J Comp Physiol B DOI 10.1007/s00360-015-0901-0

REVIEW

Stress physiology in marine mammals: how well do they fit the terrestrial model? Shannon Atkinson1 · Daniel Crocker2 · Dorian Houser3 · Kendall Mashburn1 

Received: 19 February 2014 / Revised: 23 March 2015 / Accepted: 9 April 2015 © Springer-Verlag Berlin Heidelberg 2015

Abstract  Stressors are commonly accepted as the causal factors, either internal or external, that evoke physiological responses to mediate the impact of the stressor. The majority of research on the physiological stress response, and costs incurred to an animal, has focused on terrestrial species. This review presents current knowledge on the physiology of the stress response in a lesser studied group of mammals, the marine mammals. Marine mammals are an artificial or pseudo grouping from a taxonomical perspective, as this group represents several distinct and diverse orders of mammals. However, they all are fully or semiaquatic animals and have experienced selective pressures that have shaped their physiology in a manner that differs from terrestrial relatives. What these differences are and how they relate to the stress response is an efflorescent topic of study. The identification of the many facets of the stress response is critical to marine mammal management and conservation efforts. Anthropogenic stressors in marine ecosystems, including ocean noise, pollution, and fisheries interactions, are increasing and the dramatic responses of some marine mammals to these stressors have elevated concerns over the impact of human-related activities on a diverse group of animals that are difficult to monitor. This Communicated by G. Heldmaier. * Shannon Atkinson [email protected] 1

School of Fisheries and Ocean Sciences, Juneau Center, University of Alaska Fairbanks, 17101 Pt. Lena Loop Road, Juneau, AK 99801, USA

2

Department of Biology, Sonoma State University, 1801 East Cotati Avenue, Rohnert Park, CA 94928, USA

3

National Marine Mammal Foundation, 2240 Shelter Island Drive, Suite 200, San Diego, CA 92106, USA





review covers the physiology of the stress response in marine mammals and places it in context of what is known from research on terrestrial mammals, particularly with respect to mediator activity that diverges from generalized terrestrial models. Challenges in conducting research on stress physiology in marine mammals are discussed and ways to overcome these challenges in the future are suggested. Keywords  Stress response · Stress physiology · Stressor · Hypothalamo-pituitary adrenal axis (HPA axis) · Cortisol · Corticosterone

Introduction The study of stress and the stress response has roots in the concept of homeostasis and the development of the general adaptation syndrome (Cannon 1932; Selye 1936). The definition of stress and its conceptual application to the biological sciences has evolved since the foundational work of Cannon and Selye; it has undertaken a diversity of meanings, yet has lacked a commonly accepted definition within the scientific community (see Levine and Ursine 1991; Koolhaas et al. 2011, for extensive reviews). Current debates on the definition of stress and the stress response (e.g., in the context of allostasis and/or the reactive scope model) will certainly continue to refine the meaning and frame of reference in relation to homeostasis and adaptive processes (Romero et al. 2009; McEwen and Wingfield 2010). However, from the perspective of many biologists, it is the variation in behavior and the physiological mediators of a response to a stressor that are of interest, particularly in regards to the proximate and ultimate consequences of the response (Kirk 2014).

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Stressors are commonly accepted as the causal factors or stimuli, occurring in either the animal’s internal or external environment, that evoke a response in a physiological mediator (e.g., glucocorticoids, cytokines, or thyroid hormones). Some stressors may be predictable and associated with daily, seasonal, or life cycles (St. Aubin and Dierauf 2001; Atkinson et al. 2009). These stressors are often coupled with behaviors specific to a life history state, and the response benefits the organism by permitting its systems of physiological regulation to operate at altered and adaptive levels. The responses are beneficial in that they allow the organism to achieve some important biological objective, (e.g., lactation, hibernation, mating). Other factors may be unpredictable, requiring additional action or interaction of physiological processes above those already engaged for predictable events. The type, magnitude and duration of a stress response have a cost to the animal, the accumulation of which has been termed the allostatic load (McEwen and Wingfield 2007). When stressors are chronic or severe, the accumulated costs associated with the response(s) become an allostatic overload, which can contribute to physiological dysfunction and increase the probability of disease and other pathologies (Turnbull and Cowan 1998). A large percentage of mammal research on the stress response and the costs incurred to an animal has focused on terrestrial species, including humans (see reviews by McEwen and Wingfield 2003; Korte et al. 2009; Everly and Lating 2013). The focus of published literature is the various systems that respond to stressors (e.g., neuroendocrine system) and the type and action of specific mediators (e.g., cortisol). This review focuses on a lesser studied group of mammals, the marine mammals. Although many aspects of the stress response are assumed to be conserved across taxa, marine mammals have evolved from terrestrial ancestors to have either a fully or semi-aquatic existence (Uhen 2007). The selective pressures experienced by marine mammals differ significantly from that of their terrestrial relatives at the most fundamental levels (Uhen 2007). For example, marine mammals must cope with a separation between prey and oxygen sources, deal with differences in available sensory information (e.g., light is more rapidly attenuated than sound in seawater; Urick 1983; Dushaw et al. 1993), and overcome the substantially greater heat conductivity of seawater. They mostly consume diets that are nearly devoid of carbohydrates but rich in lipid and protein, yet many marine mammal species undergo periods of fasting from both food and water while maintaining energetically costly activities, thus invoking a predictable nutritional stress. As an example, this combination of physiological demands is exemplified by lactational fasting in northern elephant seals (Mirounga angustirostris; Houser et al. 2007). Lactating females produce copious amounts of energy rich milk (>4 kg/day) and meet their own metabolic demands from

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the same endogenous lipid stores with which they begin the lactational fast. Cortisol increases with the progression of the fast and presumably contributes to the efficient management of carbohydrate metabolism, potentially by contributing to insulin resistance (see Houser et al. 2013 for review). In addition, due to salinity of seawater, the water most marine mammals live in is typically hyperosmotic relative to their internal milieu (Lewis 2009). Each of these factors, as well as others unmentioned, require morphological and adaptive modifications to specific physiological systems and potentially in the kinetics and mode of action of biochemical mediators. How these morphological and physiological differences impact the stress response is largely unknown in marine mammals, yet identifying mediators of the stress response and understanding their behavioral and physiological consequences is important to marine mammal management and conservation efforts. These issues have come to the forefront of conversations regarding the combined impact of oil spills, environmental contaminants, deleterious fisheries interactions, and ocean noise on the ability of marine mammal populations to maintain growth or sustainability (Fair and Becker 2000; National Academy of Sciences (2005). Marine mammals are subject to numerous natural and anthropogenic stressors (Table 1). Multiple studies have been conducted on natural stressors such as oscillatory events (e.g., El Niño), storms, climate change, predation and disease, and others (Table 1). Additional research is taking place on anthropogenic stressors, such as ocean noise, pollution, and fisheries interactions (Table 1). Over the last century, the growth in ocean noise has been substantial, primarily due to increased shipping, and has been demonstrated to alter the communication behavior of marine mammals (Hildebrand 2005, 2009; Tyack 2008; Weilgart 2007; Holt et al. 2008; Parks et al. 2011). Dramatic behavioral responses of some marine mammals to underwater sound have elevated concerns over the cumulative impact of anthropogenic activities on a group of animals that is difficult to monitor (Jepson et al. 2003; Fernández et al. 2005). Indeed, the NAS requested that efforts be undertaken to model the population consequences of underwater noise to marine mammals (National Academy of Sciences (NAS) 2005), and has since broadened the scope to include all forms of anthropogenic stressors. Fundamental to addressing this effort is determining how marine mammals respond to particular types of environmental stressors and then linking these responses to costs at the individual level (Wingfield 2013). In acknowledgement of this need, the Office of Naval Research (ONR) convened a workshop to discuss the topic of stress in marine mammals and develop research priority recommendations (ONR 2009). The purpose of this review is to present work that has been performed on marine mammals related to a myriad of

Steller sea lions, manatee Multiple ice-dependant species

Harbor seal, gray seal Steller sea lions, California sea lions, harbor seals

Multiple species

Multiple species Multiple species Multiple pinnipeds

 Storms

 Climate change

 Interspecific competition

 Predation

 Disease

 Harmful algal blooms  Breeding competition

 Aggressive male breeding behavior (mobbing)

Multiple species Coastal cetaceans

Fin whales, humpback whales, north/south Atlantic right whale, manatees, Australian fur seals, northern elephant seal

 Fisheries interactions (e.g., entanglement)  Wildlife viewing tours

 Increased anthropogenic noise

Beluga Long-fin pilot whales, killer whales, sperm whales, beaked whales, humpback whales

