Shelter availability, stress level, and digestive performance - Journal of ...

J Exp Biol Advance Online Articles. First posted online on 15 November 2012 as doi:10.1242/jeb.078501 Access the most recent version at http://jeb.biologists.org/lookup/doi/10.1242/jeb.078501

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Shelter availability, stress level, and digestive

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performance in the aspic viper

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The Journal of Experimental Biology – ACCEPTED AUTHOR MANUSCRIPT

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Xavier Bonnet, Alain Fizesan, Catherine Louise Michel

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1. CEBC-CNRS, 79360 Villers en Bois, France

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Correspondance : Xavier Bonnet ; Tel +33 549 097 879 ; Fax +33 549 096 111 ; email

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[email protected]

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Submitted for consideration in Journal of Experimental Biology

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Keywords: corticosterone, digestive performance, ectothermy, heliotheria, reptiles,

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safety, shelter, snake, stress, thermoregulation

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Short title: Impact of lack of shelter on snakes

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Abstract

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The lack of shelter can perturb behaviors, increase stress level, and thus alter

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physiological performances (e.g. digestive, immune, or reproductive functions).

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Although intuitive, such potential impacts of lack of shelter remain poorly

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documented. We manipulated shelter availability, environmental and physiological

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variables (i.e. access to a heat source, predator attack, feeding status) in a viviparous

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snake. We assessed sun-basking behavior, digestive performance (i.e., digestive

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transit time, crude estimate of assimilation, regurgitation rate) and plasma

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corticosterone levels (a proxy of stress level). Shelter deprivation provoked a strong

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increase in sun-basking behavior and thus elevated body temperature; even in unfed

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individuals for which energy savings would have been otherwise beneficial. The lack

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of heat was detrimental digestive performance (i.e. all the metrics used to assess it).

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Simulated predator attacks worsened the situation and entailed a further

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deterioration of digestion. The combination of the lack of shelter with cool ambient

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temperatures markedly elevated basal corticosterone level and was associated with

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low digestive performance. This hormonal effect was absent when only one negative

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factor was involved, suggesting a threshold response. Overall, our results revealed

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important non-linear cascading impacts of shelter availability on stress-hormone

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levels, behaviors and physiological performance. These results infer shelter

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availability is important for laboratory studies, captive husbandry, and possibly

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conservation plans.

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Introduction

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In most animals, individuals spend a considerable amount of time in refuges, and

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shelter availability is often essential for the persistence of populations (Wright and

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Shapiro 1990, Armstrong and Griffiths 2001, Souter et al. 2004, Bonnet et al. 2009,

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Grillet et al. 2010, Lagarde et al. 2012). Shelters provide protection against predators

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and buffer environmental conditions (Anderson 1986, Schwarzkopf and Alford 1996,

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Sih 1997; Roper et al. 2001, Berryman and Hawkins 2006). The benefits of retreat site

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selection have been examined in species belonging to various lineages (arachnids,

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fish, reptiles, mammals or amphibians for instance; Bulova 2002, Kearney 2002,

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Goldsbrough et al. 2004, Kotler et al. 2004, Millidine et al. 2006, Hossie and Murray

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2010, 2011). However, previous investigations focused on constraining temperature

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conditions: hibernation, aestivation or sun-scorching periods (Seebacher and Alford

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2002, Beck and Jennings 2003, Lagarde et al. 2002, 2012, Cooper and Withers 2005).

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Yet, many animals intensively use their refuges for other reasons than to escape

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severe climatic conditions: for instance to sleep, for reproduction or digestion (Siegel,

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2005, Pike et al. 2010, 2011). Consequently, important behavioral and physiological

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consequences unrelated to overheating or dehydration should be associated with

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shelter availability; yet, this eco-physiological issue is poorly documented.

