Response to capture stress involves multiple corticosteroids and is ...

MARINE MAMMAL SCIENCE, 00(00): 00–00 (Month 2018) C 2018 Society for Marine Mammalogy V

DOI: 10.1111/mms.12517

Response to capture stress involves multiple corticosteroids and is associated with serum thyroid hormone concentrations in Guadalupe fur seals (Arctocephalus philippii townsendi) EUGENE J. DERANGO,1 Department of Biology, Sonoma State University, 1801 E Cotati Avenue, Rohnert Park, California 94928, U.S.A.; DENISE J. GREIG, Ornithology and Mammalogy Department, California Academy of Sciences, 55 Music Concourse Drive, San Francisco, California 94118, U.S.A.; CASANDRA GA´LVEZ, Centro Interdisciplinario de Ciencias Marinas, Instituto Politecnico Nacional, Avenida IPN s/n Col. Playa Palo de Santa Rita, 23096, La Paz, Baja California Sur, Mexico; TENAYA A. NORRIS, LORRAINE BARBOSA, The Marine Mammal Center, 2000 Bunker Road, Sausalito, California 94965, U.S.A.; FERNANDO R. ELORRIAGA-VERPLANCKEN, Centro Interdisciplinario de Ciencias Marinas, Instituto Politecnico Nacional, Avenida IPN s/n Col. Playa Palo de Santa Rita, 23096, La Paz, Baja California Sur, Mexico; DANIEL E. CROCKER, Department of Biology, Sonoma State University, 1801 E Cotati Avenue, Rohnert Park, California 94928, U.S.A.

ABSTRACT Guadalupe fur seals are a threatened species with few breeding locations, which potentially makes them sensitive to environmental or anthropogenic stressors. We present the first study to quantify adrenal and thyroid function in this species in an effort to measure their stress response to capture. We analyzed a suite of corticosteroid hormones released over time during capture in both adult females (n 5 10) and weanling pups (n 5 26) during March 2016. Multiple corticosteroids were released during capture, and aldosterone was associated with the response to stress in adults only. These results suggest the regulation of aldosterone secretion in association with the HPA axis in otariids as reported in other marine mammals. Individuals varied markedly in the magnitude of their endocrine response to capture. A lower total integrated stress response to capture for both cortisol and corticosterone was associated with decreased concentrations of thyroid hormone T3 and elevated concentrations of reverse T3 (rT3), suggesting parallel downregulation of adrenal and thyroid endocrine axes in some individuals. A scaled body condition index was negatively associated with T3 and positively associated with rT3 in adults. Together these findings suggest utility in using endocrine responses to capture stress to evaluate individual and population health. Key words: Guadalupe fur seal, stress, corticosteroids, thyroid, capture.

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Corresponding author (e-mail: [email protected]).

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MARINE MAMMAL SCIENCE, VOL. 00, NO. 00, 2018

