Influence of Supraphysiological Cortisol Manipulation on Predator

Integrative Zoology 2018; 13: 206–218 doi: 10.1111/1749-4877.12282

1 ORIGINAL ARTICLE 1 2 2 3 3 4 4 5 5 6 Influence of supraphysiological cortisol manipulation on predator 6 7 7 8 8 9 avoidance behaviors and physiological responses to a predation 9 10 10 11 threat in a wild marine teleost fish* 11 12 12 13 13 14 Michael J. LAWRENCE,1 Erika J. ELIASON,1,2 Jacob W. BROWNSCOMBE,3 Kathleen M. 14 15 4 4,5 1 1 15 16 GILMOUR, John W. MANDELMAN, Lee F.G. GUTOWSKY and Steven J. COOKE 16 17 1Fish Ecology and Conservation Physiology Laboratory, Department of Biology, Carleton University, Ottawa, Ontario, Canada, 17 18 2Department of Ecology, Evolution & Marine Biology, University of California, Santa Barbara, California, USA, 3Department of 18 19 Biology, University of Ottawa, Ottawa, Ontario, Canada, 4School for the Environment, University of New England, Biddeford, 19 20 5 20 Maine, USA John H. Prescott Marine Laboratory, New England Aquarium, Boston, Massachusetts, USA 21 21 22 22 23 Abstract 23 24 The stress axis in teleost fish attempts to maintain internal homeostasis in the face of allostatic loading. Howev- 24 25 er, stress axis induction has been associated with a higher predation rate in fish. To date, the physiological and 25 26 behavioral factors associated with this outcome are poorly understood. The purpose of the present study was to 26 27 investigate the impact of experimental cortisol elevation on anti-predator behavior and physiological responses 27 28 to predator presence. We hypothesized that semi-chronic cortisol elevation would increase susceptibility to pre- 28 29 dation by increasing stress-induced risk-taking behaviors. To test this hypothesis, schoolmaster snapper were 29 30 given cocoa butter implants without cortisol (sham) or with cortisol (50 mg/kg body weight) and tethered to 30 31 cover. Fish were exposed to either a lemon shark or control conditions for 15-min. Space use and activity were 31 32 recorded throughout and fish were terminally sampled for blood. Cortisol implantation, relative to shams, result- 32 33 ed in higher blood glucose and plasma cortisol concentrations with a lower plasma lactate concentration. Shark 33 34 exposure, relative to controls, elicited higher blood glucose and lactate concentrations but had no effect on plas- 34 35 ma cortisol concentration. No interactions were detected between shark exposure and cortisol treatment for any 35 36 physiological trait. Behavioral metrics, including shelter use and activity, were unaffected by either cortisol im- 36 37 plantation or shark exposure. Physiological responses to cortisol implantation likely resulted from enhanced 37 38 gluconeogenic activity, whereas alterations under predator exposure may have been the product of catechol- 38 39 amine mobilization. Further work should address context-specific influences of stress in mediating behavioral 39 40 responses to predation. 40 41 41

42 Key words: homeostatic overload, lemon shark, predation, refuging, stress axis, teleost physiology 42 43 43 44 Correspondence: Michael J. Lawrence, Fish Ecology and 44 45 Conservation Physiology Laboratory, Department of Biology, 45 46 Carleton University, Ottawa, ON K1S 5B6, Canada. INTRODUCTION 46 47 Email: [email protected] 47 48 *M.J.L., E.J.E., J.W.B. and S.J.C. designed the project. The With increasing anthropogenic activities and distur- 48 49 experiments, data collection and analyses were done by M.J.L., bances in the marine environment (e.g. coastal develop- 49 50 E.J.E., K.M.G., L.F.G.G. and S.J.C. All authors contributed to ment, fisheries interactions, noise pollution, water quali- 50 51 the writing and editing of this work. ty degradation, environmental change; Gray 1997; Crain 51

1 et al. 2009), declines in fish populations and alterations evation can result in increases in both routine (Chan 1 2 in community and ecosystem structure and function & Woo 1978; Morgan & Iwama 1996; De Boeck et al. 2 3 have been observed (Hutchings & Baum 2005; Hutch- 2001) and resting metabolic rates (i.e. the standard met- 3 4 ings & Reynolds 2005; Halpern et al. 2007). Anthro- abolic rate; Sloman et al. 2000; O’Connor et al. 2010) 4 5 pogenic disturbances may serve as stressors, perturb- in a teleost fish, potentially leading to energetic trade- 5 6 ing the internal homeostasis of a fish and activating its offs that compromise predator avoidance capacity (Fry 6 7 stress axis (Walker et al. 2005; Busch & Hayward 2009; 1947; Priede 1977; Guderley & Portner 2010; Killen et 7 8 Wright et al. 2011; reviewed in Barton & Iwama 1991, al. 2015). 