Multiple pinnipeds

Natural stressor  El Nino

Anthropogenic stressors  Air gun  Sonar/remote sensing

Species

Documented stressor

Table 1  Documented natural and anthropogenic stressors of marine mammals References

Endocrine response Gas bubble lesions, fat emboli, altered dive behaviors, changes in song/cessation of song Mortality Population decline, reduced population fitness, reduced reproductive success, avoidance behaviors Communication masking, shifts in call frequency, abandonment of feeding/breeding ground, significant disturbance, altered vocalization, avoidance, subtle changes in dive behavior

Injury, mortality, pup mortality

Costa et al. (2003), Cox et al. (2006) Parks et al. (2011), Miksis-Olds et al. 2007, Wright et al. (2007), Clark et al. (2009), Tripovich et al. (2012)

Geraci et al. (1999), Read et al. (2006) Bejder et al. (2006), Lusseau and Bejder (2007), Tseng et al. (2011)

Romano et al. (2004) Jepson et al. (2003), Fernández et al. (2005), Sivle et al. (2012), Risch et al. (2012)

Riedman and Le Boeuf (1982), Campagna et al. (1988), Le Boeuf and Mesnic (1990), Atkinson et al. (1994), Kiyota and Okamura (2005)

Robinson and Del Pino (1985), Limberger (1990), Le Boeuf and Reiter (1991), Trillmich et al. (1991), Crocker et al. (2006), Weise et al. (2006), Melin et al. (2008) Langtimm and Beck (2003), Maniscalco et al. (2008) Shane (1995), Harvell et al. (2002), Monnett and Altered foraging, loss of prey species, increased disease, ice entrapment, mortality Gleason (2006), Laidre et al. (2008, 2012) associated with increased swim distances, atypical movement to nearshore waters Altered population demographics, altered Bowen et al. (2003), Lidgard et al. (2008) hormones, reproductive success Population decrease, increased corticosterone Baird and Stacey (1989), Nordstorm (2002), Matkin et al. (2002), Mashburn and Atkinson following pup predation, avoidance behav(2007) iors, haul out selection based on predation risks Slade (1992); Kretzmann et al. (2001), KennedyLimited genetic diversity (MHC polymorphism), mortality, possible mass strandings, Stoskopf (2001), Dunn et al. (2001), Reiderson et al. (2001), Dailey (2001), Barrett et al. (2003) increased susceptibility to novel disease Mortality, neurotoxicity Van Dolah et al. (2003), Hallegraeff (1993) Injury, mortality Reiter et al. (1981), Riedman (1990) Mortality, emigration, abnormal breeding behaviors, low birth rates, changes in foraging behaviors, altered diving, movement behaviors Pup loss, reduced survival

Effect or marker

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Cortisol, testosterone Multiple species

internal or external stressors, and the subsequent behavioral and physiological stress responses. The review examines stress-related research within the context of what is known from investigations on various terrestrial mammals, and highlights mediator activity in marine mammals that differs from that of their land-based counterparts. This will be accomplished by initially presenting those premises from terrestrial animals and humans that can be accepted and built on, followed by the physiology of stress responses that have been measured in marine mammals, and including unique behaviors that outwardly reflect stress physiology. The expected outcome of this effort is the promotion of a scientific consensus on the foundational research required to better understand stress physiology and related behaviors of marine mammals. This foundation and the concluding recommendations should be considered by resource managers and scientists to better address marine mammal conservation issues.

Stress response in terrestrial mammals Neuroendocrine system

Each stressor listed has an effect or marker and references

 Captivity restraint and handling

Mellish et al. (2006), Lidgard et al. (2008), Fair et al. (2014)

Energy balance, metabolic rate, hormones, birth rate Steller sea lions and killer whales

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 Induced nutritional stress

Helle et al. (1976), Subramanian and Bray (1987), Ross et al. (1993), Kannan et al. (2000), Fossi et al. (2003), Wang et al. (2005, 2007, 2010) du Dot et al. (2009), Calkins et al. (2013), Atkinson et al. (2008), Rosen and Kumagai (2008), Gerlinsky et al. (2014) Endocrine disruption, vitamin imbalances, subclinical effects, reproductive impacts, mass mortalities Multiple pinnipeds and cetaceans  Contaminants and pollution

References Effect or marker Species

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Documented stressor

Table 1  continued



The mammalian neuroendocrine response to a stressor is well documented (Sapolsky et al. 1986; Schulkin 1999; Sapolsky et al. 2000). In response to stimulation at the level of the sensory systems in the brain, the sympathetic nervous system (SNS) stimulates the adrenal medulla to release the catecholamines, epinephrine and norepinephrine. This is an immediate response and may occur in milliseconds. Co-activated with the SNS is the hypothalamopituitary-adrenal (HPA) axis. Activation of the HPA axis begins at the level of the hypothalamus, which releases corticotrophin releasing factor (CRF) into the hypophysial portal veins. The target of CRF is the anterior pituitary, which subsequently releases adrenocorticotropic hormone (ACTH). ACTH then stimulates secretion of the glucocorticoids (GCs), mainly cortisol and corticosterone, from the zona fasciculata of the adrenal glands. While this is a secondary response it is initiated quickly (within seconds) and occurs rapidly (within minutes). Corticosteroids The primary action of the GCs is to alter behavior, increase blood glucose concentrations, inhibit growth and reproduction, and inhibit the immune system (Romero 2004). GCs provide negative feedback at the hypothalamic and pituitary levels to suppress further production and release of the GCs as well as gonadotrophins and growth hormone. The predominant GC in most mammals is cortisol, and in some systems corticosterone, although one or the other is

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typically dominant (Koren et al. 2012). Cortisol stimulates gluconeogenesis by increasing the enzymes responsible for converting amino acids to glucose in the liver, affecting the metabolism of fats and proteins, and mediating inflammatory processes. These processes are typically believed to have adaptive value and aid an organism in dealing with a stressor (McEwen 1999). However, chronic elevations of cortisol, when no recovery from the stressful stimulus occurs, have been shown to be potentially detrimental to an animal’s endocrine function and overall health (Sapolsky et al. 2000). The magnitude and duration of the cortisol responses across a broad range of domestic and wild terrestrial animals have been studied to determine their response to both acute and long-term natural and anthropogenic stressors (Mӧstl and Palme 2002; Wingfield 2013). The magnitude and the duration of the response have been observed to vary by species, age, gender, body condition, time of day, social environment, and experience. The broad variability in response underscores the need for targeted studies that not only address specific stressors, but also the variability that occurs normally within a species due to changes in season, life history or individual sensitivities (Delehanty and Boonstra 2012; McEwen 2012; Cockrem 2013). The other class of corticosteroids sometimes considered in the cascade of the HPA-axis is the mineralocorticoids, which function in the maintenance of electrolyte balance (Aguilera and Rabadan-Diehl 2000). Aldosterone is the major mineralocorticoid and is primarily responsible for increasing sodium reabsorption from the renal tubules. However, prolonged or excessive exposure to aldosterone can lead to increases in arterial pressure and extracellular fluid volume, hypokalemia, and fatigue (Guyton and Hall 2000). In humans, an overabundance of aldosterone has been linked to cardiovascular damage and increased oxidative stress (Queisser and Schupp 2012). It is important to note that, although aldosterone shares portions of a synthetic path with cortisol, it is primarily regulated by the action of angiotensin II. This topic is covered below in the discussion of the rennin–angiotensin–aldosterone system (RAAS). Binding proteins and receptors The physiological response to circulating hormones is ultimately determined by their interactions with tissue receptors (McEwen 2000; Sapolsky et al. 2000). In general, mammalian GCs exert influence utilizing both glucocorticoid and mineralocorticoid receptors that demonstrate different affinities for circulating GCs with the former exhibiting a tenfold greater affinity than the latter. The net result of the involvement of both receptors types is that GCs initiate a variety of permissive, stimulatory, and suppressive