Physiological response to stress is a fundamental process for all animals and is associated with an internal or external disturbance to homeostasis (Sapolsky et al. 1986). In vertebrates, stress activates the hypothalamic-pituitary-adrenal (HPA) axis, which ultimately releases glucocorticoid hormones that mediate a diverse array of physiological responses, including permissive, suppressive, and stimulatory effects (Sapolsky et al. 2000). Some of these effects enhance and mediate the stress response while others limit the stress response and enhance the recovery from it (e.g., Khudyakov et al. 2017). These acute responses can include interactions with other endocrine hormone axes (Ensminger et al. 2014). The release of hormones by the HPA axis and associated physiological changes allow animals to respond in the short term through allostasis, or maintaining internal stability in the event of change or stressors in the environment (McEwen and Wingfield 2003). The response to stress is mediated by a variety of mechanisms, and the dominant corticosteroid released varies among taxa. In most mammals, cortisol is the main glucocorticoid hormone, whereas corticosterone release dominates in amphibians, reptiles, birds, and rodents (Wasser et al. 2000). The release of a third adrenal corticoid, aldosterone, is primarily regulated via the renin-angiotensin system (RAS), and plays an active role in regulation of blood pressure and volume by controlling electrolyte and water balance (St. Aubin 2001). Marine mammal taxa have evolved physiological adaptations to cope with stressors that differentiate them from terrestrial animals (Atkinson et al. 2015). Several investigations have suggested an unusually strong degree of HPA axis association with aldosterone in phocids, odontocetes and mysticetes. Substantial release of aldosterone has been shown to result from induced stress in northern elephant seals (Mirounga angustirostris, Ensminger et al. 2014, Champagne et al. 2015), dolphins (Ortiz and Worthy 2000, Houser et al. 2011, Hart et al. 2015, Champagne et al. 2017) and beluga whales (Delphinapterus leucas, Schmitt et al. 2010). Fecal aldosterone was strongly associated with fecal glucocorticoids in right whales (Eubalaena glacialis, Burgess et al. 2017). Together these findings suggest that aldosterone is an important stress responsive hormone in many marine mammals, but to date no evidence has been presented for otariids. Conservation physiologists are increasingly using measurements of glucocorticoids after capture to provide insight into the ability of individuals to mount a stress response, which can have implications for population or whole species dynamics (Wikelski and Cooke 2006). It has been shown that individuals within a species can vary greatly in their ability to respond to a stressor (Cockrem 2013b). Release of glucocorticoids can exert immediate effects on metabolism, while chronic exposure can have deleterious effects such as suppression of immune response (McEwen et al. 1997) and reproduction (Breen et al. 2005) Animals may have a reduced ability to respond to additional stressors during stressful periods when corticosteroid levels are already increased to support energy mobilization. However, elevated corticosteroid concentrations cannot always be used as a proxy for chronic stress. In some study species, chronic stress can decrease basal corticosteroid levels through reduced HPA sensitivity and significantly reduce response to an acute stressor (Rich and Romero 2005). Similarly, the size of the total integrated corticosteroid response to the stress of capture can be highly repeatable within individuals and may provide great insight into variation in HPA sensitivity during chronic stress (Cockrem and Silverin 2002). The hypothalamic-pituitary-thyroid (HPT) axis is another endocrine system that that can respond to stress. The HPT axis regulates the release of thyroid hormones (thyroxine, T4; and triiodothyronine, T3), which primarily regulate metabolism

DERANGO ET AL.: GUADALUPE FUR SEAL STRESS RESPONSE

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(Danforth and Burger 1984). T4 is primarily a prohormone and a reservoir for the more active thyroid hormone T3. Both acute and chronic stress can alter thyroid hormone secretion and cortisol and corticosterone have been suggested to play a role in the regulation of the HPT axis (Helmreich et al. 2005). Elevated corticosteroids can reduce thyroid function by suppressing release of thyroid stimulating hormone, inhibiting enzymatic deiodination of T4 into T3 and increasing concentrations of reverse T3 (rT3), an inactive form of the T3 hormone and a thyroid receptor blocker (Charmandari et al. 2005, Cooper and Ladenson 2011). While the responses of the HPT axis to acute stress are highly variable and context dependent (Helmreich et al. 2005), chronic stress, such as disease or nutrient limitation, suppresses thyroid function and reduces energy expenditure (Chrousos 2009). Therefore, the chronic effects of elevated cortisol are likely associated with low levels of thyroid hormones (Oppenheimer et al. 1987). Life-history stage can have dramatic effects on hormone concentrations, which vary depending on age, sex, season, reproductive status, and nutritional stress. For example, due to the separation of terrestrial reproduction from marine feeding, many pinnipeds undergo food deprivation during periods of energetically costly activities such as nursing, development, breeding or molting. These events at various ages are associated with variation in adrenal and thyroid activity that alter circulating baseline hormone concentrations (Guinet et al. 2004, Mashburn and Atkinson 2004, Greig et al. 2007, Ensminger et al. 2014, Champagne et al. 2015, Jelincic et al. 2017). Thus, contextual information about changes in hormones with life-history is critical to interpreting their implications for individual and population health. Fur seals and sea lions, members of the pinniped family Otariidae, are notoriously difficult to capture and handle due to logistics such as capture location, large numbers of conspecifics present, ease of mobility on land, and aggressive behavior (Loughlin et al. 2010). In some cases capture is facilitated through darting (Baylis et al. 2015), but for many species capture involves pursuit and capture of individuals in nets. Due to a need to manually capture and restrain for blood sampling or subsequent chemical immobilization, measuring baseline glucocorticoid concentrations can be difficult in most species within this family (Atkinson et al. 2015). The capture process can evoke a variable stress response depending on the methodology used, including length of capture time and type of equipment (i.e., nets, head bags). Due to these constraints, most previous work on otariid endocrinology has used captive animals (e.g., Mashburn and Atkinson 2007). Stress has been measured in response to an acute or chronic stressor, such as short-term captivity with rehabilitated free-ranging sea lions (Petrauskas et al. 2008) or during branding procedures required for identification of individual animals (Mellish et al. 2007). Studies which measured serum cortisol in free-ranging sea lions showed that concentrations varied by geographic region in Steller sea lion (Eumetopias jubatus) pups (Myers et al. 2010) and between islands in California sea lion (Zalophus californianus) pups in the Gulf of California (Padernera-Romano et al. 2010). Within the otariid family, Guadalupe fur seals (Arctocephalus philippii townsendi, GFS) are a threatened species with high sensitivity to environmental stress because of their reduced population and small, isolated breeding area (Aurioles-Gamboa et al. 2010). These fur seals are generally reproductively isolated to a small volcanic island (Guadalupe Island) off the coast of Baja California, which poses logistical challenges to the study of their ecology, behavior, and reproduction. The species can also be seasonally found at a small archipelago (San Benito) 250 km south of

