Manuscript Click here to download Manuscript Rosen et al diving review REVISED.docx Click here to view linked References 1 2 3 4 5 6 Physiological constraints and energetic costs of diving behaviour in marine 7 mammals: a review of studies using trained Steller sea lions diving in the open 8 ocean 9 10 11 12 David A.S. Rosen*, Allyson G. Hindle, Carling D. Gerlinsky, Elizabeth Goundie, Gordon 13 D. Hastie, Beth L. Volpov, Andrew W. Trites 14 15 16 Corresponding author: Marine Mammal Research Unit, Institute for the Oceans and 17 Fisheries, University of British Columbia, 2202 Main Mall, Vancouver, BC, Canada, 18 V6T 1Z4 19 20 rosen@zoology.ubc.ca 21 22 Ph: 604-822-8181 23 FAX: 604-822-8182 24 25 Keywords: diving physiology, Steller sea lions, metabolism, foraging 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 Diving metabolism of Steller sea lions 1 64 65 1 2 3 4 1 Abstract 5 6 7 8 2 Marine mammals are characterised as having physiological specializations that 9 10 3 maximize use of oxygen stores to prolong time spent under water. However, it has 11 12 13 4 been difficult to undertake controlled studies to determine the physiological 14 15 5 limitations and trade-offs that marine mammals face while diving in the wild under 16 17 18 6 varying environmental and nutritional conditions. For the past decade, Steller sea 19 20 7 lions (Eumetopias jubatus) trained to swim and dive in the open ocean away from 21 22 23 8 the physical confines of pools participated in studies that investigated the 24 25 9 interactions between diving behaviour, energetic costs, physiological constraints, 26 27 28 10 and prey availability. Many of these studies measured the costs of diving to 29 30 11 understand how they vary with behaviour and environmental and physiological 31 32 33 12 conditions. Collectively, these studies show that the type of diving (dive bouts or 34 35 13 single dives), the level of underwater activity, the depth and duration of dives, and 36 37 38 14 the nutritional status and physical condition of the animal affect the cost of diving 39 40 15 and foraging. They show that dive depth, dive and surface duration, and the type of 41 42 43 16 dive result in physiological adjustments (heart rate, gas exchange) that may be 44 45 17 independent of energy expenditure. They also demonstrate that changes in prey 46 47 48 18 abundance and nutritional status causes sea lions to alter the balance between time 49 50 19 spent at the surface acquiring oxygen (and offloading CO2 and other metabolic by- 51 52 53 20 products) and time spent at depth acquiring prey. These new insights into the 54 55 21 physiological basis of diving behaviour furthers understanding of the potential 56 57 58 22 scope for behavioural responses of marine mammals to environmental changes, the 59 60 61 62 63 Diving physiology of Steller sea lions 2 64 65 1 2 3 4 23 energetic consequences of these adjustments, and the consequences of approaching 5 6 7 24 physiological limits. 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 Diving metabolism of Steller sea lions 3 64 65 1 2 3 4 25 The need to study diving metabolism 5 6 7 8 26 Marine mammals are well known for being able to remain submerged for extended 9 10 27 durations. Early studies of marine mammals (mainly phocid or “true” seals) 11 12 13 28 investigated the anatomical features by which they managed to do so. These include 14 15 29 adaptations for withstanding the intense pressures experienced at depth and 16 17 18 30 greater relative on-board oxygen stores than their terrestrial counterparts, which 19 20 31 allows them to remain active during submergence breath-holding. For example, 21 22 23 32 elevated oxygen storage is present in both circulating haemoglobin and the 24 25 33 myoglobin of their muscle tissues (Kooyman 1985). 