effects that depend on circulating concentrations (de Kloet et al. 1993; Sapolsky et al. 2000). Most circulating cortisol is bound to carrier proteins— primarily corticosteroid-binding globulin (CBG). Only free (unbound) cortisol is thought to be biologically active and capable of interacting with target tissue receptors. Under chronic stimulation by a stressor, high concentrations of free corticosteroids can be detrimental to key body functions (Desantis et al. 2013). Thus, the metabolic influence of cortisol on target tissues is mediated by carrier protein expression, primarily that of CBG (Fleshner et al. 1995; Dhillo et al. 2002). The exact role of CBG is unknown at this time—it may enhance transport and delivery of corticosteroids to target tissues or, conversely, act as a buffer in regulating alterations in circulating free corticosteroids (Romero 2002; Delehanty and Boonstra 2012). Nevertheless, assessment of the circulating CBG concentrations and its variability over time permits far greater understanding of circulating corticosteroid levels and the magnitude of effect on target tissues (Dhillo et al. 2002). CBG may, in fact, be an accurate marker of long-term stress as it does not seem to vary with acute stress, like capture shock, in some species (Chow et al. 2010). Thyroid hormones Another potential response pathway to acute or chronic stress in mammals may involve alteration of the hypothalamo-pituitary-thyroid (HPT) axis. Generally, stress tends to inhibit the HPT axis (Danforth and Burger 1984), particularly stressors involving food restriction (Eales 1985; van der Heyden et al. 1986). Thyroid hormone produced in the thyroid gland is primarily stimulated by thyroid stimulating hormone (TSH), which is biosynthesized by the pituitary in response to thyrotropin releasing hormone (TRH) being released from the hypothalamus (Eales 1988). Thyroid hormones (thyroxine, T4; triiodothyronine, T3) have a profound regulatory effect on many metabolic pathways, stimulating both catabolic and anabolic reactions, upregulating mitochondrial proliferation, and strongly influencing whole-animal metabolic rate. Most thyroid hormone is released from the thyroid gland as T4 (Cooper and Ladenson 2011). At target tissues, transmembrane deiodinases D1 and D2, convert T4 into the more biologically active T3. A third deiodinase, D3, inactivates T3 but may also convert T4 into an inactive form—reverse T3 (rT3). The rT3 binds to T3 receptors without upregulating gene expression, thus blocking most thyroid hormone action. Both T4 and T3 occur as free and bound forms and are typically measured in both forms (Demers and Spencer 2003; Gar-Elnabi et al. 2013). The transport protein thyroxine-binding globulin, (TBG) carries T4 to the surface of the target tissue and its conformation allows for the binding and release of the

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hormone in a modulated and targeted delivery (Zhou et al. 2006). D1, activity can be decreased by elevated cortisol and catecholamine levels, as well as by food limitation and illness (Weissman 1990). TSH is inhibited during the early phase of fasting, which is thought to be driven by acutely elevated free T4 (Spencer et al. 1983). The stress-induced decrease in D1 activity causes increased rT3, which likely leads to reductions in metabolic rate by blocking T3 receptors. The resulting decrease in energy use is an important mechanism animals may use to endure stressful periods, such as reduced foraging success, prolonged fasts, or hibernation (Wenberg and Holland 1973; Eales 1988; Thomasi et al. 1998; Nicol et al. 2000). Catecholamines The catecholamines, epinephrine and norepinephrine, are produced in the adrenal medullae in response to SNS stimulation (Cannon and Lissak 1939; von Euler 1946; Goldstein 1995). They have organ-specific effects that are similar to the direct sympathetic stimulation of the organs, but which operate over a longer time course. Epinephrine and norepinephrine are vasoconstrictive, with epinephrine providing greater cardiac stimulation and potent metabolic effects (e.g., increased metabolic rate, enhanced glycogenolysis, and increased release of glucose into the blood stream). Norepinephrine plays key roles in homeostasis of blood volume and of blood pressure. However, because of the short-term nature of the catecholamine response, it is difficult to integrate the consequences of its occurrence into an energy-based model that accommodates long-term impacts of repeated or sustained stressors (Romero et al. 2009). Nonetheless any tissue with adrenergic receptors can respond to the catecholamines and the response is generally immediate (Goldstein 2003). Renin–angiotensin–aldosterone system (RAAS) The renin–angiotensin–aldosterone system (RAAS) is a powerful controller of blood pressure and volume (Hall 1986; Levy and Tasker 2012). However, sympathetic activation of RAAS is also an important stress response in numerous mammals (Aguilera et al. 1995). Angiotensin II release in response to a stressor is a potent promoter of oxidative stress and is also a secretagogue, or stimulant, for ACTH release. Vasopressin, also a secretagogue for ACTH, is stimulated by angiotensin II via aldosterone and has been shown to have an active role in stress response behaviors (Aguilera and Rabadan-Diehl 2000). For example, plasma renin activity and angiotensin II (or both) have been shown to increase in response to physical (cold exposure, immobilization/restraint) and psychological stressors (exposure to predators, novel environments, pain avoidance) in a

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variety of terrestrial mammals, including humans (see Carrasco and Van de Kar 2003; Groeschel and Braam 2011, for extensive reviews). In addition, increases in RAAS activity under conditions perceived as stressful occurred regardless of salt-loading (Clamage et al. 1976; Dimsdale et al. 1990; Aguilera et al. 1995), indicating a significant role for the RAAS in the stress response that is stimulated through perceived stressors, as well as in response to a physical stressor. Other endocrine responses Other stress responses have been measured in peptide hormones and demonstrated in mammals, such as the release of prolactin, and the suppression of both growth hormone (GH) and gonadotropic releasing hormone (GnRH; Sapolsky et al. 2000). Suppression of GnRH leads to reduced circulating concentrations of the sex steroids. In addition, the adrenal gland has been shown to affect the reproductive system of domestic animals independently of gonadotropins (Atkinson and Adams 1988; Adams et al. 1990). Species specificity of peptide hormones (Aumüller et al. 1990; Leob 2000) presents a methodological challenge, as most commercially available systems of measurement have been developed for humans and laboratory/domestic animals. The lack of viable and easily accessible means of protein hormone detection for non-domestic mammals hampers the ability to precisely characterize pituitary function or activity in other peptide producing tissues (Minton 1994; Ehrhardt et al. 2000).

Stress response in marine mammals Neuroendocrine system While it is assumed that the HPA axis functions in the same manner in both marine and terrestrial mammals, a few gaps in our knowledge stand out. Much of the neuroendocrine response is facilitated by peptide hormones, such as epinephrine and norepinephrine (Romano et al. 2004; Romero and Butler 2007), and these tend to be species specific in their constitution. (Dvorakova and Kummer 2005; Romero and Butler 2007). However, the amino acid structure of ACTH is strongly conserved across species, [e.g., the structure of ACTH from fin whales (Balaenoptera physalus) is identical to that of human ACTH, Kawauchi and Sasaki (1978)]. The use of commercial ACTH preparations has yielded responses in marine mammals that are consistent with there being some homology across ACTH from a variety of marine mammal species [e.g., beluga (Delphinapterus leucas), Steller sea lion (Eumetopias jubatus), harbor seal (Phoca vitulina), northern elephant seal,

J Comp Physiol B

and ringed seal (Phoca hispida)] and the terrestrial preparations (Thomson and Geraci 1986; Aubin DJ and Geraci 1990; Mashburn and Atkinson 2004, 2007; Ensminger et al. 2014). In general, the chemical structure of steroid hormones such as the sex steroids, glucocorticoids, and mineralocorticoids is more conserved than the peptide hormones across taxa (Lasley and Kirkpatrick 1991; LabradaMartagon et al. 2014). Thus, more research on steroid than peptide hormones has been conducted, due to the availability of commercially prepared testing reagents for steroid hormones. Indeed, ACTH stimulation tests have been performed to evaluate the adrenocortical response under conditions of hyponatremia in ringed seals (St.Aubin and Geraci 1986), to characterize differences in the adrenocortical response of wild and rehabilitating harbor seals (Gulland et al. 1999), to determine seasonal and gender variability in the adrenocortical response of Steller sea lions and harbor seals (Mashburn and Atkinson 2004, 2008; Keogh and Atkinson 2015), to characterize the response of the HPA-axis to acute stressors in the bottlenose dolphin (St. Aubin and Geraci 1990), and to evaluate changes in HPA axis sensitivity and metabolic impacts of cortisol during breeding in free-ranging adult male northern elephant seals (Ensminger et al. 2014). Overall, the neuroendocrine system in marine mammals appears to respond largely in a manner similar to that of terrestrial mammals. This is of particular importance when considering or preparing to undertake investigation of neuroendocrine function in marine mammals. As with all studies in captive animals, it needs to be recognized that individual differences in response to study environment, captive setting and research-related contacts may all have an impact on the response of an animal to a stressor. In addition, environment and capture/handling are two different and possibly mutually exclusive stressors that can have a cumulative effect and must be taken into account when reporting results or planning procedures. Capture stress in wild animals and handling stress in captive animals (e.g., associated with unconditioned medical procedures) are known to rapidly increase ACTH (Gulland et al. 1999; Desportes et al. 2007) This phenomenon has been observed in several marine species, including manatees (Trichechus manatus), belugas, and pan-tropical spotted dolphins (Stenella attenuata) (Schmitt et al. 2010; Tripp et al. 2011; St. Aubin et al. 2013), and is an important consideration given that only a handful of species are available to be trained for voluntary sampling. Likewise, the social setting of captured harbor seal pups has been shown to significantly buffer the stress response depending on whether or not the pups were captured and held with their mothers (Di Poi et al. 2014). In the following sections, the impact of handling and surrounding environment should be considered as it may influence the interpretation of results.