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Guadalupe (Hambrecht et al. 2016), where recolonization is currently taking place, but with no significant breeding (Elorriaga-Verplancken et al. 2016a). GFS inhabit the California Current ecosystem and likely share similar strategies with other fur seal species by foraging in oceanic frontal structures or continental shelf-edges with upwelling regions (Amador-Capitanachi et al. 2017). Published foraging data for free-ranging GFS are limited to three lactating females which were tracked using satellite telemetry on Guadalupe Island (Gallo-Reynoso et al. 2008). Analysis of stable isotopes of carbon and nitrogen in fur has provided evidence for oceanic foraging in individuals from both sites (Amador-Capitanachi et al. 2017). Despite their threatened population status, basic life history traits for the current breeding population such as type of prey consumed and the growth rates of neonate pups have only recently been described (Gallo-Reynoso and Figueroa-Carranza 2010, GalloReynoso and Esperon-Rodrıguez 2013). Although minimal data exist for the current Guadalupe Island population of GFS, aspects of their life history, including timing of breeding, weaning, and attendance patterns have been studied (Gallo-Reynoso 1994). GFS on Guadalupe Island congregate and give birth between June and early August (Gallo-Reynoso 1994). Once pups are approximately 1 wk old, adult females begin to take longer foraging trips to regain lipid stores lost during milk production (Gallo-Reynoso 1994). During late spring, adult females are weaning dependent pups while opportunistically foraging (Gallo-Reynoso 1994, Melin et al. 2000). During this time, 9– 10-mo-old pups typically remain ashore and transition to independent foraging while simultaneously beginning to develop important swimming skills in small, shallow tide pools (Gallo-Reynoso 1994). These life history events for both adults and pups are likely associated with changes in endocrine response and development (Atkinson et al. 2015). In recent years, GFS have stranded in increased numbers along the Pacific coastline from east Alaska to Mexico due to malnutrition and subsequent disease (NOAA Fisheries 2015). Stranding numbers of both live and dead GFS were eight times the historical average. Due to their amphibious and pelagic lifestyle, GFS also experience a variety of anthropogenic stressors in both aquatic and terrestrial environments, such as biotoxin exposure, contaminants, vessel traffic and collisions or entanglement in marine debris (Bossart 2011, Kovacs et al. 2012). It has been shown that exposure to anthropogenic stress can cause changes in susceptibility to infectious disease and high energetic costs (Brock et al. 2013). In extreme cases, acute exposure to capture stress in South American fur seals has been documented to cause cardiomyopathy or even death (Seguel et al. 2014). Understanding how these animals respond to stressors and its implication for other health systems is a crucial step towards management and conservation efforts. We present the first study to quantify adrenal and thyroid function in this species in our initial effort to measure the stress response to capture. Although Guadalupe Island is geographically isolated in the California Current ecosystem, the region supports both commercial and small fisheries, ecotourism via recreational fishing and shark cage diving, and an established naval base in close proximity to the fur seal rookery. Each of these has the potential to evoke physiological responses to mediate the impact of the stressor, which may alter metabolism. In a threatened species, such as GFS, physiological trade-offs during chronic stress may have significant downstream effects on metabolism and ultimately manifest in species level consequences. We present evidence that reduced stress responses to capture, potentially caused by chronic stress, are associated with downregulated thyroid function and