26 27 28 29 34 Marine mammals also possess a series of physiological specializations that allow 30 31 35 them to maximize the use of these oxygen stores and prolong their time submerged 32 33 34 36 (Davis and Williams 2012; Andersen 1966; Butler and Jones 1997). These are 35 36 37 traditionally grouped into a set of physiological responses known as the “dive 37 38 39 38 response”, comprising apnea, peripheral vasoconstriction, and bradycardia. In the 40 41 39 face of low blood oxygen pressure (PO2), apnea maximizes the use of oxygen stores 42 43 44 40 by delaying the impulse to return to the surface to breathe. The reduction of blood 45 46 41 flow to “non-essential” tissues limits their local rates of oxygen consumption (and 47 48 49 42 may also assist in myoglobin store utlization within constricted muscle groups; 50 51 43 Davis et al. 2004). Bradycardia is required to maintain central blood pressure in 52 53 54 44 light of these adjustments to peripheral blood flow. These physiological and 55 56 45 anatomical adaptions are widespread among marine mammals — although not 57 58 59 46 always to the same degree (Mottishaw et al. 1999; Davis 2014). 60 61 62 63 Diving metabolism of Steller sea lions 4 64 65 1 2 3 4 47 The common response of reduced heart rate and restricted blood flow observed in 5 6 7 48 early studies of diving physiology suggested that energy expenditure (i.e., 8 9 49 metabolism) while diving was low — certainly lower than for active terrestrial 10 11 12 50 mammals. The diving metabolic rate (DMR) has been directly measured in a few 13 14 51 pinniped species, but rarely under conditions that approach natural diving in the 15 16 17 52 wild, and often under conditions that are euphemistically characterized as “forced 18 19 53 diving” (e.g., Irving et al. 1935; Scholander et al. 1942). Although forced diving 20 21 22 54 experiments provided novel and useful insight into marine mammal diving 23 24 55 physiology, they likely represented an extreme example of the dive response that 25 26 27 56 does not occur frequently in nature (in fact, some have referred to these 28 29 57 experiments as demonstrations of the “fear response”). We now appreciate that the 30 31 32 58 dive response is not an automatic reflex, but rather a graded adjustment that is 33 34 59 under some degree of voluntary control (Butler 1988; Butler and Jones 1997). 35 36 37 38 60 Quantifying the metabolism of marine mammals while diving is important beyond 39 40 61 the realm of comparative physiology. The rate at which oxygen stores are depleted, 41 42 43 62 together with the extent of those stores, allows of an animal’s aerobic dive limit 44 45 63 (ADL) to be calculated. The ADL is the maximum time that an animal can remain 46 47 48 64 submerged relying solely on aerobic metabolism (Kooyman et al. 1983). Although 49 50 65 the transition between aerobic and anaerobic metabolism during diving is likely not 51 52 53 66 a distinct switch, the ADL is a critical comparative parameter for investigating the 54 55 67 ecological physiology and behaviour of marine mammals. It not only defines the 56 57 58 68 physiological constraints and costs of different foraging patterns, but also is integral 59 60 61 62 63 Diving metabolism of Steller sea lions 5 64 65 1 2 3 4 69 to formulating optimal foraging strategies (Carbone and Houston 1996; Houston 5 6 7 70 and Carbone 1992; Thompson and Fedak 2001). 8 9 10 71 In theory, determining the diving metabolism of a marine mammal is relatively 11 12 13 72 straightforward; it can be estimated from the rate of oxygen consumption (V O2) and 14 15 73 carbon dioxide production (V CO2) over a dive event using standard techniques of 16 17 18 74 flow-through respirometry. This entails having the animal breathe into a semi- 19 20 75 enclosed container with a constant flow-through airstream upon returning to the 21 22 23 76 surface after completing a dive. Consequently, the V O 2 and V C O 2 during the dive are 24 25 77 inferred from data obtained during the post-dive surface interval (Hastie et al. 26 27 28 78 2006a; Reed et al. 1994; Reed et al. 2000; Scholander 1940). 29 30 31 79 However, actual measures of diving metabolism are sparse. This is partly because it 32 33 34 80 is highly challenging to measure in wild animals — a notable exception being 35 36 81 Weddell seals (Leptonychotes weddellii) that reliably surface in ice holes (Kooyman 37 38 39 82 et al. 1980). It is equally logistically difficult to obtain these measures from trained 40 41 83 animals in aquariums and similar facilities, given that pool depths cannot 42 43 44 84 accommodate natural diving behaviour. While some novel tests have been 45 46 85 undertaken measuring the metabolism of harbour seals (Phoca vitulina) swimming 47 48 49 86 for extended linear distances in a pool, it is questionable whether this invokes the 50 51 87 same physiological responses as diving to depth (Gallon et al. 2007; Sparling