8 9 Wendelaar Bonga 1997, and Schreck & Tort 2016). The The metabolic consequences of HPI axis activation 9 10 duration and magnitude of these perturbations can have also may be problematic on a behavioral level with re- 10 11 a significant influence on the ability to respond to a -fu spect to predation risk. Foraging behavior and gener- 11 12 ture stressor as well as contributing to the animal’s al- al activity are highly dependent on the energetic status 12 13 lostatic load: the concept incorporating the physiologi- of the animal. Energetically compromised individuals 13 14 cal “costs” of sustained stress axis stimulation (reviewed are more likely to take on a greater burden of predation 14 15 in Korte et al. 2005; Romero et al. 2009). risk (e.g. higher activity and foraging duration) to satis- 15 16 In teleost fish, one arm of the stress axis, the hypo- fy metabolic demands (reviewed in Lima 1998). For ex- 16 17 thalamic–pituitary–interrenal (HPI) axis, regulates the ample, parasitized three-spine stickleback (Gasteros- 17 18 biosynthesis and secretion of cortisol, the primary cor- teus aculeatus) exhibited greater activity levels (Gilles 18 19 ticosteroid (reviewed in Mommsen et al. 1999; Schreck 1987; Godin & Sproul 1988) and quicker behavioral re- 19 20 & Tort 2016). The re-establishment of internal homeo- covery from a predator encounter (e.g. latency to re- 20 21 stasis during a stress response is an energetically de- sume feeding; Giles 1983, 1987; Godin & Sproul 1988), 21 22 manding process (Chan & Woo 1978; Barton & Schreck and foraged within close proximity to a potential preda- 22 23 1987; Sloman et al. 2000; Lankford et al. 2005; O’Con- tor (Milinski 1985; Godin & Sproul 1988). Thus, duress 23 24 nor et al. 2010; Schreck & Tort 2016) and, as such, the (i.e. parasite load) enhanced predation risk in stickle- 24 25 glucocorticoid function of cortisol is important in ini- back (Godin & Spoul 1988), with parasitism likely act- 25 26 tiating an upregulation of energy mobilizing process- ing to increase both cortisol (Ross et al. 2000; Costello 26 27 es. Consequently, increases in plasma cortisol levels are 2002) and metabolic load (Fry 1971). In Atlantic salm- 27 28 often accompanied by an elevation of circulating glu- on (Salmo salar), energetic stress corresponded with 28 29 cose concentrations, thus meeting the enhanced energet- a reduced latency to resume feeding activities follow- 29 30 ic requirements under a stressor (reviewed in Barton & ing a predation event (Gotceitas & Godin 1991), as well 30 31 Iwama 1991; Wendelaar Bonga 1997; Mommsen et al. as foraging at greater distance from cover (Dill & Fras- 31 32 1999; Schreck & Tort 2016). er 1984), suggesting a greater degree of risk-taking be- 32 33 33 While cortisol’s actions are generally considered to havior in stressed individuals. Furthermore, the dura- 34 34 be beneficial to the organism in surviving a stressor tion of time spent in this refuge is highly dependent on a 35 35 (Wendelaar Bonga 1997; Schreck & Tort 2016), stress number of factors, including the animal’s energetic sta- 36 36 axis stimulation can be problematic in other aspects of tus and body condition, with poor body conditions and 37 37 a fish’s life history, including its responses to a pred- increasing hunger levels corresponding with reduced re- 38 38 ator. Teleosts stressed through air exposure, handling, fuging activity (Sih 1992, 1997; Kraus et al. 1998). Giv- 39 39 or exposure to toxicants suffer higher rates of preda- en the role of the stress axis in mediating energy metab- 40 40 tor-induced mortality relative to unstressed counter- olism and budgeting, it is reasonable to hypothesize that 41 41 parts, with effects occurring over varying timescales a stressed teleost fish would accept an elevation of pre- 42 42 and stressor types (Brown et al. 1985; Jarvi 1989; Olla dation risk to optimize energy intake (Sokolova 2013; 43 43 & Davis 1989; Olla et al. 1992, 1995; Mesa et al. 1994, Schreck & Tort 2016; Lawrence et al. 2017). 44 44 1998; Danylchuk et al. 2007). The specific physiologi- The objective of the present study was to investigate 45 45 cal mechanisms associated with the influence of stress the impact of HPI axis activity in modulating the behav- 46 46 on predator–prey dynamics is currently unknown, but a ior and physiology of a teleost fish in response to a pre- 47 47 role for cortisol itself warrants investigation. The met- dation threat. Because the most visible outcome of HPI 48 48 abolic consequences associated with continued HPI axis activation is a rise in circulating cortisol titres, cor- 49 49 axis stimulation could be a contributing factor (Guder- tisol levels were manipulated and the consequences of 50 50 ley & Portner 2010). Specifically, sustained cortisol el- elevated cortisol levels on predator–prey interactions 51 51