Corticosteroids GCs have been measured in a number of marine mammals and substantial work has been done defining circadian and seasonal patterns (St. Aubin et al. 1996; Gardiner and Hall 1997; Suzuki et al. 1998; St. Aubin and Dierauf 2001; Oki and Atkinson 2004), age and life history related changes (Engelhardt and Ferguson 1980; Raeside and Ronald 1981; Petrauskas and Atkinson 2006; Myers et al. 2010; Burgess et al. 2013), responses to nutritional stress and regulation of lipolysis in fasting (Chow et al. 2010), and the effects of handling, captivity, or rehabilitation on glucocorticoid production (Thomson and Geraci 1986; St.Aubin and Geraci 1989; Engelhard et al. 2002; Petrauskas et al. 2008; Harcourt et al. 2010; Pedemera-Romano et al. 2010; Bennett et al. 2012; Champagne et al. 2012; Spoon and Romano 2012; Trumble et al. 2012; St. Aubin et al. 2013). Thus a substantial number of studies have been conducted, albeit it, mainly on pinnipeds and cetaceans, resulting in general descriptions of baseline GC activity under different conditions. In response to a challenge or stimulation with ACTH, marine mammals appear to respond similarly to terrestrial mammals, in that there is a short-term increase in cortisol concentration from baseline concentrations within an hour of stimulation, which is then cleared from the animal’s system within 1–2 days (Thomson and Geraci 1986; Aubin DJ and Geraci 1990; Gulland et al. 1999; Mashburn and Atkinson 2004, 2007, 2008). Based on the limited number of ACTH stimulation tests done, differences in the magnitude of the response between cetaceans and pinnipeds may exist (Thomson and Geraci 1986; Aubin DJ and Geraci 1990). In cetaceans, maximum responses are ~110 nmol/L in bottlenose dolphins (Thomson and Geraci 1986), and >198 nmol/L in beluga whales (Schmitt et al. 2010) postACTH injection. In pinnipeds, the maximum response is much higher, ~645 nmol/L in harbor seals (Gulland et al. 1999) and ~900 nmol/L in Steller sea lions (Mashburn and Atkinson 2007) and ~825 nmol/L in elephant seals (Ensminger et al. 2014). Thus, although there are relatively few comparative studies between the groups, evidence gathered so far suggests that the level of GC expressed following an ACTH challenge are greater within the pinnipeds than in the limited odontocete cetaceans physiologically challenged to date. It must be noted, however, that ACTH studies have typically been performed with captive animals that are generally maintained in controlled, stable environments and may not be representative of wild populations. In addition, as is true in free-ranging animals, handling by humans or transport may be perceived as additional stressors (Desportes et al. 2007; Noda et al. 2007). Further, the role of binding globulins on the regulation of corticosteroid activity has largely been ignored in marine mammals,

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and CBG may be modulated in response to various stressors, e.g., to mitigate protein catabolism during periods of fasting (Chow et al. 2010). Metabolic responses to ACTHinduced elevation in cortisol concentrations varied widely among life-history stages in adult male northern elephant seals, suggesting different tissue responses to cortisol depending on physiological demands of the specific life history stage (Ensminger et al. 2014). Variation in CBG levels is one mechanism by which tissue responsiveness to cortisol might be regulated. A limited number of studies suggest that diurnal variations of GCs exist in marine mammals, similar to that observed in terrestrial mammals. For example, Gardiner and Hall (1997) reported that harbor seals exhibited a significant mean diel concentration difference of 49.8 nmol/L in cortisol, with the highest concentrations occurring at night, but this circadian rhythm was only found during the summer (Oki and Atkinson 2004). In belugas and killer whales (Orcinus orca), cortisol levels were lower between noon and midnight than during the rest of the day (Suzuki et al. 1998). Thus the circadian pattern appears to be species-specific, making it necessary to characterize the pattern for a given species before initiating new studies on that species. Aldosterone is known to regulate sodium levels (Ortiz 2001) and this likely remains a key function in marine mammals that either intentionally or incidentally consume sea water. It may also play several other important roles in marine mammals, including regulation of water retention during extended natural fasts (Ortiz et al. 2000, 2006). Aldosterone secretion is typically elevated coincident with cortisol increases in a variety of situations, including cold water exposure (Houser et al. 2011), restraint and handling (St. Aubin and Geraci 1989; St. Aubin et al. 1996; Ortiz and Worthy 2000; Schmitt et al. 2010; Champagne et al. 2012), and with an ACTH challenge (St.Aubin and Geraci 1986; Thomson and Geraci 1986; Gulland et al. 1999; Ensminger et al. 2014; Keogh and Atkinson 2015). Thus, aldosterone appears to serve a role in the stress response in marine mammals (St. Aubin and Dierauf 2001) and may be a very useful indicator of the stress response, particularly in regard to potential impacts on salt balance. Corticosteroids have been measured in a number of free-ranging marine mammals; however, interpreting the results presents a significant challenge. Capture of wild marine mammals for research purposes may require periods of chase and restraint (St. Aubin et al. 2013). Results of GC analysis in free-ranging marine mammals most often dependent on a single sample and under these conditions can be conflicting. For example, following the chase and capture of fin whales, no significant correlation between cortisol concentrations and chase duration was found (N = 15, p = 0.305; Kjeld 2001). Ortiz and Worthy (2000)

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reported that bottlenose dolphins chased for at least 36 min exhibited a mean cortisol concentration of 74.49 nmol/L, a concentration well within normal ranges for the species (St. Aubin 2001). In contrast, free-ranging spotted dolphins sampled following chasing events exhibited a mean cortisol concentration ~139 nmol/L, a concentration higher that that reported for a similarly sized cetacean at peak following an ACTH challenge (~110 nmol/L, Thomson and Geraci 1986). In harbor seals, animals infected with phocine distemper displayed a mean cortisol concentration of 1076 nmol/L, a value well above that reported by Gulland et al. (1999) following an ACTH challenge in the same species. Harbor seal pups that were captured with their mothers had significantly lower cortisol concentrations than dependent pups that were captured without their mothers (Di Poi et al. 2014). These studies suggest that there is the potential for confounding factors to affect GC concentrations in free-ranging marine mammals, despite best efforts to minimize capture and handling stress. Using a cautionary approach, care must be taken when relying solely on GCs as an indicator of a mounted stress response. Thyroid hormones The regulation of metabolism by thyroid hormones in marine mammals is consistent with the general role of this class of hormones. Baseline studies have characterized variations in circulating thyroid hormones by season in Steller sea lions (Myers et al. 2006), life history in harbor seals (Oki and Atkinson 2004), and geographic location in bottlenose dolphins (Fair et al. 2011). Thyroid hormones have been shown to influence field metabolic rate during fasting (Crocker et al. 2012; Kelso et al. 2012), diving metabolism (Weingartner et al. 2012), and fluctuate in response to changes in environmental temperature (Aubin et al. 2001; Fair et al. 2011). However, relatively little research has been done on TSH or TRH challenges in marine mammals to monitor the time course and magnitude of response of the HPT axis (Aubin 1987; Aubin and Geraci 1992; Yochem et al. 2008; Atkinson and Myers, unpublished data), likely due to uncertainty of the speciesspecific chemical structure of TSH or TRH. Likewise, little work has been done on the impact of handling stress on these hormones (Keogh et al. 2013), although a suppressive effect on thyroid hormones in belugas was identified and thought to be mediated by GCs (St. Aubin and Geraci 1988, 1992). Furthermore, at least within the odontocetes, a limited amount of information suggests that the suppressive effect of a chronic stressor on thyroid hormone concentration varies by species (Ridgway and Patton 1971). The impact of nutritional stress on thyroid hormone production has been measured in a number of marine mammal systems (Table 2). Reductions in T3 have been observed in

Mediators are upregulated in response Antioxidants (e.g., 8-isoprostanes or Vazquez-Medina et al. (2010, 2012) to oxidative stress nitrotyrosine) Northern elephant seals Antioxidant enzymes [e.g., superoxidase dismutase (SOD), catalase, glutathione peroxidase (GPx)]

Thyroid hormone

Renin–angiotensin–aldosterone system (RAAS)

