Stress in wild and captive snakes : quantification , effects and the importance of management Stress bij in het wild en in gevangenschap levende slangen : kwantificatie , gevolgen en het belang van management

All vertebrates respond to unpredictable and stressful conditions with the so-called ‘stress response’ (Wingfield, 2005). Stressors can be defined as stimuli that put the individual in a state of uncertainty, lack of information and/or lack of control (Brown et al., 1991). Snakes faced with such stressful stimuli launch a stress response, not only through secretion of catecholamines, such as adrenaline and noradrenaline into the blood, but also through an endocrine stress response with activation of the hypothalamic-pituiBSTRACT


INTRODUCTION
All vertebrates respond to unpredictable and stressful conditions with the so-called 'stress response' (Wingfield, 2005).Stressors can be defined as stimuli that put the individual in a state of uncertainty, lack of information and/or lack of control (Brown et al., 1991).Snakes faced with such stressful stimuli launch a stress response, not only through secretion of catecholamines, such as adrenaline and noradrenaline into the blood, but also through an endocrine stress response with activation of the hypothalamic-pitui-

BSTRACT
As in other animals, distress and impaired welfare have a deleterious effect on the mental, physical and behavioral health of snakes in the wild and in captivity.Besides anthropogenic disturbance, the availability of food and shelter, the presence of predators, and environmental factors, such as seasonality and climatological changes, are important factors that affect the stress level and subsequent welfare in wild snake populations.In captive snakes, inappropriate management is the most prominent cause of chronic stress and impaired welfare.Chronic stress can be assumed by looking at the snake's behavior, but there is need for a standardized quantification method to pin-point more accurately (chronic) stress levels.The biomarker suitable in this framework is the level of corticosterone in plasma, feces and shed skin.
Determination and quantification of stress are pivotal because of its relation to the general performance and health of the individual.Behavioral changes indicate disturbance, injury or disease, but may also lead to physical lesions and infections if prolonged.Higher corticosterone levels caused by chronic stress lead to immune suppression, making the individual more susceptible to viral, bacterial and parasitic infections (Dantzer et al., 2014;Neuman-Lee et al., 2015;Silvestre, 2014).However, the perception of stressors is highly individual; whereas one individual does not experience stress from a specific stimulus, the welfare of another individual could be strongly impaired.This individual perception is caused by intrinsic factors, such as sex, age, reproductive condition and former experiences (Dantzer et al., 2014) (Figure 2).
A plethora of stimuli may influence the welfare of snakes.The most common stressors for snakes in the wild are food deprivation, habitat loss due to anthropogenic activity and changes in climate (Ajtić et al., 2013;Gregory, 2016;Robert and Bronikowski, 2009).Other potential stressors are the presence of predators and non-indigenous species in the same habitat (Burger, 1998;Gregory, 2016;Pasmans et al., 2017;Šukalo et al., 2014).Snakes in captivity are mainly stressed by management related aspects, such as temperature, humidity, enclosure size and crowding (Silvestre, 2014).
The quantification of chronic stress in a wild snake population is pivotal for their managment and habitat, while for snakes in captivity, it is important regarding welfare.However, this is challenging due to the individual nature of the chronic stress response, but also because of the lack of a uniformly used and standardized quantification method and matrix in the pertinent literature.In the literature, various matrices such as blood, feces and shed skin have been described to measure corticosterone as a biomarker for chronic stress, but comparing results of these studies using different methods and matrices is challenging (Dantzer et al., 2014;Sheriff et al., 2011).In the last section, the commonly used matrices to quantify corticosterone, i.e. blood, feces and shed skin are discussed.