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Table 1. Mean 6 standard deviation of scaled mass index and analytes measured from the first sample taken after capture in adult and pup Guadalupe fur seals

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Scaled mass index Time first sample (min) Cortisol (mg/dL)a Corticosterone (mg/dL) Aldosterone (pg/mL)a Total T4 (mg/dL)a Total T3 (ng/dL) Reverse T3 (ng/mL) a

Adults 49.5 6 5.6 963 10.66 6 6.77 0.829 6 0.564 580 6 300 3.10 6 1.82 261 6 110 0.923 6 0.297

Pups 16.6 6 2.3 864 5.97 6 3.26 0.617 6 0.34 980 6 580 4.89 6 2.26 208 6 100 1.11 6 0.280

P 0.05) and pup individual variation accounted for 92.3% of the variance in aldosterone. Cortisol and corticosterone concentrations were associated for both adults and pups (Fig. 3A; y 5 0.064x 1 0.233, R2 5 0.70, F1,61.9 5 146.7, P < 0.0001). Both cortisol (Fig. 3B) and corticosterone (Fig. 3C) were associated with aldosterone in adults only (cortisol, y 5 37.5x 1 66.0, R2 5 0.59, F1,18.3 5 25.7, P < 0.0001; corticosterone, y 5 441.7x 1 153.3, R2 5 0.68, F1,20.5 5 45.6, P < 0.0001). No associations were found between either initial cortisol or initial corticosterone and initial


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Figure 2. Corticosterone (A) increased with time after capture in 10 GFS adults and 26 pups (R2 5 0.53, P < 0.0001). The fitted regression line is derived from the LMM fit, which includes both age classes. Aldosterone (B) increased with time after capture in adults only (R2 5 0.50, P 5 0.0003). The fitted regression line represents adult fur seals, whereas time after capture had no association with aldosterone in pups (P > 0.05).

aldosterone in pups (P > 0.05). Initial cortisol was higher in adults than pups (Table 1, t 5 2.82, df 5 34, P 5 0.008), but initial aldosterone was higher in pups than adults (Table 1, t 5 2.67, df 5 31.1, P 5 0.011). Total integrated stress responses (TSR) for each corticosteroid calculated to 30 min after capture also varied greatly amongst individuals. TSR for cortisol (n 5 19) ranged from 26.3 to 299.3 (mg/dL)/min (mean 5 188.5 6 80.0 SD), TSR for corticosterone ranged from 3.7 to 45.2 (mg/dL)/min (mean 5 15.8 6 10.8 SD), and TSR for aldosterone ranged from 602.0 to 13,975.6 (pg/mL)/min (mean 5 5,054.1 6 4,332.8 SD). No differences in age class were found in TSR of all corticosteroids (Table 2, P > 0.05). TSR was also not associated with hormone concentrations in the first sample after capture for cortisol (F1,16 5 0.06, P 5 0.81), corticosterone (F1,16 5 4.0, P 5 0.07), or aldosterone (F1,16 5 0.06, P 5 0.09). Body Condition and Thyroid Function Scaled mass index (SMI) varied by age class due to large differences in body size (t 5 25.1, df 5 33, P < 0.0001). SMI was not associated with TSR for any of the

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Figure 3. Associations between corticosteroids during repeated samples in GFS adults and pups. Cortisol was associated with corticosterone (A) in both adults and pups combined in a single fitted regression line derived from the LMM fit (R2 5 0.70, P < 0.0001). Both cortisol (R2 5 0.59, P < 0.0001) and corticosterone (R2 5 0.59, P < 0.0001) were associated with aldosterone (B and C) in adults only (pups, P > 0.05). The fitted regression lines in B and C represent adult fur seals only.