Catecholamines

Multiple marine mammals (e.g., bot- Increased release and rate of decline Adaptation to high Na intake and heat Aubin and Geraci (1986), St Aubin tlenose dolphins) of aldosterone response conservation (2001) In dolphins maybe due to unihemi- Hance et al. (1982), Cabanac et al. Indo-Pacific dolphin, Weddell seals, No diurnal variation, but possible harbor seals, hooded seals seasonal variation; increased activity spheric sleep; may be an adaptation (1989), Hochachka et al. (1995), Hurford et al. (1996), Suzuki et al. to diving in epinephrine may vary inversely (2012) with heart rate during diving Likely evolved in response to hyper- Zenteno-Savin and Castellini (1998a, Northern elephant seals, bottlenose Complex control, reduced secretab), Ortiz et al. (2003), Vasqueztonic environments in response to dolphins, harbor seals gogue by vasopressin, RAAS may Medina et al. (2010), Houser et al. replace HPA axis as primary stimu- fasting (2011) lus for aldosterone Reduction in metabolically active T3; Reduced T3 in circulation in times of St Aubin et al. (1996a, b), St Aubin Spotted dolphin, northern elephant seals, bottlenose dolphin, beluga increased activity of rT3, nutritional stress and Dierauf (2001), Ortiz et al. (2003), St Aubin et al. (2013) Aldosterone

Effects

Table 2  Differences between marine and terrestrial species in their stress responses

Responses

References Species Mediator

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response to nutritional stress in killer whales (Ayres et al. 2012) and Steller sea lions (Rosen and Kumagai 2008). Reductions in T4 were noted during the early stages of fasting in subantarctic fur seals (Arctocephalus tropicalis) with subsequent modest increases during the prolonged stage II of fasting (Verrier et al. 2012). Differences in thyroid hormone concentrations of fasting phocid seals as a function of gender, body condition, and duration of fasting have been reported (Ortiz et al. 2001; Bennett et al. 2012; Kelso et al. 2012), but have also been observed to be stable in different age classes of the same species under similar periods of food deprivation (Crocker et al. 2012). Therefore, when evaluating thyroid hormone concentrations, it is important to take into account distinct age groups, sexes, and life history states such that differences in thyroid hormone activity can be accurately used in comparisons (Meyers et al. 2006). Investigations targeting the effect of environmental stressors on thyroid activity in both marine and terrestrial mammals include the impact of contaminants on thyroid hormone production and thyroid morphology (Isanhart et al. 2005; Sonne 2010; Kirkegaard et al. 2011), studies which collectively suggest the disruption of thyroid function from the accumulation of certain contaminants. Free triiodothyronine (T3) and the ratios of free-to-total T3 have been observed to be higher in ringed seals (Phoca hispida) in waters more polluted with persistent organic pollutants (Routti et al. 2010), whereas free and total T3 were lower in gray seals (Halichoerus grypus) from the same regions and with elevated levels of blubber organochlorines (Sormo et al. 2005). Chlorinated hydrocarbons were inversely related to total and free T3 (but not T4) in spotted (Phoca largha) and ribbon seals (Phoca fasciata) (Chiba et al. 2001) and PCBs have been correlated with lower free and total T4 and total T3 in harbor seals and bottlenose dolphins (Brouwer et al. 1989; Tabuchi et al. 2006; Schwacke et al. 2012). The effects of organohalogenated compounds on thyroid hormones concentrations may be via an endocrine disruption mechanism whereby the phenolic metabolites, such as hydroxylated PCB, attach to binding sites on the transthyretin retinol-binding protein complex in plasma, thereby disrupting the normal transport of hormones and vitamins to their target tissues (Brouwer et al. 1989; Rolland 2000). In polar bears, thyroid gland lesions were recorded in 40 % of bears exposed to organohalogens (Sonne et al. 2011), and in two multivariant studies, organohalogens were found to possibly disrupt thyroid homeostasis (Villanger et al. 2011; Bechshøft et al. 2012). Given that thyroid hormones are important regulators of metabolism and can vary in the face of environmental stressors, it is important to understand how the thyroid response can be disrupted by contaminants and how such a disruption affects the animal’s ability to effectively deal with current and future stressors.

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The thyroid response to other forms of acute and chronic stress in marine mammals is not well understood. The relationship between T3 and T4 and rT3 are of particular interest (Table 2). A few studies suggest that rT3 levels and rT3:T3 ratios are unusually high and unique to marine mammals (St. Aubin et al. 1996; Ortiz et al. 2003; St. Aubin et al. 2013), suggesting a novel mode of regulating T3 binding in these species. In belugas, capture and handling were associated with an acute rise in rT3 followed by a persistent reduction in T3 (St. Aubin and Geraci 1988) and variations in T4 suggested a change in the conversion rate of T4 to rT3 (St. Aubin and Dierauf 2001). Similarly, a 48-h elevation in cortisol after an ACTH stimulation test resulted in suppression of diiodothyronine (T2) and increased rT3 in adult male northern elephant seals (Ensminger et al. 2014). Whether this response is common to marine mammals as a whole remains to be determined, as does the purpose of the high levels of rT3 across all marine mammals surveyed to date. Catecholamines Catecholamines have been measured in pinnipeds and cetaceans under various experimental conditions, including diving physiology, response to noise, and handling (Hance et al. 1982; Thomas et al. 1990; Hochachka et al. 1995; Hurford et al. 1996; Romano et al. 2004; Champagne et al. 2012; Spoon and Romano 2012). However, little work has been conducted to understand normal baseline variations in marine mammals, although work with Indo-Pacific dolphins suggests seasonal variations in catecholamines with elevations in epinephrine, norepinephrine, and dopamine occurring in the winter (Suzuki et al. 2012). Diurnal variation in the catecholamines was not observed and the authors hypothesized that this may be due to the unihemispheric sleep of the dolphin, (i.e., the physiology of sleep in dolphins is not the same as in terrestrial mammals, which demonstrate a circadian rhythm in catecholamine levels: Table 2). Handling effects on catecholamine production have been observed in pinnipeds and cetaceans, similar to those seen in terrestrial mammals. Transport of belugas from one facility to another produced statistically significant increases in both epinephrine and norepinephrine that was coupled to an increase in cortisol (Spoon and Romano 2012). A study of handling techniques in elephant seals demonstrated a significant acute increase in epinephrine that accompanied manual restraint, and a lack of a response when chemical sedation was used for immobilization (Champagne et al. 2012). Similarly St. Aubin et al. (2013) used chasing and encirclement of pan-tropical spotted dolphins to demonstrate the elevation of dopamine and enzymes indicative of muscle damage. Catecholamines have also been

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monitored in some cetaceans to determine whether an acute stress response is produced when exposed to anthropogenic sound. In captive belugas, no increase in catecholamines and no behavioral reactions were observed in the presence of playbacks of oil drilling noise (Thomas et al. 1990). Conversely, a significant increase in epinephrine, norepinephrine, and dopamine was observed in a beluga exposed to high levels of impulsive noise from a seismic water gun (Romano et al. 2004); however, the shift in the catecholamine concentrations was relatively minor. In the same study, no change in catecholamine concentration was observed in a bottlenose dolphin exposed to either the same sound or to high amplitude tonal sounds. In both studies, blood collections were made 1 h (Romano et al. 2004) or 8–40 min (Thomas et al. 1990) following the exposure to the acoustic stimulus, which is potentially problematic with the interpretation of the data given the short half-life of the catecholamines (i.e., on the order of a few minutes). Thus, determination of the maximum level of stimulusinduced catecholamine production and the time course of the response warrant additional study. Despite the uncertainty of the role that catecholamines play in the acute stress response phase, catecholamines have been shown to be an integral part of the dive response. In pinnipeds, dramatic changes in circulating catecholamines are associated with splenic contraction and circulatory adjustments during diving (Hance et al. 1982; Cabanac et al. 1989; Hochachka et al. 1995; Hurford et al. 1996). Indeed, the progression of bradycardia with dive duration that occurs in parallel with increases in epinephrine/ norepinephrine suggests a complex regulatory interaction between vagally mediated responses and adrenergic stimulation of cardiac function (Hochachka et al. 1995). This interaction can potentially produce counterintuitive interpretations of an acute stress response (i.e., epinephrine may vary inversely with heart rate during diving, not directly as might be expected during the classic fight or flight response). As such, the activity of catecholamines during a dive requires additional investigation before the role in mediating responses to rapid-onset stressors can be determined. RAAS Marine mammals have evolved under conditions that require marked physiological adaptations for success in a life at sea, including osmotic homeostasis in a hypertonic milieu (Atkinson et al. 2009). Beginning with evolutionary adaptations in kidney structure, pinnipeds, cetaceans, and sea otters possess reniculate kidneys with hundreds of individual lobes, discrete cortical tissue, and increased medullary thickness to concentrate urine (Bester 1975; Ortiz 2001). The kidney structure of Sirenians varies