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When they leave their refuge, animals are exposed to both challenging abiotic and

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biotic factors (Sih 1997, Kearney 2002, Langkilde et al. 2003, Berryman and Hawkins

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2006). Assessing the consequences of the absence of shelter on stress level, behaviors

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and physiological performances is thus crucial to understand how behavioral trade-

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offs are mediated. Notably, many animals retreat into their shelter between foraging

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episodes. Such shuttling activity offers an appropriate context to test behavioral 3

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trade-offs. For example, individuals deprived from a shelter may be forced to

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undertake displacements to escape unfavorable and/or dangerous conditions with

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possible negative consequences on other activities. A lack of shelter may also

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generate physiological stress, which in turn may perturb immunity, sexual

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behaviors, and possibly many other fitness related processes (Rabin 1999).


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To examine these issues, we manipulated shelter availability, ambient

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temperature, feeding status, and predatory threat in a snake species. We assessed the

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consequences on three major traits: thermoregulation, digestive performance, and

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plasma corticoid levels (Möstl and Palme 2002, Bassett and Buchanan-Smith 2007).

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We measured corticoid hormones as they influence the process of acquisition and

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allocation of resources, the mobilization of body reserves, metabolism, and various

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behaviors (DeNardo and Sinervo 1994, Guillette et al. 1995, Romero 2004).

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We expected that snakes would display contrasting behavioral and

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physiological responses to different environmental conditions. We notably

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hypothesized that unfavorable conditions (e.g. lack of shelter, low ambient

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temperature) would provoke an elevation of plasma corticoid levels and would

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impede digestive performances. By contrast, favorable conditions (presence of a

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shelter plus high ambient temperature) should be associated with low plasma

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corticoid level and high digestive performance. Finally, intermediate situations

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(combination of favorable and unfavorable factors) should produce intermediate

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stress level and average digestive performance. Overall, these predictions can be

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expressed through one main hypothesis: environmental conditions should exert an

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additive effect (either positive or negative) on physiological parameters associated

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with stress and resource assimilation. Further, we expected state dependent effects

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where individual physiological status influences response (Aubret and Bonnet 2005).

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Notably, recently fed snakes should exhibit different behaviors compared to unfed

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snakes (second hypothesis). Indeed, as digestion requires elevated body

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temperatures, fed snakes should exhibit a positive thermoregulation shift to select

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hot microhabitat more often than unfed snakes.


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Snakes are suitable organisms to test these hypotheses. Most species are

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secretive and individuals spend considerable amounts of time sheltered (Bonnet and

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Brischoux 2008). They usually leave their refuge to feed on large prey items, and they

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tend to cease moving during long digestive episodes. Therefore, foraging and

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digestion can be easily distinguished and monitored (by comparison these processes

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overlap in herbivorous animals, rendering analyses complicated). Digestion speed

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depends on body temperature and is often associated with prolonged phases of sun

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basking (at least in temperate climates) that increases predation risk (Garland and

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Arnold 1983, Beck 1996); consequently, there is a conflict between rapid digestion

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associated to sun exposure versus survival provided by the refuge (Sih 1997). The

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experiments presented in this study are largely based on this trade-off.

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Material and methods

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Ethics Statement

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The authors attest their adherence to NIH standards. No snake was mistreated and

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the mice were euthanized following rapid cervical dislocation. Permit to collect the

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snakes were issued by the DIREN Poitou-Charentes (# 09/346/DEROG). All the

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experiments were performed under permit A79-001 and 79-157. 5


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Study species

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Previous studies provided important background (Saint Girons 1957, Bonnet et al.

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1999, 2000, 2001, 2002; Bonnet 2011; Ladyman et al. 2003; Zuffi et al. 2009; Michel and

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Bonnet 2010). Aspic vipers feed mainly on rodents and digestion is impeded when

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body temperature falls below 15°C (Naulleau 1983). In the field, animals are active

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under ambient temperatures ranging from 10°C to more than 30°C (depending upon

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cloudy layer). Vipers shuttle between periods of heliotheria (i.e. “sun-basking behavior

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to warm up the body”, Bailly 1894) and periods sheltered in their refuge. This shuttling

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activity is a central element of their thermoregulation.

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The snakes involved in the experiment were captured in the vicinity of the

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laboratory (between spring 2003 and autumn 2008; forest of Chizé, 46°08’49”N,

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0°25’31”W). Prior to the experiment the individuals were maintained in captivity

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(during at least 3 months) in standard boxes (42cm*34cm*19cm; L*l*h), with artificial

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grass as substratum, clean water ad libitum, a thermal gradient (20-35°C), and a roof-

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tile as a refuge. All the snakes accepted their food regularly (dead mice

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approximately every two weeks). The experiments were performed during the

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normal activity period of the species (May – August).