Table 2. Mean 6 standard deviation of total integrated stress response (TSR) for corticosteroids calculated from adult and pup Guadalupe fur seals after 30 min of a capture event. No significant differences were found between age classes

n TSR Cortisol (mg/dL)/min TSR Corticosterone (mg/dL)/min TSR Aldosterone (pg/mL)/min

Adults 9 202.7 6 22.8 16.65 6 4.22 5,020.5 6 4,415

Pups 8 170.78 6 32.0 14.67 6 2.60 5,096.1 6 4,530

P 0.41 0.71 0.97


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Figure 4. Associations between scaled mass index (SMI) and thyroid hormones TT3 (A, r2 5 0.43, P 5 0.039), TT4 (B, r2 5 0.50, P 5 0.023) and rT3 (C, r2 5 0.47, P 5 0.029) in 10 GFS adults. The inverse relationship between TT4 and TT3 likely reflects conversion of TT4 to TT3 during energy expenditure in foraging adults. There was no association between SMI and thyroid in pups (P > 0.05).

corticosteroids or hormone concentrations in the first sample after capture in either age class (P > 0.05). When analyzed separately, SMI was associated with concentrations of TT4, TT3 and rT3 in adult fur seals (Fig. 4). SMI was positively associated with TT4 (y 5 0.23x 2 8.3, r2 5 0.50, F1,8 5 7.89, P 5 0.023), negatively associated with TT3 (y 5 896.5 2 12.8x, r2 5 0.43, F1,8 5 6.12, P 5 0.039) and positively associated with rT3 (y 5 0.034x 2 0.75, r2 5 0.47, F1,8 5 7.11, P 5 0.029). SMI was not associated with any thyroid hormone in pups (P > 0.05). No association was found between TT4 and TT3 in either age group (P > 0.05). The mean ratio of TT4:TT3 was highly variable among individuals (26.3 6 21.4) but did not vary between age classes (P > 0.05). There was a

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Figure 5. Associations between the total integrated stress response (TSR) and thyroid hormones TT3 and rT3 in GFS. The fitted regression lines derived from the LMM include both adults and pups, which were combined due to sample size. TSR for cortisol (A, R2 5 0.39, P 5 0.006) and corticosterone (B, R2 5 0.35, P 5 0.01) was positively associated with TT3. TSR for cortisol (C, R2 5 0.55, P 5 0.0007) and corticosterone (D, R2 5 0.35, P 5 0.0126) was negatively associated with rT3.

strong negative association, however, between TT3 and rT3 (y 5 1.52x 2 0.0002, r2 5 0.57, F1,30 5 39.7, P < 0.0001). The mean ratio of TT3:rT3 was also highly variable (2.48 6 1.84 SD). TT4 was higher in pups than adults (t 5 2.25, df 5 31, P 5 0.032) but no differences were found in TT3 or rT3 (P > 0.05). Of all indicators of stress, only TSR for cortisol and corticosterone were associated with thyroid function (TT3, Fig. 5). TSR for cortisol (y 5 93.9 1 0.768x, R2 5 0.39, F1,16 5 10.0, P 5 0.006) and corticosterone (y 5 153.6 1 5.41x, R2 5 0.35, F1,16 5 8.63, P 5 0.01) were positively associated with TT3 in adults and pups (Fig. 5A, B). TSR for cortisol (y 5 1.39 2 0.002x, R2 5 0.55, F1,15 5 18.0, P 5 0.0007) and corticosterone (y 5 1.16 2 0.014x, R2 5 0.35, F1,15 5 8.03, P 5 0.0126) were negatively associated with rT3 in adults and pups (Fig. 5C, D). TSR for aldosterone did not predict TT3 in adults or pups (P > 0.05). No relationships were found between any stress marker and TT4 (P > 0.05), and no


