muscle and more fat reserves from their tail, and were virtually indistinguishable from those held without food nor water. Thus, despite providing no significant net intake of water, high quality food and strong food consumption were important to recoup some of the energy costs of water deprivation and reduce the body condition loss induced by water stress. These results highlight that feeding is important to maintain body condition during water restriction, including muscular mass and reserves that are essential for future survival. Thus, while food may be important to cope with limited rainfall and water availability, it does not primarily provide common lizards with water. Rather, food intake enables them to survive longer by maintaining a higher body condition, and improves their future prospects for reproduction by preserving muscles and reserves. Of course, this hypothesis will require confirmation through more accurate assessments of the lizards' condition and improved tests with prey that are both hydrated and less hydrated since our manipulation of crickets did not yield reliable results in this regard (Table 2). In this context, we predict that shifting diet toward higher water content prey would not allow this generalist lizard to avoid dehydration, but it can reduce the shortterm costs of drought and long-term fitness consequences [START_REF] Dezetter | Behavioural hydroregulation protects against acute effects of drought in a dry-skinned ectotherm[END_REF].

Unexpectedly, we found a significant difference between hydrated and dehydrated control groups in their plasma glucose concentration levels but no effect of food provisioning and water deprivation on plasma concentrations of ketone bodies (β-Hydroxybutyrate). It is predicted that ketone bodies' concentration should elevate during the second stage of fasting when carbohydrate stores are depleted and animals mobilize lipid stores instead [START_REF] Mccue | Physiological Responses to Starvation in Snakes: Low Energy Specialists[END_REF]. However, previous studies have shown that plasma ketone concentrations do not necessarily increase during fasting in reptiles, especially when fasting is too short, and may even decrease when starvation is extremely long [START_REF] Pontes | Concentrations of ketone bodies in the blood of the green lizard Ameiva ameiva (Teiidae) in different physiological situations[END_REF]. Conversely, the glucose levels in water-deprived lizards were maintained at relatively high levels, regardless of their feeding status. In addition, lizards with neither water nor food had much higher glucose concentration at the end of the manipulation than those from the hydrated control group. In vertebrates exposed to periods of fasting or intense exercise, maintenance of high glucose concentrations in the blood is ensured by gluconeogenesis in the liver (a metabolic pathway that generates glucose from non-carbohydrate carbon substrates; [START_REF] Zain-Ul-Abedin | Gluconeogenesis in the kidney and liver slices of a lizard, Uromastix hardwickii[END_REF] and glycogenolysis (the degradation of glycogen stored in adipose tissues or muscles; [START_REF] Storey | Metabolic adaptations supporting anoxia tolerance in reptiles: Recent advances[END_REF]. Gluconeogenesis involves glucose production in the liver from transported blood glycerol that is released by triglyceride hydrolysis in the tail or other abdominal reserve tissues. Glycerol levels and liver gluconeogenesis activity can increase during periods of fasting in snakes [START_REF] Price | The physiology of lipid storage and use in reptiles[END_REF]. Thus, high glucose concentrations in the blood of food and water-deprived lizards may represent a higher capacity for gluconeogenesis due to lipid catabolism induced by fasting and dehydration. Similarly, water deprivation in the absence of food led to stronger muscle wasting, which may end up in muscle glycogen degradation and increased glucose production. This conversion of muscle stored glycogen into plasma circulating glucose was previously demonstrated in turtles [START_REF] Emerson | Preliminary Study on Seasonal Liver Lipids and Glycogen, and Blood Sugar Levels in the Turtle Graptemys pseudogeographica (Gray) from South Dakota[END_REF] and in amphibians and lizards after vigorous exercise, which is close to simulated conditions of fasting [START_REF] Gleeson | Lactate and glycogen metabolism during and after exercise in the lizard Sceloporus occidentalis[END_REF][START_REF] Gleeson | Patterns of metabolic recovery from exercise in amphibians and reptiles[END_REF]. These observations are interesting because our understanding of how stored macronutrients are utilized during fasting in reptiles still remains limited compared to our knowledge of the physiology of fasting in mammals and birds [START_REF] Price | The physiology of lipid storage and use in reptiles[END_REF].