1 were investigated. dure of Rypel et al. (2007). Anesthesia was not used on 1 2 these animals in an attempt to minimize handling stress 2 3 MATERIALS AND METHODS as well as to avoid physiological perturbations result- 3 4 ing from anesthesia usage (Wagner & Cooke 2005). An- 4 5 Experimental animals imals were allowed to recover for 24 h in a small mesh 5 6 chamber that was maintained under ambient seawater 6 7 Juvenile schoolmaster snapper (Lutjanus apodus conditions (as above). This 24-h period also allowed for 7 8 Walbaum, 1792; 53.6 ± 2.1 g; N = 57), selected for their cortisol to reach a homeostatic overload state to mim- 8 9 commercial, recreational and ecological importance ic a semi-chronic stressor. All procedures were in ac- 9 10 (Allen 1985), were collected using minnow traps from cordance within the standards of the Canadian Council 10 11 a mangrove nursery habitat (Page Creek, Eleuthera Is- on Animal Care (CCAC) under authorization from Car- 11 12 land, Bahamas; 24°49′04″N, 76°18′51″W) in Novem- leton University’s Animal Care Council (AUP-100612). 12 13 ber and December 2014. Fish were transported to The 13 14 Cape Eleuthera Institute (Eleuthera Island, Bahamas) Behavior trials 14 15 and held in a raceway style tank (519 L) containing sim- Behavioral assessment trials were conducted in a 15 16 ulated cover. Juvenile lemon sharks (Negaprion brevi- large, outdoor circular tank (approximately 6420 L) 16 17 rostris Poey, 1868; 602 ± 11 mm; N = 6) were collect- that was shielded from the elements by a roof. A san- 17 18 ed by seine net from a nearby mangrove system (Kemp’s dy substrate was placed on the bottom of the tank, with 18 19 Creek, Eleuthera Island, Bahamas; 24°48′41.45″N, a trio of conch shells (10-cm spacing) being located 19 20 76°18′16.83″W). Sharks were held in a large, circu- 84-cm from the center of the tank, where a stand pipe 20 21 lar tank (approximately 6420 L) with a sandy substrate. (8.9-cm outer diameter) was located. The tank was di- 21 22 Fish were collected under a scientific collection per- vided in half with a fine mesh seine net. Prior to behav- 22 23 mit provided by the Bahamian Department of Marine ior trials, the tank was maintained with water on a flow- 23 24 Resources. All tanks were supplied with aerated natu- through arrangement using ambient filtered seawater (see 24 25 ral seawater on an overflow system (dissolved oxygen above). Water was allowed to flow through the system 25 26 >85%; temperature 24.5 ± 0.7°C; pH 8.16 ± 0.04; sa- overnight, and water flow was stopped before any ex- 26 27 linity 33.9 ± 0.1 ppt). Both species were maintained on perimental procedures began. 27 a natural photoperiod (13 D: 11 L) and were fed dai- 28 In preparation for behavior trials, a snapper was fitted 28 ly to satiation on chopped sardines. Snapper were fasted 29 with a 1.5-m long tether as described in Lawrence et al. 29 overnight (approximately 16-h) in advance of cortisol 30 (2017). Use of the tether was necessary to complement 30 manipulation and were not fed during the experimental 31 previous stress-predation work that had been carried 31 series (approximately 40-h fasting total). 32 out in this species. The fish was moved to the behavior- 32 33 Fasted snapper were given an intraperitoneal injec al arena (see above) with the tether being secured to the 33 34 tion of hydrocortisone 21-hemisuccinate (50 mg/kg outer conch shell. The fish was allowed to acclimate in 34 35 body mass; N = 30; Sigma-Aldrich, Oakville, ON, Can- the arena for 5-min prior to the experiment. The snapper 35 36 ada) suspended in cocoa butter (5 mL/kg body mass) was then exposed, for 15-min, to 1 of 2 possible scenar- 36 37 warmed to be in liquid form; sham-treated animals (N ios: control conditions or the presence of a lemon shark. 