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relationships were found between thyroid hormones and corticosteroid concentrations in the first sample after capture (P > 0.05). DISCUSSION Glucocorticoid Response to Capture All serum corticosteroids measured increased with time after capture in adult GFS, reinforcing the importance of considering handling and capture effects when interpreting stress hormone measurements in free-ranging pinnipeds. Individual patterns of corticosteroid release varied greatly, with some individuals showing declines from peak values by 60 min. Aldosterone also increased with time in adult GFS, with some animals showing declines before 40 min. In contrast, pups did not show a relationship between time to sample and concentrations of aldosterone. The release of cortisol and corticosterone in response to capture was strongly associated in both adult and pup fur seals. Similar studies in otariids that measure glucocorticoids typically analyze serum cortisol and correlate fecal glucocorticoid metabolites using antibodies for corticosterone or cortisol (Atkinson et al. 2015). Our study quantified the association between cortisol and corticosterone within an individual serum sample after capture stress. Corticosterone was released in biologically significant amounts relative to cortisol (mean cortisol: corticosterone mass ratio 5 11.1:1, ranging from 6.5:1 to 16.2:1). This range is similar to that reported for 18 other mammalian species where corticosterone release in response to stress has been evaluated (Koren et al. 2012). That study, however, did not include marine mammal taxa. Previous studies found that sea otters also release corticosterone in biologically significant concentrations in tandem with cortisol in response to stress (Larson et al. 2009). Both glucocorticoids compete for binding globulins, and the dominant glucocorticoid likely binds with greater affinity, allowing for differences in circulating concentrations of either (Westphal 1986). The association of cortisol and corticosterone may suggest differences in second messenger system activation during acute stress that can perform different physiological or behavioral functions (Koren et al. 2012). Regulation of Aldosterone by the HPA Axis Marine mammals consist of several monophyletic taxa that evolved from terrestrial ancestors which underwent intense selective pressure to return to an aquatic environment (Berta et al. 2005). For example, cetaceans and pinnipeds have evolved greater diving capacity and therefore physiological adaptations to oxygen metabolism and blood circulation (Ponganis 2015). Although not entirely understood, the regulation of aldosterone release by the HPA axis may be especially advantageous in a diving mammal. The RAS system is activated in response to reductions in renal tubular flow and involves a conversion step that is thought to occur predominantly in the pulmonary capillaries (Paul et al. 2006). Therefore, while alterations in renal or pulmonary blood flow during diving (Ponganis 2015) could potentially impact typical regulation of the RAS system, enhanced HPA control of aldosterone release in otariid species would prove valuable (Atkinson et al. 2015). Several previous studies have suggested that aldosterone is released as part of the stress response in marine mammals. For example, aldosterone is released in response to adrenocorticotropic hormone (ACTH) challenges and stress tests (Gulland et al.