Our findings suggest that muscle breakdown (catabolism) in reptiles may commence earlier than expected in the starvation process, particularly in cases of water scarcity [START_REF] Brusch | Muscles provide an internal water reserve for reproduction[END_REF]. Further physiological investigations would aid in elucidating these pathways and how they may be influenced by hydration status.

The effect of environmental temperatures on ectotherm metabolic rates is now wellstudied [START_REF] Schulte | The effects of temperature on aerobic metabolism: towards a mechanistic understanding of the responses of ectotherms to a changing environment[END_REF] but we know little about the other abiotic factors involved in the regulation of metabolism and energetic functions of the organisms. If digestion is strongly dependent on body temperature regulation in reptiles (e.g. [START_REF] Harlow | The effect of temperature on digestive efficiency in the herbivorous lizard, Dipsosaurus dorsalis[END_REF][START_REF] Naulleau | The Effects of Temperature on Digestion in Vipera aspis[END_REF], how it is related to water balance and how water availability impacts nutrient assimilation is poorly studied. When body temperature increases, evaporative water losses also increase, and these losses are further accentuated by higher metabolism and respiration rates (Reilly et al., 2007), which are closely linked to food intake. It is therefore crucial to account for potential effects of food consumption and digestion on temperature and water balance regulation in reptiles. Changes in diet have been observed in other ectothermic species, where omnivores increase their herbivory in response to higher temperatures [START_REF] Carreira | Warm vegetarians? Heat waves and diet shifts in tadpoles[END_REF][START_REF] Carreira | Can heat waves change the trophic role of the world's most invasive crayfish? Diet shifts in Procambarus clarkii[END_REF][START_REF] Zhang | Ectothermic omnivores increase herbivory in response to rising temperature[END_REF]. While these studies primarily focus on aquatic ectotherms, the impact of environmental factors on the diet of terrestrial ectotherms remains poorly known [START_REF] Zimmerman | Interactions between the Environment and Ectothermy and Herbivory in Reptiles[END_REF]. Our findings indicate that insectivorous species face trade-offs between water and energy balance, and their survival in water-deprived regions may depend on food intake. This has potential implications for ecosystems, such as increased top-down regulation of intermediate levels on primary producers with global change [START_REF] Zhang | Ectothermic omnivores increase herbivory in response to rising temperature[END_REF]. Further research is needed to gain a deeper understanding of how [START_REF] Munsey | Water loss in five species of lizards[END_REF] Table 2: Water content and nutritional quality (mean ± SD) of invertebrates used in the experiment. N=20 individuals for water content, N = number of pools used for energy content, with each pool containing tens of individuals, and N = number of lizards in each food treatment for quantity of food eaten. The total quantity of food provided during the 6 days of manipulation was 1.2 g on average. *** indicates that the group is significantly different from the others.

Hydrated Crickets

  Fig 2B), whereas a significant and similar reduction in tail width was observed in the

Figure 1 :

 1 Figure 1: A) Timeline of the experimental design and of measurements performed between

Figure 2 :

 2 Figure 2: Morphological changes A) Change in body mass (g, mean initial value: 3,81 ± 0,05

Figure 3 :

 3 Figure 3: Changes in blood parameters A) Change in triglyceride concentration in plasma

Table 1 :

 1 Summary of empirical studies of dietary water intake in squamate reptiles. Field measures of water influx rates suggest that water is mostly provided from a diet including succulent plants. Lizards apparently did not drink water but lost body mass. Field measures of water influx rates suggest that water is mostly provided from diet and lizards maintain a constant hydration state during the year, but shift to different plant materials during the summer. Food consumption allows to recoup part of the body mass loss induced by a chronic water deprivation experiment in the laboratory. Osmolality and body hydration were not measured.