37 38 = 27) received the cocoa butter vehicle alone. The cor- A single lemon shark randomly selected from the pool 38 39 tisol dose was based on that in Cull et al. (2015) for of animals was added to the behavioral arena on the op- 39 40 use in a tropical teleost as well as being a common dos- posite side of the net from the tethered snapper. Sharks 40 41 age used in the teleost literature (reviewed in Gamperl were never in a fasted state during trials to avoid active 41 42 et al. 1994; Mommsen et al. 1999). Fish were fasted to hunting by the animals. The behavioral responses of the 42 43 standardize hunger status in the animals given that hun- snapper were monitored during this time using a Go-Pro 43 44 ger is an important trait regulating risk assessment (Mi- Hero camera (Go-Pro, San Mateo, CA, USA; Struthers 44 45 linski 1993). Because the work occurred at a remote et al. 2015) mounted directly above the tank. 45 46 field site, an a priori validation study could not be con- 46 After the behavior trial, snapper were killed by cere- 47 ducted so we relied on the doses in the literature. At the 47 bral percussion and a blood sample (approximately 200 48 same time, an anchoring point for a tether was made by 48 µL) was withdrawn by caudal venipuncture into a hep- 49 creating a small hole on the lower jaw with a fine su- 49 arinized (Na+ heparin, 10 000 USP units/mL; Sandoz 50 turing needle (1/2 circle, cutting edge, size 14; Integ- 50 Canada, Boucherville, QC, Canada) 1-mL syringe us- 51 ra Miltex, Plainsboro, NJ, USA) according to the proce- 51

1 ing a 23-G needle taking no more than 3 minutes (Law- (N). Blood and plasma parameters as well as activity 1 2 rence et al. 2018). Glucose and lactate concentrations scores were assessed using 2-way analysis of variance 2 3 were measured immediately, and the remaining blood (ANOVA) followed by Tukey’s post-hoc tests when P < 3 4 was centrifuged (2000 g; Mandel Scientific, Guelph, 0.05. Because of the supraphysiological levels of plas- 4 5 ON, Canada) for 1-min. Plasma was decanted, frozen ma cortisol in cortisol-treated fish, Student’s t-tests were 5 6 and stored at −20°C for later analysis of plasma cortisol used to compare cortisol concentrations between corti- 6 7 and ion concentrations. sol-treated and sham-treated fish within an exposure se- 7 8 ries as well as to compare sham-treated fish between 8 Behavioral and blood analyses 9 predator exposure groups. 9 10 Concentrations of blood glucose (Accu-Chek Com- Analysis of the percentage of time spent in cover was 10 11 pact Plus, Hoffman-La Roche, Mississauga, ON, Can- performed within the R statistical environment (R Core 11 12 ada) and lactate (Lactate Plus, Nova Biomedical Can- Development Team 2016). Given the nature of the data, 12 13 ada, Mississauga, ON, Canada) were measured using we used a negative binomial generalized linear model 13 14 medical-grade, hand-held analyzers previously validat- (MASS package, Venables & Ripley 2002) where time 14 15 ed for use in teleost fish (Wells & Pankhurst 1999; Ser- spent in cover (number of seconds + 1) was the response 15 16 ra-Llinares et al. 2012; reviewed in Stoot et al. 2014). variable. Factors included cortisol treatment (cortisol 16 17 Plasma cortisol concentrations were measured using a vs sham), predator treatment (shark present vs control), 17 18 previously validated (Gamperl et al. 1994), commer- and the interaction between cortisol treatment and pred- 18 19 cially available radioimmunoassay kit (ImmunoChem ator treatment. The model was validated after checking 19 20 Cortisol Coated Tube RIA Kit, MP Biomedicals, Solon, the spread of the residuals against each covariate, and 20 21 OH, USA). Intra-assay and inter-assay variation was checking the residuals for overdispersion (i.e. the occur- 21 22 − + 22 3.1% and 1.8%, respectively. Plasma Cl and Na con- rence of more variance in the data than predicted by a 23 centrations were determined using, respectively, a col- statistical model, Bolker et al. 2009). 23 24 orimetric assay (Zall et al. 1956) and flame spectropho- 24 25 25 tometry (Varian Spectra AA 220FS, Varian, Palo Alto, RESULTS 26 CA, USA). The chloride assay was carried out in tripli- 26 27 cate at room temperature (approximately 22°C) using a Blood analyses 27 28 96-well microplate reader (SpectraMax, Molecular De- 28 29 vices, Sunnyvale, CA, USA). Cortisol-treated fish exhibited significantly higher 29 30 Behavioral metrics were collected for the first 10-min plasma cortisol levels than sham fish (Student’s t-tests, 30 31 of exposure to the predator or control conditions, and P < 0.001 and P < 0.001; Fig. 1a). Plasma cortisol con- 31 32 included activity, time spent in cover, time in proximi- centrations were not affected by shark exposure in sh- 32 33 ty to the net and time spent in the open. Activity scores am-treated snapper (Student’s t-test, P = 0.143; Fig. 33 34 were determined using a line crossing analysis employ- 