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1999, Ortiz et al. 2000, Ensminger et al. 2014, Champagne et al. 2015). Here we provide data for aldosterone response to induced stress in an otariid seal. In this study, the responses of both glucocorticoids (cortisol and corticosterone) were highly associated with aldosterone in adult GFS only, while pups showed no relationship between both glucocorticoids and aldosterone. Adult females likely have greater diving ability and capacity compared to weaned pups that potentially have yet to start diving. Anecdotally, we did not observe any of the pups sampled in this study to travel far from their capture location. Although pups have access to deep water directly around Guadalupe Island, this area is likely not extensively used as the pups had not yet begun to forage. Water immersion in pups at this age is mostly confined to small shallow tide pools or in close proximity to the rocky shoreline. Our results suggest that stimulation of aldosterone release by the HPA axis may increase with the development and use of diving. Similarly, baseline cortisol and aldosterone concentrations were strongly associated in yearling elephant seals returning from diving at sea (Jelincic et al. 2017) but not associated in weanlings during the postweaning fast (Ortiz et al. 2003). Future studies could further explore this hypothesis by looking at the relationship between cortisol and aldosterone in other pinniped species of multiple age classes or life history stages that vary in their diving capacity. When compared to adult fur seals, GFS pups did show higher concentrations of aldosterone with greater individual variation measured in the first initial sample. Similarly, aldosterone concentrations measured after capture in Steller sea lion pups also showed large variation (Keogh et al. 2013). The current study took place in March when most adult female Guadalupe fur seals are making repeated intermittent foraging trips after their pups have begun to wean. These foraging trips likely become longer as pups age, and the length of the fast that pups undergo prior to and after weaning may vary similar to California sea lion life history. The RAS system is activated to maintain homeostasis and retain salts during extended fasting on land, and aldosterone increases during fasting in juvenile elephant seals (Ortiz et al. 2000). Differences in electrolyte and water balance when either nursing or fasting may contribute to the variation measured in aldosterone in these pups. Body Condition and Thyroid Thyroid hormones are important in maintaining metabolism and growth and development of neonatal and juvenile pups. Although our study did have a limited sample size of adult females captured, SMI showed a positive association with TT4 and rT3 and negative association with TT3 in adult females. These findings may reflect the underlying drivers of maternal body condition. The increase in TT4 may be due to the need for a large reservoir available for conversion to TT3. Adult female fur seals during late spring are typically ending lactation after a long nursing period with dependent pups. Adult females with better body condition are likely meeting metabolic demands through intermittent foraging. Adult females with decreased body condition likely have yet to build body reserves, which may contribute to lower mass and decreased metabolism. The effects of diving and intermittent foraging on thyroid hormones is largely unexplored, but elevation of TT3 and reductions in rT3 may reflect the high rates of energy expenditure found in foraging otariids (Costa 1991). Pups had higher concentrations of TT4 than adults but no differences in TT3 or rT3. These results are consistent with trends in juveniles of most species where


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enhanced metabolism reservoirs in the form of TT4 are needed for pup growth and development when compared to adults. Better body condition in neonate or young pups is likely correlated with greater energy reserves, and therefore, more active thyroid. In a previous study with Australian fur seal (Arctocephalus pusillus doriferus) pups, T4 increased during predictable stressors throughout their first year but were low at the time of weaning (Atkinson et al. 2011). Our study took place at a time when pups were weaning. Thus, younger pups likely have better body condition compared to older pups, as these animals may have nursed more recently. As otariid pups age, they will decrease in body condition as they deplete lipid stores (Jeanniard du Dot et al. 2009). Decreased body condition is also associated with a lower resting metabolic rate in subantarctic fur seal (Arctocephalus tropicalis) pups (Verrier et al. 2012) and thyroid hormone is shown to decrease when lipid stores and mass decrease in Steller sea lion pups while fasting ( Jeanniard du Dot et al. 2009). Similarly with this species, it is likely that older Guadalupe fur seal pups with lower body condition have lower metabolic rates. It is assumed that all pups in this study were approximately 9–10 mo of age due to body size, breeding cycle, and time of year. However, not knowing the exact age of pups in this study and the inability to control for these effects may have resulted in a lack of association between SMI and thyroid in our sample population. Total Integrated Stress Response (TSR) and Impacts on Thyroid We found positive associations between TSR for cortisol and corticosterone and TT3 and negative associations with rT3 in both adult females and pups. One explanation for this association is that lower TSR in response to capture reflects decreased HPA sensitivity to CRH (corticotropin-releasing hormone) and ACTH after sustained stress (Rich and Romero 2005). So, while we are unable to measure baseline cortisol levels, both the suppression of glucocorticoid release in response to stress and suppression of thyroid function through increased rT3 production are consistent with sustained elevated stress in individuals. A study that measured plasma cortisol in adult female subantarctic fur seals found that cortisol increased during onshore nursing periods while losing body mass over a 4 d period (Guinet et al. 2004). Prolonged elevation in glucocorticoid secretion in some individuals may reduce the ability to mount a stress response to capture and alter deiodinase production, downregulating TT3, and increasing in rT3, thus reducing metabolic rate to conserve energy. Baseline cortisol has been found to be positively associated with rT3 in northern elephant seals undergoing stress due to both handling and normal life history fluctuations (Ensminger et al. 2014, Champagne et al. 2015, Jelincic et al. 2017). In contrast to TT3, TSR was not associated with TT4 concentrations. Chronic stress can also reduce TT4 concentrations by suppressing release of thyroid stimulating hormone (Cooper and Ladenson 2011). This effect on TT4 was not evident in GFS suggesting stronger modulation of thyroid function by glucocorticoids through impacts on deiodination. Previous studies in pinnipeds have shown strong associations between cortisol and thyroid concentrations (Ensminger et al. 2014, Champagne et al. 2015, Jelincic et al. 2017). Concentrations of glucocorticoids during the first sample measured after capture did not predict thyroid function (TT4 or TT3), even when controlling for time to sample. TSR for each corticosteroid also greatly varied between individuals, and there was no association between total integrated stress and corticosteroids measured in the first sampling time point. In contrast, TSR measured through