	Species name	Standard diet	Climate zone	Link between diet and water balance	Reference
	Acrochordus granulatus	Carnivorous	Marine	Direct test. Dietary water intake is insufficient for water balance	Lillywhite, Heatwole,
					& Sheehy, 2014
	Agkistrodon conanti	Carnivorous	Semi-aquatic	Direct test. Food consumption increases post-prandial drinking in a	Lillywhite, 2017
				laboratory experiment.
	Crotalus atrox	Carnivorous	Semi-arid	Direct test. Eating compromises water balance (osmolality) in chronic	Murphy & DeNardo,
				laboratory experiments of food consumption under water deprivation.	2019
	Heloderma suspectum	Carnivorous	Xeric	Direct test. Eating does not correct water balance (osmolality) in acute and	Wright et al., 2013
				chronic laboratory experiments of food consumption.
	Lampropeltis californiae	Carnivorous	Semi-arid	Direct test. Food consumption increases post-prandial drinking in a	Lillywhite, 2017
				laboratory experiment.
	Nerodia fasciata	Carnivorous	Semi-aquatic	Direct test. Food consumption increases post-prandial drinking in a	Lillywhite, 2017
				laboratory experiment.
	Nerodia clarkii	Carnivorous	Marine	Indirect test. Moderate dehydration with access to food (fish) inhibits	Edwards et al., 2021
				feeding and leads to body mass loss.
	Nerodia fasciata	Carnivorous	Semi-aquatic	Indirect test. Moderate dehydration with access to food (fish) inhibits	Edwards et al., 2021
				feeding and leads to body mass loss.
	Pantherophis alleghaniensis Carnivorous	Mesic	Direct test. Food consumption increases post-prandial drinking in a	Lillywhite, 2017
				laboratory experiment.
	Varanus rosenbergi	Carnivorous	Mesic	Indirect test. Field measures of water influx rates suggest that water is	Green, Dryden, &
				mostly provided from diet during the summer but not in other seasons.	Dryden, 1991
				Free-standing water consumption unknown.
	Angolosaurus skoogi	Herbivorous	Xeric	Indirect test. Nagy et al., 1991
	Dipsosaurus dorsalis	Herbivorous	Xeric	Indirect test. Minnich &
					Shoemaker, 1970
	Dipsosaurus dorsalis	Herbivorous	Xeric	Direct test.

digestive physiology affects various aspects of an individual's life history traits [START_REF] Karasov | Interplay between Physiology and Ecology in Digestion[END_REF], as trade-offs between linked physiological traits are crucial in understanding organism responses to environmental constraints [START_REF] Urban | Improving the forecast for biodiversity under climate change[END_REF][START_REF] Riddell | Cooling requirements fueled the collapse of a desert bird community from climate change[END_REF].

Acknowledgements

The authors want to thank Jean-Patrice Robin and the IPHC UMR 7178 for their help with the analysis of the invertebrate calorimetric contents. We also thank the intern students who help us with the experiment and animal care, especially Anna Marcout and Jules Domalain, as well as the CEREEP Ecotron IleDeFrance staff.

Competing interests

No competing interests declared.

Author contributions

Funding

This study received the financial and technical support from the Centre National de la Recherche Scientifique (CNRS) and was funded by the Agence Nationale de la Recherche AQUATHERM project (ANR-17-CE02-0013 to JFLG and OL).

Data availability

Data will be deposited in Zenodo after acceptance.

Munsey, 1972

Varanus caudolineatus Omnivorous Semi-Arid Indirect test. Field and laboratory measures of water influx rates suggest that water is importantly provided from diet. Free-standing water consumption unknown. [START_REF] Thompson | Energy and Water Turnover Rates of a Free-living and Captive Goanna, Varanus caudolineatus (Lacertilia: Varanidae)[END_REF]