1a). Shark-exposed snapper had higher blood glucose 34 35 ing a 2 × 2 body length (BL) grid overlaid on the video concentrations than fish exposed to control conditions 35 36 recording. A line crossing was defined as a fish’s body (2-way ANOVA, P = 0.041; Fig. 1b). Similarly, corti- 36 37 completely crossing a line in the horizontal axis. The sol-treated fish exhibited significantly higher blood glu- 37 38 animal was considered to be in cover when it was with- cose concentrations than sham-treated fish P( = 0.032; 38 39 in 1 BL of either the outer rim of the conch shell trio or Fig. 1b); there was no interaction between shark expo- 39 40 the standpipe in the center of the tank. Proximity to the sure and cortisol implantation (P = 0.636). Blood lactate 40 41 net was defined to occur when the fish was within 1 BL levels increased in response to shark exposure (2-way 41 42 of the net but not including the 1 BL radius around the ANOVA, P = 0.041; Fig. 1c) and were higher in sh- 42 43 standpipe. Fish not occupying these regions were con- am-treated fish relative to cortisol-treated fishP ( = 0.004; 43 44 sidered to be in the open. Fig. 1c). There was no interaction between shark expo- 44 45 sure and cortisol implantation on blood lactate levels (P 45 46 Statistical analysis = 0.350). Hematocrit and plasma Na+ and Cl− concentra- 46 47 Unless otherwise noted, statistical analysis was car- tions were generally unaffected by either shark exposure 47 48 or cortisol treatment (Table 1), although cortisol-treated 48 ried out using SigmaPlot v11.0 (Systat Software, San − 49 Jose, CA, USA). The statistical limit of significance snapper exhibited significantly higher plasma Cl con- 49 50 was α = 0.05. Values are reported as the mean ± 1 SE centrations relative to shams (Table 1). 50 51 51

1 Activity patterns 1 2 2 3 Schoolmaster snapper activity was similar between 3 4 cortisol-treated and sham animals (2-way ANOVA, P 4 5 = 0.784) and between control and shark exposure (P = 5 6 0.571), with mean activity ranging between 27.5 and 6 7 43.5 line crossings during the 10-min observation period 7 8 (Fig. 2). No significant interaction was detected between 8 9 cortisol treatment and predator exposure (P = 0.450) 9 10 Cover use 10 11 11 12 While variable, snapper were generally found to asso- 12 13 ciate with cover in most instances. There were no statis- 13 14 tically significant effects of either shark exposure or cor- 14 15 tisol treatment on the percentage of time snapper spent 15 16 in cover (P > 0.05 in all cases). However, sham-treat- 16 17 ed snapper exposed to control conditions exhibited the 17 18 lowest median percent use of cover. By contrast, use of 18 19 cover in cortisol-treated animals exposed to control con- 19 20 ditions was comparable to that of predator-exposed ani- 20 21 mals (Fig. 3). 21 22 22 23 23 24 24 25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 35 35 36 36 37 37 38 38 39 39 40 Figure 1 Concentrations of plasma cortisol (a), blood glucose 40 (b) and blood lactate (c) in schoolmaster snapper, 24-h after 41 41 receiving a cocoa butter implant (sham; 5 mL/kg body mass; 42 42 white bars; N ≤ 13) or a cocoa butter implant containing corti- 43 43 sol (50 mg/kg body mass; black bars; N ≤ 12) and in response Figure 2 Activity score of schoolmaster snapper during a 10- 44 44 to exposure to a lemon shark (N = 13) or control conditions min exposure to either a lemon shark (N = 13) or control con- 45 (N ≤ 11). Samples, cortisol notwithstanding (see Methods), ditions (i.e. no predator; N ≤ 11), 24-h after receiving a cocoa 45 46 were analyzed using a 2-way ANOVA and a Tukey post-hoc butter implant (sham; 5 ml/kg body mass; white bars; N ≤ 13) 46 47 test where a significant interaction was detected. Unique let- or a cocoa butter implant containing cortisol (50 mg/kg body 47 48 ters represent statistically significant P( < 0.05) differences be- mass; black bars; N ≤ 13). Samples were analyzed using a 2-way 48 49 tween shark and control exposure groups, whereas asterisks (*; ANOVA. No significant effects of either shark exposure P( = 49 50 P < 0.05) denote statistically significant differences between 0.571) or implant treatment (P = 0.784) were found. Values are 50 51 implant groups. Values are reported as the mean ± SE (N). reported as the mean ± SE (N). 51