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Experimental design

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We performed two main experiments, and to obtain comparable data between them

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we used a single modular system (Figure 1). We built ten three-compartment cages

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fitted with artificial grass substratum (total length 150cm, 50x30x20cm per

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compartment) and with a small door (∅ 5cm) between adjacent compartments. When 6


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the doors were open, the snakes could freely move across the compartments

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(experiment 1). When the doors were shut, the snakes were restricted to one

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compartment (experiment 2). In one compartment (A, Figure 1) we placed a 50-W

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halogen lamp to create a heat source (i.e. mimicking solar radiation). Roof-tiles were

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used as shelters. The cages were placed in a temperature-controlled room set at 16°C

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(14 hours of daylight: 9:00AM-21:00PM). Ambient temperatures were measured with

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data loggers (iButtons Thermochron®). The temperature of the shelter and the

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surface body temperature of the snakes were measured from distance (20-30cm)

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using a 3 dots infrared laser thermometer (Raytek MX2, Fotronic Corporation;

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measurement diameter 19mm, range -30°C-900°C, precision ±1°C, 250ms/reading;

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see supplementary material). Surface and internal body temperature are highly

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correlated in reptiles (Lagarde et al. 2012). The front of the cages was transparent;

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hence, the observer and the snakes could see each other.

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a) Experiment 1

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We first assessed the behavioral trade-off between safety and sun basking. More

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precisely, we focused on the impact of shelter availability and feeding status on

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heliotheria. Each snake was placed in a three-compartment cage, and thus in a 16°C -

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40°C thermal gradient during the day (Figure 1-A). Two compartments, A and C,

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were fitted or not with a shelter, and we obtained four combinations:

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• No shelter available: the snake was always visible and could select either the

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hot compartment (A) versus one of the two cold compartments (B or C, figure 1).

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• One shelter was placed in the cold compartment (C) at the opposite of the hot

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spot (A). The snake could select the hot-unsheltered (A), the cold-unsheltered (B),

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or the cold-sheltered compartment (C).

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• Two shelters were provided: one in the hot compartment and one in the

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opposite cold compartment. The snake could select the hot-sheltered, the cold-

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sheltered, or the cold-unsheltered compartment.

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The medium compartment (B) was never provided with a shelter to better assess

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selection; indeed the snakes were forced to cross it to find a heat source and/or a

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shelter in the compartments A and/or C.

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To obtain comparative data, we also tested other situations. Notably, the snakes

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(fed or unfed) were given the choice between hot-sheltered versus cold-unsheltered

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compartments, or sheltered versus unsheltered compartments without any heat

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source. However, these situations do not entail any major conflict (hence, they were

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partly out of scope of the aim of the current study). Indeed, terrestrial snakes prefer

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to remain sheltered rather than to stay in the open and they prefer a warm place

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rather than a cold one. For simplicity, these specific results, although useful to test

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our protocol will be briefly presented and referred as checking tests.

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We used 32 snakes in this first experiment, balancing sex and age ratios, and

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encompassing a wide range of body sizes (Table 1). At least two weeks after their last

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meal, unfed snakes were randomly allocated to one of the four situations described

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above (following a random order). The snake was gently placed in the medium

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compartment of the cage one night before the beginning of records. It was observed

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every hour between 09h05 to 20h05 during two subsequent days (36h). Its position

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and attitude (e.g. coiled, stretched, moving, motionless) were noted (N=12

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observations per day, total number of observations per snake: N=24). Then, we

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performed again the same experiment with the same recently fed snakes. We offered

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a mouse to each snake (mouse mass represented 25±2% of snake mass, all individuals

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accepted the meal except during skin shedding [a relatively rare event] hence slight

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variations in sample sizes) and we placed the viper into the cage immediately after

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the prey was swallowed.