STRESS IN WILD SNAKES
Over the last decade, the welfare of captive as well as wild animals has become increasingly important.In this framework, Bonnet et al. (2016) studied the rapid pace of urbanization and its negative impact on biodiversity, the functioning of ecosystems and the subsequent impairment of animal welfare in snakes.Assessment of stress in wild snake populations is challenging and has been underexamined.However, having a good understanding of stressful stimuli and their effects on welfare impairment is needed in managing snake populations.
The direct effect of food resources on the stress level has been little studied; however, it is known that they influence the size of a population.In a study by Sewell et al. (2015), the population size of grass snakes (Natrix natrix) positively correlated with common frog (Rana temporaria) spawn clumps and peak counts of pool frogs (Pelophylax lessonae) on a site in eastern England restored for the reintroduction of these amphibians.This was explained by the migration of snakes when food resources in this site increased.Management of forests and heathland which damages prey species populations, has been shown to have a negative impact on the preservation of wild snake populations (Reading and Jofré, 2013).
Secondly, loss of habitat caused by urbanization also affects wild snake populations.Dantzer et al. (2014) have shown that anthropogenic disturbances are frequently correlated with increased levels of plasma baseline glucocorticoids.Bonnet et al. (2016) has shown that shrub habitats, being important shelters for several snake species, have been dramatically altered for practical and esthetical reasons.This shift results in a decrease of sufficient habitat for snake species, which in turn has a negative effect on their population size and biodiversity.In addition, the presence of snakes sometimes triggers rather negative perceptions by the public, making local authorities remove snakes from parks and green areas (Ballouard et al., 2013).For example, changes in French legislation in 2007 allowed the clearing of the aspic viper (Vipera aspis), a species previously marked as 'protected' (Bonnet et al., 2016).
Lastly, climatological changes are believed to have an impact on the overall performance of wild snake populations.Snakes, like all reptiles, are ectothermic, relying on the environment to maintain their body temperature.Snakes living in the temperate-zone are well-adapted to harsh wintering conditions as they hibernate.During this dormant period, their body temperature is generally lower than during active periods in contrast to plasma corticosterone, which is generally higher during hibernation (Dupoué et al., 2013;Nordberg and Cobb, 2016).Subsequently, it has been assumed that mild winter conditions trigger a state of chronic stress and a decrease of body condition when emerging.However, in a study by Brischoux et al. (2016), lower levels of plasma corticosterone were demonstrated when hibernation temperatures were higher, even though there was more body mass loss in snakes maintained in mild wintering conditions.Increased body mass loss may be caused by higher metabolism resulting from significant energy use during mild wintering conditions (Brischoux et al., 2016).Furthermore, it is possible that the experimental design of the study and the matrix, in which corticosterone was measured, affected the quantification of corticosterone.Therefore, further studies must be conducted to assess the effect of global warming on chronic stress levels.

STRESS IN CAPTIVE SNAKES
Inappropriate management conditions are the main reason of stress and impaired welfare in snakes in captivity (Silvestre, 2014).Factors like temperature, humidity, enclosure size and crowding all influence snake welfare, hereby restricting the physiologic needs of the animals.Persistent inadequate conditions lead to chronic stress and, with time, to dwindling (DeNardo, 2006).Raising awareness of the effect of bad management on mental, physical and behavioral health is the key to improve snake welfare in captivity (Silvestre, 2014).In some countries, guidelines for husbandry standards for reptiles are available providing a good framework of management factors for snakes kept in captivity (Table 1).
In a study by Sparkman et al. (2014) captivity of wild gravid female snakes (Tamnophis Elegans) resulted in increased plasma corticosterone levels as well as heterophile to lymphocyte (H:L) ratios over time.Maladaptation syndrome is a phenomenon occurring in reptiles that are acquired from the wild but do not adapt to captivity, subsequently leading to their death (DeNardo, 2006).