1 Table 1 Blood and plasma parameters for schoolmaster snapper implanted with either cortisol (50 mg/kg BW) or vehicle alone (sham) 1 2 and exposed to either a lemon shark or control conditions 2 3 Parameter Exposure 3 4 4 Control Shark 5 5 6 Sham Cortisol-treated Sham Cortisol-treated 6 7 Hematocrit (%) 27.6 ± 2.1 (8) 23.9 ± 1.8 (10) 26.0 ± 1.5 (12) 26.3 ± 0.6 (10) 7 8 Plasma [Na+] (mM) 166.2 ± 11.5 (8) 164.8 ± 7.8 (9) 152.4 ± 6.4 (7) 177.6 ± 4.6 (10) 8 9 − 9 Plasma [Cl ] (mM) 166.0 ± 11.0 (8) 174.1 ± 8.4* (9) 155.6 ± 5.6 (7) 183.8 ± 5.7* (10) 10 10 11 Values are presented as mean ± 1 SE (N), with numbers (N) presented in parentheses. Two-way ANOVA was used to determine sta- 11 tistical differences among treatment groups. No statistically significant effects were detected for hematocrit or plasma [Na+]. For 12 12 plasma [Cl−], implant group P = 0.031, predator P = 0.970, implant x predator P = 0.220 and asterisks (*) represent the statistically 13 13 significant main effect of the implant group. 14 14 15 15 16 16 17 17 18 including a high predation risk coefficient (i.e. lack of 18 19 food for which to forage), the relatively short duration 19 20 of cortisol elevation, the assessment of behavior during 20 21 daylight hours, and a possible disconnect between phys- 21 22 iological function and behavior. Animals appeared to re- 22 23 spond to the threat of predation through a rise in blood 23 24 glucose levels, likely mediated through the actions of 24 25 catecholamines as part of the fight-or-flight response 25 26 (Cannon 1929; Godin 1997). Future work should assess 26 27 how risk-taking behaviors are influenced by cortisol ma- 27 28 nipulation in a more ecologically-relevant setting that 28 Figure 3 Cover use (% of observation period) for cortisol-treat- 29 includes access to food. 29 ed and sham-treated schoolmaster snapper when exposed to a 30 30 shark predator or to control conditions. Dark horizontal lines Physiological validation of implants 31 are the median values. Boxes denote the interquartile range (1st 31 Snapper given cortisol implants had higher concen- 32 to 3rd quartile) with sample sizes. Whiskers are 1.5× the upper 32 33 or lower interquartile range to the highest or lowest value with- trations of blood glucose and plasma cortisol than sham 33 34 in the interquartile range. Outliers are shown as black points fish. The relationship between blood glucose and plas- 34 35 extending beyond the whiskers. ma cortisol concentrations is consistent with cortisol’s 35 36 regulation of gluconeogenic pathways (Vijayan et al. 36 37 2003; Aluru & Vijayan 2007; Choi et al. 2007; Wise- 37 38 man et al. 2007; reviewed in Mommsen et al. 1999, Al- 38 uru & Vijayan 2009; Schreck & Tort 2016). Howev- 39 DISCUSSION 39 40 er, plasma cortisol concentrations were far in excess of 40 41 Overview what has been observed in this species in response to a 41 42 stressor (30-min post-exhaustive exercise), where val- 42 43 It was hypothesized that, as a result of an expect- ues reached approximately 270 ng/mL (Lawrence et al. 43 44 ed increase in metabolic energy expenditure with 2017). Despite using a standardized implantation pro- 44 45 semi-chronic elevation of cortisol concentrations, cor- cedure (Gamperl et al. 1994), cortisol concentrations in 45 46 tisol-treated fish would demonstrate greater risk-taking the plasma of implanted fish were supraphysiological 46 47 behavior. In contrast to this hypothesis, cortisol treat- relative to other species at comparable dosages and time 47 48 ment did not influence snapper behavior patterns, with points, which may be a product of the fish’s environ- 48 49 all animals tending to associate with cover and exhib- ment and/or metabolic tendencies (reviewed in Mom- 49 50 iting consistent activity throughout the experiment. A msen et al. 1999). It should be noted that sham-treated 50 51 number of factors may have contributed to this result, fish also had higher plasma [cortisol]; a baseline cortisol 51

1 titer for this species has been reported at approximately are the primary habitat of juvenile schoolmaster snap- 1 2 67 ng/mL (Lawrence et al. 2017). This elevation likely per during daylight hours, offering shelter from pred- 2 3 reflects handling, tethering and the implantation of the ators (Nagelkerken et al. 2000a,b; Nagelkerken & van 3 4 animals (Wendelaar Bonga 1997; Schreck & Tort 2016). der Velde 2004; MacDonald et al. 2009). Prop root shel- 4 5 This cortisol elevation may also explain why there were tering constitutes 60–70% of their daily spatial use pat- 5 6 no detectable effects between shark-exposed and con- terns, with the animals also spending a significant pro- 6 7 trol-exposed sham fish. portion of their time in areas where overhead cover is 7 8 lacking (MacDonald et al. 2009). In