J Comp Physiol B

among species and may be related to the diversity of habitats they occupy ranging from freshwater (e.g., West Indian manatees, Trichechus manatus) to hypersaline (e.g., marine dugongs, Dugong dugon). Manatees possess superficially lobulated kidneys with a continuous cortex, lacking true reniculi (Maluf 1989). In addition, all marine mammals studied to date have the ability to concentrate their urine above the concentration of sea water (Costa 1982; Ortiz 2001). While sea otters are reported to commonly drink sea water (Costa 1982), and manatees freshwater (Ortiz et al. 1978, 1999), the majority of marine mammals ingest little water and rely on metabolic water and water intake incidental to prey capture for electrolyte balance (Ortiz et al. 1978; Castellini et al. 1987). Although most marine mammals inhabit hyperosmotic environments and many also practice periodic fasting, they have retained an ability to maintain electrolyte and water balance under conditions that could be considered analogous to terrestrial desert environments. An active RAAS has been identified in most groups of marine mammals, but with varying sensitivity related to salt availability (Ortiz et al. 2001). In terrestrial mammals, the RAAS is stimulated primarily by changes in blood pressure and in solute volume (Hall 1986). However, in marine mammals the RAAS seems to have a more complex system of control (Table 2. Zenteno-Savin and Castellini 1998a, b; Houser et al. 2001; Ortiz et al. 2003). In free-ranging dolphins, no correlation was found between adrenal steroids, vasopressin concentrations, and capture/restraint, implying a reduced secretagogue role for vasopressin in marine mammals, although the animals also did not exhibit a significant increase in glucocorticoids (Ortiz and Worthy 2000). In a study by Houser et al. (2011), cold stress did elicit an adrenal response, including increased aldosterone, which may support a RAAS role in the stress response. Marine mammals also activate RAAS in response to fasting (Vázquez-Medina et al. 2010), but its response to perceptual or physical stressors, or under extended fasts, are not well understood. Maintained elevations of aldosterone may primarily be due to the stressor-induced upregulation of the RAAS system rather than through the HPA axis, as has been suggested for maintenance of chronic peripheral vasoconstriction via angiotensin II production in cold-stressed bottlenose dolphins (Houser et al. 2011). Conversely, rapid stimulation of aldosterone release by ACTH suggests a potentially important role for the HPA axis in regulating aldosterone release in marine mammals (Ensimnger et al. 2014; Keogh and Atkinson 2015). Oxidative stress markers While most investigations of stress in wildlife systems have focused on neuroendocrine responses to stress, a wide

variety of other techniques are available to assess impacts of stress on marine mammal health. One important system that is critical to all marine mammals is the defense system that guards against oxidative stress. As air-breathers, marine mammals are obligated to breath-holding with associated ischemia and hypoxia during foraging. Perfusion of ischemic tissues at the end of breath-holds is associated with increased oxidant production and oxidative stress (Vázquez-Medina et al. 2012). The ability of marine mammals to avoid the consequences of oxidative stress resides in two different muscle fiber types. Type I muscle fibers are slow oxidative fibers with low aerobic capacity, enhanced myoglobin concentrations, and increased intramuscular oxygen stores (Kanatous et al. 2002; Kielhorn et al. 2013). These Type I slow twitch muscle profiles tend to reduce the oxygen consumption characteristic of oxidative stress. Type II muscle fibers are typically associated with increased glycolytic capacity for burst swimming and are analogous to a sprinter’s fiber-type profile. However, Velten et al. (2013) found that beaked whales, considered amongst the deepest diving marine mammals, exhibited a unique locomotor morphology of ~80 % Type II fibers and hypothesized that the higher number of Type II fibers decreased the energetic cost of diving in the event of a switch to anaerobic diving conditions. However, they also proposed that the high number of Type II fibers may also serve to store oxygen for use by Type I fibers, as the type II fibers have a surprisingly high content of myoglobin (Velten et al. 2013). Independent of diving, fasting seasons are associated with dramatic increases in oxidative stress (Martensson 1986). To overcome these problems, marine mammals have evolved the ability to upregulate anti-oxidant defenses (Vázquez-Medina et al. 2010, 2012). Because the stress hormones cortisol and angiotensin II are potent promoters of oxidative stress, evidence of oxidative damage may be a critical diagnostic of chronic stressor exposure in marine mammals. Markers of oxidative damage include 8-isoprostanes and 4-hydroxynonenal for lipid peroxidation, 8-hydroxy-2-deoxy guanosine for DNA oxidation, and nitrotyrosine for protein nitrosylation (Table 2). Recent work on breeding male elephant seals suggests that many of these markers covary with circulating cortisol levels (Crocker, unpublished data).

Measuring stress markers in marine mammals Logistical difficulties Most stress biomarkers are traditionally measured in blood, but in marine mammals it can be logistically difficult to obtain blood samples rapidly enough to avoid the effects of handling stress. Methods need to be developed that avoid

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the potential of creating handling artifacts (PedemeraRomano et al. 2010; Champagne et al. 2012; Keogh et al. 2013; Fair et al. 2014), which have been well identified in birds (Nilsson et al. 2008). Many marine mammal species are fully marine and some are only rarely seen at the ocean surface. Access to such species may only be available through stranding events and rehabilitation making baseline measures nearly impossible to obtain. For amphibious marine mammals (e.g., pinnipeds and polar bears), where access on land can be achieved, animals still have to be captured for direct sampling, and procedures must be completed in a time course that precedes any significant change in the marker being investigated. It should be noted that for most amphibious marine mammals their time on land is predictable and seasonal, and often linked to reproduction or molting, which also influences results of biomarkers. In some cases, the use of immobilizing agents may minimize the impact of handling stress (Champagne et al. 2012), but the potential effects of the immobilizing agents themselves have not been fully investigated (Mashburn and Atkinson 2004). Two possible solutions to minimizing handling artifacts in the sampling of marine mammals exist: (1) less invasive sampling methods (e.g., fecal collection) or, (2) for those species which are available, the use of trained animals for voluntary sampling. In contrast to free-ranging marine mammals, those in well-managed facilities are afforded protection from predators, reliable and predictable food sources, and the presence of veterinary care. Training for routine husbandry procedures can ensure that sample collection is not perceived as a stressful event. Unfortunately, there are only a few species (e.g., bottlenose dolphins or harbor seals) that are maintained in sufficient numbers to provide adequate sampling of stress biomarkers to characterize variability as a function of age, gender, seasonality, and/or reproductive status (i.e., baseline measures). Marine mammals under human care are not directly comparable in other facets of life, they likely have a modified social structure which is not dictated by conspecific interactions, rather their social structure and dynamics are under human control. Biological samples A number of other biological samples and sampling techniques can be utilized for analyzing biomarkers in marine mammals to better understand the stress response to natural and anthropogenic stressors, thereby using the best available science to improve marine mammal conservation efforts. Potential matrices include feces, urine, blubber, respiratory collections (e.g., whale blow), and saliva (Hunt et al. 2013). For obvious reasons, the complications with sampling each of these depends largely on the behavior

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J Comp Physiol B

of the marine mammal sampled. Of primary importance in using any sample media is conducting both analytical and biological validations of the measurement protocol, typically assay systems. Analytical validations have long been established (Robard 1974), and include (1) ensuring that the presence of the biological sample itself does not impact measurement or efficacy of testing components; (2) the concentration of the biomarkers is likely going to be measured in the middle of the standard curve, and (3) that the assay is linear such that the addition of the biomarker or increasing amount leads to a corresponding increase in its measurement. Biological validations are also important and can be as simple as comparing known stressed versus unstressed individuals. However, a complete biological validation may include various life history or physiological states, as well as assessing circadian and seasonal cycles, trophic hormone challenges, or introduction of hormonal precursors (e.g., progesterone) to assess metabolites. Blood Biological samples appropriate for measurement of peptide biomarkers of stress typically are blood and urine, both of which are difficult to obtain without capture in free-ranging animals. However, blood remains the historical standard largely based on research of terrestrial animals. Peptide biomarkers are often pulsatile in secretion, rapidly cleared and the involvement of many peptides with normal behaviors make it difficult to accurately define peptide baselines, even when blood can be obtained. Without such baselines, characterization of a peptide-based stress response cannot be precise. Most steroid hormones are easily measured in the blood and methods have been developed for the measurement of cortisol or its metabolites in other media, e.g., feces, urine, and hair (Wasser et al. 2000; Gow et al. 2010; Meyer and Novak 2012). The broad number of biological samples that cortisol or its metabolites can be measured in has increased the utility of cortisol as one measure of the stress response in wildlife systems. Because blood has notoriously been difficult to collect from free-ranging marine mammals, substantial effort has been invested in finding other biological materials that can be less invasively collected. Fecal samples Feces or scat can be collected non-invasively from wild and captive animals through incidental field collections and can be obtained through voluntary participation of some species under human care (Biancani et al. 2009). Fecal glucocorticoids have been assayed in North Atlantic right whales (Eubalaena glacialis) from feces collected at the ocean surface (Hunt et al. 2006). Variations in fecal glucocorticoids