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repeated samples were strongly associated with TT3 and rT3. Together these findings suggest the importance of using a time series of repeated samples rather than a single time point to assess glucocorticoid status after capture and restraint in a pinniped. Significance Physiological responses to stressors are increasingly being used as indicators of both animal and ecosystem health. Anthropogenic stressors can impact the ability of free- ranging pinnipeds to adaptively respond to additional environmental stress (Romero et al. 2009). Studies have also used proxies for fitness, such as the ability to respond to a stressor, to predict which otariid species will be vulnerable in a changing climate or geographic location with increased anthropogenic pressures (Gallagher et al. 2015). As top predators, fur seals associate with areas of high marine productivity and can be sentinel species if there are disruptions to lower trophic levels or changes in ocean patterns, such as El Ni~no-Southern Oscillation (ENSO) events that create warm water and affect prey abundance near their breeding grounds (Costa et al 1991, Crocker et al. 2006, Trillmich and Ono 2012). In past years with similar ENSO events, there is a general trend towards increasing stranding numbers in fur seals and sea lions each year, and malnutrition was a significant cause of stranding (Greig et al. 2005). Our data provide information regarding corticosteroid and thyroid levels during an Unusual Mortality Event that can be used as a comparison to future studies with more normal environmental conditions. Recent studies of GFS at San Benito Archipelago in Mexico during the 2015–2016 ENSO event showed declines in abundance and an increase of isotopic niche area, as a probable result of increase in foraging effort (Elorriaga-Verplancken et al. 2016a). Additionally, during this period, unprecedented records of emaciated GFS from the southern Gulf of California were recorded (Elorriaga-Verplancken et al. 2016b). Recent data suggest that large scale warming anomalies within the California Current ecosystem will likely be persistent (Peterson et al. 2015), and have the potential to impact sea lion and fur seal populations in the future. We collected physiological data during an anomalous year and quantified how these animals respond to acute stress via capture with implications for thyroid function. Conclusion Multiple corticosteroids were released during a stress response in GFS, and aldosterone was likely associated with the response to stress in adults only. These results offer further support of the enhanced regulation of aldosterone secretion by the HPA axis in otariid seals in addition to other marine mammal taxa. Low concentrations of glucocorticoids released due to capture stress were likely indicative of chronic stress and downregulation of the HPA axis. Body condition index had positive relationships with thyroid hormones T4 and rT3 and negative relationships with T3 in adults. This association may have been driven by differences in recent foraging activity and fasting duration. Lower total integrated stress response for both cortisol and corticosterone was associated with decreased production of T3 and increased levels of inactive rT3. Together these findings suggest utility in using endocrine responses to capture stress to evaluate individual and population health.


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ACKNOWLEDGMENTS Research activities with Guadalupe fur seals were performed under the research permit of Dr. Fernando Ellorriaga-Verplancken of Instituto Politecnico Nacional, Baja California Mexico (SEMARNAT SGPA/DGVS/00050/16). Huge gratitude goes to the Mexican Navy for transportation to and housing on Guadalupe Island and CONANP for logistical support during fieldwork. Financial support was from the Office of Naval Research (#N000141410393, to DEC), Sonoma State University, the American Cetacean Society – Monterey Bay, and those that contributed via crowdfunding on Experiment.com Scientific Grants page. Dr. Shawn Johnson of The Marine Mammal Center also provided field equipment for safe capture and anesthesia.

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