addition, foraging 8 Behavioral responses to a predation threat 9 during daylight hours constitutes only 2% of their to- 9 10 In contrast to the hypothesis, risk-taking behaviors tal activity budget; this species is predominately a noc- 10 11 were not influenced by cortisol treatment but some ev- turnal feeder (Nagelkerken et al. 2000a; MacDonald et 11 12 idence of assessment of predation risk was present. al. 2009). Thus, the conditions of the present study may 12 13 Shark-exposed, sham-treated fish exhibited increased not have been optimal for detecting differences in pred- 13 14 refuge use relative to their control-exposed counter- ator-avoidance behavior in the context of foraging-risk 14 15 parts, although the difference was not statistically sig- management. 15 16 nificant. The apparent lack of cortisol treatment effect Memory may play an important role in mediating 16 17 on snapper behavior may have been a consequence of predator–prey interactions. Stress and cortisol can have 17 18 the duration of the implant. The relatively short dura- a significant influence over cognitive functions in tele- 18 19 tion of elevated cortisol (24 h) may not have had sub- ost fishes, including memory and associated processes 19 20 stantial consequences for the energetic status of the an- (Ellis et al. 2012; Sorensen et al. 2013; Noakes & Jones 20 21 imal. Indication of depleted energy stores with cortisol 2016). Indeed, memory, from both a predator’s hunt- 21 22 implantation has been reported over more chronic dura- ing performance and from a prey’s predator-avoidance 22 23 tions (e.g. hepatosomatic index; Davis et al. 1985; Bar- capacity, is deeply rooted in experience from previous 23 24 ton et al. 1987; Davis et al. 2003). As such, the snap- encounters and can modulate the interaction between 24 25 per here likely had sufficient reserves to draw upon and the two organisms (Mitchell & Lima 2002; Wcisel et 25 26 may not be expected to assume additional predation risk al. 2015). Although not investigated here, cortisol may 26 27 (reviewed in Lima 1998). However, fasting over a 24-h have modulated the cognitive function of the snapper, 27 28 period has been shown to elicit significant changes in affecting memory-related anti-predator responses and 28 29 teleost risk-taking behaviors when exposed to a preda- resulting in altered behavioral dynamics. This possibili- 29 30 tion threat (Godin & Sproul 1988; Gotceitas & Godin ty remains speculative at this time but presents an inter- 30 31 1991), demonstrating that predator–prey interactions, esting avenue for future research. 31 32 in the context of energetic budgeting, is a complex sys- 32 33 tem involving the interaction of a number of physiologi- Physiology and behavior: A complex relationship 33 34 cal processes (Lima & Dill 1990). Furthermore, glucose The lack of behavioral responses to cortisol admin- 34 35 was mobilized in fish when exposed to experimental istration in the present study adds to a growing body of 35 36 cortisol elevation, which suggests either increased syn- literature that has failed to detect direct effects of corti- 36 37 thesis (i.e. gluconeogenesis) or elevated turnover of gly- sol on a range of behaviors (Crossin et al. 2015; Sopin- 37 38 cogen stores (glycogenolysis), which are both metabol- ka et al. 2015). For example, cortisol treatment failed to 38 39 ic consequences of elevated cortisol exposure (reviewed alter the locomotory activity of largemouth bass (Micro- 39 40 in Mommsen et al. 1999). As risk represents an interac- pterus salmoides; O’Connor et al. 2010) and creek chub 40 41 tion of a number of endogenous (i.e. physiological state) (Semotilus atromaculatus; Nagrodiski et al. 2012). Sim- 41 42 and exogenous (e.g. predation threat, food availability ilarly, anti-predator behaviors in checkered pufferfish 42 43 and cover) factors (Lima & Dill 1990), outcomes in risk (Sphoeroides testudineus) were not influenced by cor- 43 44 taking and the associated behaviors become difficult to tisol treatment, despite significant physiological effects 44 45 predict. As such, snapper could be behaving in a manner (Cull et al. 2015; Pleizier et al. 2015). These observa- 45 46 that minimizes risk while maximizing fitness under its tions suggest that the interaction between physiology 46 47 current set of conditions. and behavior is inherently complex and likely requires 47 48 The activity patterns of snapper may also have played a number of physiological inputs other than just plas- 48 49 a significant role in determining its behavioral responses ma cortisol concentrations to induce a change (Crossin 49 50 in the present study. The prop roots of mangrove trees et al. 2015; Sopinka et al. 2015). It is also possible that 50 51 51