J Comp Physiol B

were subsequently used to demonstrate the potential impact of ocean noise as a stressor to the right whale (Rolland et al. 2012). Similar methods were used with the endangered southern resident killer whales (Orcinus orca) to determine if variations in fecal GC and fecal thyroid hormones corresponded to nutritional stress (i.e., lack of primary prey species) or a high degree of interaction with ocean vessels (Ayres et al. 2012). Within pinnipeds, fecal GC and thyroid hormones have been measured in Steller sea lions to assess adrenal and thyroid function under chemical stimulation as well as the impact of rehabilitation and surgical procedures on glucocorticoid production (Mashburn and Atkinson 2004; Petrauskas et al. 2008; Keech et al. 2010). Assessment of stress markers in scat has the potential to remove or minimize artifacts due to handling, pursuit, or capture. However, it might not be suitable for all marine mammals. Not all marine mammals produce feces sufficiently buoyant enough that they float at the surface and/or with enough cohesion to allow collection of an adequately representative sample. This may necessitate being in close proximity to the animal at the time of defecation, creating a potential disturbance artifact, to enable fecal collections. For species that congregate on rookeries (e.g., sea lions, elephant seals), collection of feces from the ground may be easy, but relating the collection to an individual animal may be difficult or impossible to do. However, specific metabolites within the feces may be usable for identifying gender, age class, and reproductive state of the individual (Hunt et al. 2006; Mashburn and Atkinson 2004, 2007). Concern needs to be given for the types of metabolites that are excreted, the effects of weather on metabolite degradation and the potential for cross-reactivity with other compounds during the immunoassays thus far employed (Hunt et al. 2006). Additional research is also required to determine the time course of metabolite excretion given that there is potentially considerable variability in the gut transit times of digesta and in the rates of luminal excretion of the metabolites across marine mammal species (GoodmanLowe et al. 1997; Bodley et al. 1999; Kastelein et al. 2003; Larkin et al. 2007; Keech et al. 2010). Blubber Blubber can be sampled from marine mammals for the analysis of steroid hormones and this approach has been used to determine reproductive status in several cetacean species (Mansour et al. 2002; Kellar et al. 2006, 2009). Extension of this process to GC is currently underway and will likely provide a viable means of assessing cortisol levels in free-ranging cetaceans (i.e., via biopsy darts). Several considerations must be given to the potential for the structure of the blubber layer and its vascularization to influence deposition of steroid hormones, as the blubber layer has

been shown to be stratified in a number of marine mammals, including harbor porpoise (Koopman et al. 1996), Weddell seals (Wheatley et al. 2007), fin whales (Aguilar and Borrell 1990), and minke whales (Olsen and GrahlNielsen 2003). A determination of whether the distribution of a targeted hormone is homogenous or heterogeneous throughout the blubber depth and across the animal’s body needs to be made, and the time course of deposition and flux needs to be determined relative to circulating hormonal variations to better enable interpretation of blubber hormone values. Saliva and whale blow Cortisol in saliva has been shown to strongly correlate with serum cortisol (Teruhisa et al. 1981) and α-amylase has been shown to correlate with variations in catecholamines in humans (Chatterton et al. 1996). In marine mammals, saliva has been used to monitor sex hormones and reproductive state (Pietrazek and Atkinson 1994; Theodorou and Atkinson 1998; Hogg et al. 2005); however, not every study has found measurements from saliva to be a suitable proxy for serum hormone values (Atkinson et al. 1999). Nevertheless, based on success in other systems and in the isolation of other steroid hormones in marine mammals, there is potential for using both saliva and whale blow to be direct measures of stressor exposure and possible impacts. This would enable researchers to assess some of the hormones involved at different levels of the stress response. For captured wild animals, saliva may provide a non-invasive alternative to blood sampling and could be obtained from many marine mammals under human care provided adequate husbandry training to enable voluntary sampling. Recent work attempting to quantify sex steroids in whale blow has demonstrated the potential for using this collection method for the analysis of other steroid hormones (Hogg et al. 2005, 2009). However, there are a considerable number of hurdles that need to be overcome to make this an effective approach to monitoring and measuring stress biomarkers in marine mammals. Although easily collected with cetaceans under human care, procedures for capturing blow in wild cetaceans need to be developed that permit adequate samples to be collected for analysis (AcevedoWhitehouse et al. 2010; Hunt et al. 2013). Analytical methods for identifying and quantifying stress response mediators need to be developed and validated such that differing proximate compositions of whale blow do not compromise the interpretation of results. If that can be standardized, concentrations of various hormones can be spatially and temporally compared across individuals and populations. Provided these challenges can be met, the approach has the potential to enable non-invasive sampling on free-ranging

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animals that might not otherwise be achieved, particularly for large mysticete whales (Hunt et al. 2013). Urine Urine has been used to monitor reproductive processes in cetaceans (Walker et al. 1987, 1988; Robeck et al. 1993). Urinary measurements of hormones in terrestrial mammals are often standardized to creatinine levels to account for differences in urinary concentration (Atkinson and Williamson 1987). This process may be complicated in marine mammals due to high variation in renal filtration and resultant creatinine excretion rates compared to terrestrial mammals (Crocker et al. 1998). Captive marine mammals have been the subjects of most of the urinary endocrine work, and because protein hormones, such as the catecholamines can be passed in urine (Li et al. 2014), this biological sample can allow for a more detailed characterization of marine mammal physiology with the additional benefit of noninvasive collections.

Discussion Marine mammals returned to the sea many millions of years ago from terrestrial relatives, and they represent a divergent group of species, e.g., canid–ursid descent for pinnipeds (Berta and Wyss 1994; Arnason et al. 2006), ungulate ancestry for cetaceans (Gatesy et al. 1996; Shimamura et al. 1997) and a common heritage for elephants and sirenians (Nishihara et al. 2005; Kjer and Honeycutt 2007). In comparing the stress response between odontocetes and elk subjected to chasing from boats/helicopters and snowmobiles, respectively, spotted dolphins did not show significant increases in cortisol but did exhibit other signs of a normal stress response such as glucose, ACTH and catecholamines (St. Aubin et al. 2013). Elk had higher corticosterone levels during the snowmobile season after controlling for age and snow depth (Creel et al. 2002). In Florida manatees, cortisol concentrations >10 ng/ml were diagnostic of chronic stress (Tripp et al. 2010), and similarly, cortisol metabolites were elevated after transport and relocation in an Asian elephant. Fecal GCs were elevated in mature males and pregnant female dugongs with elevated progesterone concentrations (Burgess et al. 2013), but these findings contrast with captive African elephants, where circulating cortisol was negatively correlated with progesterone concentrations or remained unchanged during pregnancy in captive Asian elephants (Brown and Lehnhardt 1995). Cooperatively breeding African wild dogs and wolves of both sexes exhibit higher GC concentrations than their subordinate