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Overall, most of the snakes were tested either fed or unfed, and for each digestive

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status they experienced four different situations (4 combinations of shelter(s)). We

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collected a total of 4,752 focal observations. In addition, we collected 816 focal

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observations for the checking tests on a random subsample of 25 snakes.

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b) Experiment 2

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In the second experiment, we examined the consequences associated with the

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selection of one of the three compartments available to the snakes in the first

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experiment. Thus, we shifted from measuring heliotheria (i.e. sun basking behavior)

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and sheltering behaviors to digestive performance and stress levels.

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During two weeks, we imposed the four combinations of heat source and shelter

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availability to recently fed snakes: cold-unsheltered; cold-sheltered; hot-unsheltered;

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hot-sheltered. The doors of the boxes were thus shut (total 30 separate

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compartments, Figure 1). Half of the compartments were fitted with a shelter and

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half were not (following a random procedure), water was provided in each

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compartment. The heat source was available during the diurnal phase in the 10 hot

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compartments, the 20 other compartments remained cold all the time (16°C). We

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placed one snake per compartment during 16 consecutive days and the

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compartments were inspected three times per day (9h00, 13h00, 17h00). The feces

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were rapidly collected, weighed and dried (see below).


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We first tested 88 recently fed snakes (32 vipers of the first experiment plus 56

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vipers, Table 1), immediately after the prey was swallowed. The snakes were

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weighed twice: before feeding and 16 days later.

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Second, we analyzed the impact of repeated simulated predator attacks on

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recently-fed snakes. We randomly selected and tested 56 vipers (among the 88

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available). We followed the same procedure as above but every day we touched each

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snake with a leather glove (10 times, total duration 15 seconds, the glove was

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attached to a 50cm rod, standard movements, same observer), and this was repeated

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three times per day (9h00, 13h00, 17h00). The snakes subjected to the simulated

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attack displayed typical defensive behaviors (e.g. hissing, striking). As above, the

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snakes were monitored during 16 days.

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Third, we assayed plasma corticoid levels in unfed vipers (N=63 vipers large

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enough for blood sampling) that experienced the same treatment from above, but

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without predatory attack simulations. These vipers were then not involved into any

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experiment for at least two weeks before being tested. They were not fed and not

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attacked to limit potential confounding effects of digestion and predatory threat of

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basal plasma corticosterone level. Indeed, our objective was to assess the impact of

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shelter and heat source availability on endocrine stress levels. All individuals were

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blood sampled 10 days after the beginning of the test. This duration was selected to

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limit the effect of initial handling stress (i.e. placing snake into the box) and to offer

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to the snakes enough time to “adapt” to experimental condition.


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Digestive performance

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Experiment 2 provided sufficient amount of time (16 days) to monitor digestion: the

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production of feces requires one week on average (Michel and Bonnet 2010). We

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considered the date for the first feces as an index of transit time; we also counted the

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other feces and noted their date of production.

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We used a crude estimate of assimilation rate: the proportion of ingested prey in

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relation to snake body mass variation. For example, a 20g mouse inducing a 4g

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increase in snake mass after digestion provides a value of 20% (see Michel and

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Bonnet 2010 for details). We emphasize that such calculation does not provide

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accurate values of digestion efficiency (e.g. other factors such as metabolism, energy

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content of feces, or specific body composition are involved), but is a useful index for

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inter-individual or inter-group comparisons.

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Several individuals regurgitated their prey. Snakes tend to regurgitate when

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threatened or exposed to unfavorable ambient temperatures (Greene 1988; Naulleau

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1983). The date of regurgitation was noted, the vomited items were examined,

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weighed (fresh and dry mass). In case of regurgitation, to estimate the proportion of

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the prey digested (not assimilated) by the snake we divided the dry mass of the

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regurgitated prey by the estimated dry mass of the corresponding ingested mouse.

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This index reflects the proportion of the prey wasted versus digested (we note that

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actual calculation of digestive efficiency requires passage through the gut). The dry

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masses of vomited prey were obtained after two weeks in a 60°C autoclave. The 11


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estimated dry mass of the ingested prey was derived from the linear correlation

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between fresh and dry masses measured in a range of euthanized mice (body mass

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ranged from 2.8g to 55.0g; N=7; r²=0.99, P