1 cortisol may not play a direct role in mediating preda- a high-risk situation deterring movement outside the ref- 1 2 tor–prey interactions in wild fish, although the current uge (i.e. no food present coupled with the animal having 2 3 body of literature does suggest a role for the stress axis sufficient energy reserves) in addition to the fact that, 3 4 at large in mediating these responses (reviewed in Mesa during daylight hours, this species usually remains un- 4 5 et al. 1994). der cover. Despite the absence of significant effects in 5 6 the present study, stress is believed to be an important 6 7 Physiological responses to a predation threat and highly relevant factor in mediating behavioral, pop- 7 8 Acute predation stress in teleosts has been associat- ulation and ecological level effects in wild fish (Hawlena 8 9 ed with increases in circulating glucocorticoids (Rehn- & Schmitz 2010; Boonstra 2013). Indeed, the ecology of 9 10 berg et al. 1987; Woodley & Peterson 2003; Remage- stress is becoming ever more relevant in today’s world 10 11 Healey et al. 2006; Barcellos et al. 2007; Schreck & where anthropogenic activities may enhance both the 11 12 Tort 2016), blood [glucose] (Rehnberg and Schreck frequency and magnitude of stressful events in aquatic 12 13 1987; Jarvi 1990) and tissue-specific heat shock pro- systems (Boonstra 2013; Crespi et al. 2013; Wingfield 13 14 teins (Kagawa et al. 1999), in addition to an elevation 2013). Given the potential importance of stress in medi- 14 15 in cardiorespiratory variables (e.g. heart rate, ventila- ating predator–prey interactions, further work on the re- 15 16 tion, cardiac output; Holopainen et al. 1997; Cooke et lationship between stress and predator–prey interactions 16 17 al. 2003; Sundstrom et al. 2005; Sunardi et al. 2007). is warranted (Schreck et al. 1997; Guderley & Portner 17 18 These physiological responses support the animal’s en- 2010; Hawlena & Schmitz 2010; Lawrence et al. 2017). 18 19 ergetic and locomotory needs as it flees from a predator 19 20 (Wendelaar Bonga 1997; Wingfield 2003; Hawlena & ACKNOWLEDGMENTS 20 21 Schmitz 2010; Schreck & Tort 2016). In schoolmaster 21 22 snapper, blood [glucose] was significantly increased in M.J. Lawrence is supported by an NSERC PGS-D. 22 23 response to shark exposure. Because sham-treated fish S.J. Cooke is supported by NSERC and the Canada Re- 23 search Chairs Program. E.J. Eliason was supported by 24 had no change in plasma cortisol in response to shark 24 an NSERC PDF. J.W. Brownscombe is supported by 25 exposure, the glucose response likely was mediated by 25 NSERC and The Berkeley Marine Conservation Fel- 26 the actions of catecholamine hormones rather than cor- 26 lowship from The American Fisheries Society. K.M. 27 tisol. In most vertebrates, catecholamines act as the pri- 27 Gilmour is supported by NSERC. J.W. Mandelman is 28 mary hormone in mediating acute anti-predator respons- 28 supported by the New England Aquarium. The authors 29 es (Cannon 1929; Hawlena & Schmitz 2010; Perry & 29 would like to thank the Cape Eleuthera Institute, Edd 30 Capaldo 2011). 30 31 Brooks and Zach Zuckerman for the support and re- 31 Blood lactate levels increased in response to a pred- sources to make this project possible. The authors would 32 ator. However, the change in blood lactate was quite 32 33 also like to thank Jean-Guy J. Godin for input on behav- 33 small and may have been associated with transient hy- ioral metrics and analyses used in this study. In addition, 34 poxia generated through a freeze response: a behavioral 34 35 the authors would like to thank Petra Szekeres for assis- 35 adaptation that induces bradycardia and reduced venti- tance in collecting the fish used in this project. 36 lation to lessen the prey’s conspicuousness to a predator 36 37 (Cooke et al. 2003; Shingles et al. 2005). 37 38 REFERENCES 38 39 39 Allen GR (1985). FAO species catalogue vol. 6 snappers 40 CONCLUSIONS 40 of the world: An annotated and illustrated catalogue 41 41 Cortisol implantation in schoolmaster snapper was of Lutjanid species known to date. Food and Agricul- 42 42 sufficient to elevate plasma [cortisol] to supraphysiolog- ture Organization of the United Nations, Rome. 43 ical levels. This effect corresponded with higher blood 43 Aluru N, Vijayan MM (2007). 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