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counterparts (Creel et al. 1997; Sands and Creel 2004). This is a phenomenon also observed in captive and freeranging Steller sea lion males (Mashburn and Atkinson, 2007), despite the fact that they employ what is typically considered a harem breeding strategy, which differs from most canids. Because of the paucity of comparative data, particularly longitudinal endocrine data from free-ranging marine mammals, it is difficult to draw detailed parallels between related terrestrial and marine species. Nevertheless, the expression of glucocorticoids, particularly cortisol, appears to be a maintained characteristic of the stress response across marine and terrestrial mammals. Evidence in marine mammals suggests that mineralocorticoids may also play an important part of the stress response, but little to no information on variations in mineralocorticoids exists for terrestrial relatives. If similar patterns are not observed in terrestrial mammals, the differences in mineralocorticoid expression may largely be due to the apneustic lifestyle of most marine mammals and their incidental/intentional consumption of ocean water. Differences in the patterns of catecholamine release in response to acute stress between terrestrial and marine mammals are also intriguing. The relationship of catecholamine release to decreased heart rate and splenic supply of blood during diving, both of which serve to conserve oxygen consumption, is counter to the response observed in terrestrial mammals. Again, the apneustic requirements of diving may be a driving force in modifying pathways of action for the catecholamines from that observed in terrestrial counterparts. Escape strategies in the marine mammals may have less to do with fight or flight and more to do with dive deep and the need to be quiet. Despite varied evolutionary lineages, the constraints associated with being air-breathing endotherms in the marine environment may have selected for common alterations to the highly conserved mammalian stress response within marine mammals. The necessity of managing breathholds while exercising may have associated aspects of the stress response with features that promote dive capacity, including bradycardia, selective ischemia and vasoconstriction of working skeletal muscle, and maintenance of centralized blood pressure (Kanatous et al. 2002; Velten et al. 2013). Evidence suggests that the typical response to an acute, potentially life-threatening stressor within marine mammals differs from that of terrestrial mammals. Simply put, the need by these air-breathing mammals to separate many biological functions from their source of oxygen has resulted in what appear to be significant modifications to the action and release of the catecholamines. These hormones, which typically serve to increase oxygen consumption and cardiac output, appear to have nearly opposite effects in the diving marine mammals (Hance et al. 1982;

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Hochachka et al. 1995; Hurford et al. 1996). Life within a hyperosmotic environment, consumption of prey potentially isotonic with sea water (e.g., squid), and incidental or intentional consumption of sea water likely increases the reliance on mineralocorticoids for maintaining electrolyte balance (Ortiz 2001). High fat, low carbohydrate diets and natural extended fasts seen in many species of marine mammals have selected for alterations in metabolism that prioritize use of lipids for fuel metabolism over carbohydrates (Crocker et al. 1998; Kelso et al. 2012; Verrier et al. 2012), including an enhanced glycolytic activity in muscles with high proportions of fast twitch (Type II) fibers (Kielhorn et al. 2013; Velton et al. 2014). These features may be associated with differences in the metabolic impacts of both catecholamines and GC and their roles in altering energy availability during stress (Guinet et al. 2004; Champagne et al. 2012). Together these disparities may have led to novel regulation of corticosteroid release by the HPA axis (e.g., aldosterone release) and more diverse tissue responses to stress hormones when compared to terrestrial systems. The terrestrial model for stress physiology is a useful foundation for studying stress response of the marine mammals, but there are discrepancies that must be taken into account (Table 2). These differences are critical to understanding how marine mammals have evolved to the constraints of a fully aquatic or amphibious existence, and the field of stress physiology in marine mammals needs to concentrate on patterns of stress biomarker production related to various life history stages unique to these species (Landys et al. 2006), responses to natural environmental stressors, particularly within the oceans, and responses to anthropogenic stressors (Wright et al. 2007; Tyack 2008). The linkages between life history states and known stressors and the stress response in terrestrial mammals are abundant in the literature (Boonstra et al. 1998; Touma et al. 2001, 2003; Monclus et al. 2006), but marine mammal experience selective pressure imposed by their environment. For example, the need for extended breath-holding during foraging coupled with osmoregulatory challenges are not often shared with terrestrial relatives. These environmental constraints across life history states can multiply to acute vulnerable conditions that may be cumulative. The cumulative effects of stressors can lead to population-level consequences through impacts on vital rates. From a conservation perspective, whether these impacts result from an acute triggering event (e.g., unusual sonar in an area) or through unknown chronic events (e.g., climate change or chronic nutritional stress), requires an understanding of the baseline variability and the impact on hormones typically involved in mediating the response to external stressors. To date, this work remains largely incomplete.

Conclusions and recommendations Physiological indices of stress commonly measured in terrestrial mammals, such as GC or ACTH, have been measured in many marine mammal species and in general indicate that the HPA axis functions similarly to terrestrial mammals. Beyond the stereotypical adrenal GC activity, there are numerous variations in associated endocrine and behavioral responses (Table 2). Aldosterone, for example, appears to clear more rapidly following ACTH stimulation, which may be an indication of feedback regulatory mechanisms that are more sensitive or complex in marine mammals than in terrestrial mammals. Marine mammals seem to diverge from the terrestrial mammal norm in the complexity of regulatory systems that increase activity under perceived or physical stressors. In particular, the activity of the RAAS and catecholamines in typical marine mammal behaviors (i.e., fasting or diving) appears different from that of terrestrial mammals. Typically in mammals the activation of RAAS leading to aldosterone release is initiated by reductions in renal perfusion and tubular flow rates. The key conversion of angiotensin I to the active form, angiotensin II, by angiotensin-converting enzyme occurs mainly in the lung capillaries. Changes in renal perfusion (Murdaugh et al. 1961) and pulmonary shunts (Kooyman and Sinnett 1982) that occur during normal diving would potentially impact typical RAAS regulation. This problem may have led to increased HPA axis regulation of aldosterone release in breath-hold divers like marine mammals. It is clear that there is much work to be done in the field of stress physiology and behavior in marine mammals. The use of terrestrial models has been an effective means of detecting differences in marine mammals, and the use of model species representative of different classes of marine mammals (e.g., phocids, otariids, odontocetes, mysticetes, sirenians) can aid in our further understanding of the physiology and behavior of stress responses in marine mammals. Specifically, we present five recommendations that vary in scope from analytical methodologies to population modeling that we believe will promote the field of stress physiology and its impacts on marine mammal conservation and management. 1. Incorporate measurements of CBG, such that the true physiological role of corticosteroid measurements can be ascertained. Substantial work on CBG has been done for terrestrial mammals; however, there are a paucity of studies on the binding proteins and receptors for corticosteroids in marine mammals. Because CBG influences the transport and delivery of corticosteroids, it likely can help to explain the paradoxical situations where researchers might expect elevations of corti-

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coids, but the response varies from that expectation (e.g., differential responses in northern elephant seals, Ensminger et al. 2014). Similarly, CBG may become a key indicator of the presence of chronic stressors, as it does not seem to vary with acute stress (Chow et al. 2010). 2. Obtain contextual data on natural variations in a suite of stress hormones to provide baselines that can be used for comparisons, and consider model species that can represent others that are difficult to access. Many studies that have taken place to date have not been able to control for the influences of season, time of day or natural physiological states. These influences may be altering the baseline concentrations of key mediators of the stress response. For species that are logistically difficult to find or study, the use of a representative proxy, or model species, could facilitate research that otherwise may not occur. Our knowledge of the full context under which samples are collected and the species they might represent will aid in the interpretation of results, and enable better comparisons between studies. 3. Spend more effort on understanding the relationship between corticosteroids and the reproductive and immune systems of marine mammals, including the role of GC in influencing natality. While corticosteroids are widely recognized as bioindicators of a stress response, their ultimate impact on the reproductive and immune systems of marine mammals requires greater understanding. Specifically the biosynthetic pathways that are altered during an acute or chronic stress response need to be confirmed, or in some cases elucidated. Some of this work has been initiated, and there are many human and domestic animal studies to serve as the background for these studies. 4. Use long-term monitoring of individuals relative to their reproductive, social, and environmental surroundings to better understand life-history influences on variations in stress responses. There is no doubt that a myriad of factors in an animal’s environment can dictate the response of that individual to internal or external changes that may be perceived as stressors. Without an understanding of the role that social dynamics, reproductive states, or physiological surroundings play, the interpretation of physiological data can be limited or even impaired. Our ability to understand variations in stress responses will be enhanced by most forms of long-term monitoring to gain knowledge of how that response benefits the organism. 5. The field of stress physiology needs to be linked to population-level consequences of disturbances of multiple origins. This will allow the study of stress physiology to be connected to marine mammal conservation and management issues. In order

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to truly link physiological studies to conservation and management a series of intermediate steps need to be defined, such that the population-level consequences of a given stressor can be evaluated. Behavioral response studies are an important intermediate step. Acknowledgments  We thank Mike Weise and the US Office of Naval Research (ONR) for highlighting the lack of knowledge about stress physiology in marine mammals. The participants of two workshops, one at ONR in Virginia, and the other at the 15th Biennial Conference on the Biology of Marine Mammals, provided many insightful discussions on evaluating the stress response in marine mammals. Numerous field crews and lab technicians have also added to our understanding. Drs. Kathleen Hunt and Frances Gulland provided friendly reviews that substantially improved the manuscript, as did four anonymous reviewers. Ms. Angela Kameroff-Steeves provided help with the preparation of the manuscript. This work was funded by a grant from the Office of Naval Research to S. Atkinson (ONR Award No. 00014121-290).

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