Assessment of chronic stress
In the pertinent literature, chronic stress is measured in various ways (Tables 2 and 3).In some studies, the snake's behavior is observed, especially when studying snakes in captivity (Silvestre, 2014).If the snake is unable to flee from its stressor, which is mostly the case in captivity, the animal will be unable to respond in an appropriate manner and will therefore express abnormal, non-functional behavior (DeNardo, 2006;Silvestre, 2014).Behavioral change in captive reptiles may indicate disturbance, injury or disease (Warwick et al., 2013).These changes can be used to evaluate their condition and welfare, but must be considered carefully in their context.A normally active reptile may become lethargic due to stress, whereas an individual with a calm nature may become very active.Also physical lesions and topical infections, e.g.rostral lesions incurred by rubbing their heads against transparent boundaries, are regarded as indicators for impaired welfare.However, in a study by Claunch et al. (2017), corticosterone administered to the Southern Pacific rattlesnake (Crotalus helleri) by an implant with 3.6 mg (snakes less than 800 g) or 6.1 mg (snakes of more than 800 g) did not mediate their behavioral trait expression.Therefore, observing snake's behavior might not be the best way to ascertain chronic stress.
Levels of circulating cortisol and corticosterone are widely accepted as biomarkers for stress in vertebrates.Both are pleiotropic having besides their role as a stress hormone, various functions in non-stress related physiological processes (Dantzer et al., 2014;Silvestre, 2014;Sparkman et al., 2014).In addition, individual differences in HPA-axis reactivity related to intrinsic factors, such as sex and age, make the interpretation of corticosterone levels challenging (Sparkman et al., 2014).Corticosterone can be determined by means of radioimmunoassay (Palacios et al., 2012;Sparkman et al., 2014), enzyme immunoassay in blood or skin (Berkvens et al. 2013), or by measuring fecal glucocorticoid metabolite (FGM) using the referred methods (Berkvens et al., 2013).
Plasma corticosterone is used to quantify acute stress in snakes.The samples can be obtained quickly, and only a small volume (ca.25-50 μL) is required to quantify glucocorticoids (Tarlow and Blumstein, 2007) (Figure 3).The response time of plasma corticosterone to acute stress caused by capture and handling is fast; hence, biasing may occur when samples are not taken in time (Sparkman et al., 2014).Palacios et al. (2012) showed that a difference of approxima-tely 300 ng/mL corticosterone can occur between blood sampling of garter snakes (Thamnophis elegans) at capture time and 50 minutes later.Nonetheless, an elevation in baseline blood corticosterone can be used to determine chronic stress (Dantzer et al., 2014;Palacios et al., 2012).An elevated baseline of blood corticosterone may be caused by acute stressful stimuli and/or the reduced ability to terminate the stress response.This principle is based on the assumption that chronically stressed individuals will initially respond greater to acute stimuli such as the blood sampling itself (Dantzer et al., 2014), which is an invasive method (Tarlow and Blumstein, 2007).In addition, plasma levels of corticosterone are influenced by circadian as well as other rhythmicities making plasma unsuitable for chronic stress quantification (Silvestre, 2014).
Measuring FGM levels is a way to assess HPAaxis activity over a specific time lapse, and has gained interest for the quantification of stress in multiple species.In snakes, specifically, fecal corticosterone metabolite (FCM) is measured (Berkvens et al., 2013).It is assumed to be a cumulative exposure of corticosterone reflecting the average of the blood corticosterone over a specific duration (Dantzer et al., 2014;Sheriff et al., 2011).Measuring FGM levels is non-invasive, and therefore not altering results due to sampling (Dantzer et al., 2014;Palme et al., 2005).However, this matrix has its limitations.In mammals and birds,   causing lesions and differences dietary differences infections dietary differences and metabolic rate have been shown to have an influence on FGM levels (Dantzer et al., 2011;Goymann et al., 2006).In a study of Dantzer et al. (2011), higher dietary fiber consumption seemed to increase fecal cortisol metabolite (FCM) concentration in North American red squirrels (Tamiasciurus hudsonicus).Moreover, microbial interconversion, degradation after defecation or wash-out by rainfall may biase results (Tarlow and Blumstein, 2007;Washburn and Millspaugh, 2002).
Although little studied, corticosterone in shed skin originated by ecdysis could be the future matrix for chronic stress quantification.Ecdysis is the process by which an old outer keratinized epithelial layer is shed, revealing new keratinized epidermal layers underneath (Maderson, 1965).Blood corticosterone is presumed to incorporate in the skin, but this level is possibly biased by extra-adrenal production as is seen in human skin (Taves et al., 2011).Berkvens et al. (2013) showed a positive correlation between fecal and shed skin corticosterone in African house snakes (Lamprophis fuliginosus) and Eastern Massasauga rattlesnakes (Sistrurus catenatus catenatus), indicating that the status of the HPA-axis over a specific time frame influences fecal and skin corticosterone in a similar manner.Just as in feces, chronic stress results in a higher cumulative exposure of corticosterone in skin; and as corticosterone in shed skin represents the status of the HPA-axis over a time lapse, it could be used to determine chronic stress (Dantzer et al., 2014;Sheriff et al., 2011).However, up till now, positively correlated levels of corticosterone in shed skin with stressful environmental stimuli have not been reported yet (Berkvens et al., 2013;Sheriff et al., 2011).

CONCLUSION
Frequently, wild as well as captive snakes are confronted with chronic stress, affecting their overall performance and health.The quantification of stress in snakes, particularly of chronic stress, is challenging and quantification of the stress hormone corticosterone in different matrices, such as plasma, feces and skin, has its advantages and disadvantages.Regardless of the matrix used, the interpretation of results is difficult as the perception of stressors differs between individuals and is influenced by intrinsic factors, such as age and sex.Therefore, further research is needed to provide a scientifically validated quantification method for chronic stress in snakes.

Figure 1 .
Figure 1.HPA-axis and its effect on systemic processes and the negative feedback response under acute stress compared with response under chronic stress (Sheriff et al., 2011).

Figure 3 .
Figure 3. Blood collection from the heart of a snake.Figure 2. Influence of the intrinsic factors of the individual on its stress response (Dantzer et al., 2014).