et al. 52 53 54 88 2007b). In particular, the effect that pressure has on the respiratory system may 55 56 89 significantly alter physiology, including how gases are managed (Scholander 1940; 57 58 59 90 McDonald and Ponganis 2013; Fahlman et al. 2009). Further (as discussed later), 60 61 62 63 Diving metabolism of Steller sea lions 6 64 65 1 2 3 4 91 once these operational impediments have been solved, the actual calculation of 5 6 7 92 metabolic rate while diving from gas exchange data is not as straightforward as it 8 9 93 would first appear and there are still varying opinions on how to experimentally 10 11 12 94 estimate the actual energy cost. 13 14 15 95 To attempt to address this paucity of data, we undertook a risky venture: we 16 17 18 96 decided to study the diving metabolism of trained Steller sea lions (Eumetopias 19 20 97 jubatus), away from the confines—and security—of a traditional laboratory or 21 22 23 98 aquarium setting. Sea lions (Otariids) had, of course, been trained to operate freely 24 25 99 in the open ocean before. However, these were California sea lions (Zalophus 26 27 28 100 californianus), and most endeavours had been undertaken by military organizations 29 30 101 for security rather than research purposes (although a group at Moss Landing 31 32 33 102 started a research program in 2005). In 2004, we began relocating trained female 34 35 103 Steller sea lions from the Vancouver Aquarium (where they had been raised since 36 37 38 104 pups) to the University of British Columbia’s Open Water Research Station, based at 39 40 105 a marina in Port Moody, British Columbia. The concept was to bridge the gap 41 42 43 106 between the types of largely uncontrolled studies that could be conducted with wild 44 45 107 animals in their natural environment and controlled experiments conducted with 46 47 48 108 trained animals in an artificial (and physically restricted) environment. 49 50 51 109 The Open Water Research Station is unique in that it is the only public research 52 53 54 110 facility that can measure physiological, behavioural, and ecological variables in a 55 56 111 controlled experimental set-up on sea lions diving unrestrained in the open ocean 57 58 59 112 environment. Although under trainer control, the sea lions perform dives to depths 60 61 62 63 Diving metabolism of Steller sea lions 7 64 65 1 2 3 4 113 and for durations similar to wild animals (Merrick and Loughlin 1997), and can 5 6 7 114 make foraging and oxygen management decisions analogous to their wild 8 9 115 counterparts. 10 11 12 13 116 The objectives of the research program were to: 14 15 16 117 1. Measure the energetic cost of diving. 17 18 19 118 2. Investigate the relationship between individual physiology and diving 20 21 119 capacity. 22 23 24 120 3. Investigate the physiological mechanisms that underpin dive behaviour. 25 26 121 4. Test specific optimal foraging theories for diving mammals. 27 28 29 122 5. Develop and test proxies for energy expenditure that could be used to 30 31 123 measure the energy expenditure of diving in wild sea lions. 32 33 34 35 124 It might seem that the first goal — measuring the energetic cost of diving — was the 36 37 125 most straightforward to address; but as we describe, defining and quantifying an 38 39 40 126 animal’s metabolism while submerged is complex. As will become apparent (and 41 42 127 which was clear in hindsight), the actual cost of diving is variable, and can be 43 44 45 128 affected by the intensity and type of subsurface activity, the dive depth, the dive and 46 47 129 surface duration, and the physiological and nutritional state of the individual, all of 48 49 50 130 which are often interacting factors. Hence, the major goal of our program developed 51 52 131 into gleaning an understanding of how these parameters affect the cost (and 53 54 55 132 underlying physiology) of diving, rather than being focused on producing a single, 56 57 133 unrepresentative value for “the” cost of diving. 58 59 60 61 62 63 Diving metabolism of Steller sea lions 8 64 65 1 2 3 4 134 This following review is not a synopsis of the field of diving physiology, as many 5 6 7 135 excellent reviews already exist (e.g., Kooyman 2002; Davis 2014; Butler and Jones 8 9 136 1997). Rather, it attempts to describe the progress and discoveries made in 10 11 12 137 exploring the complex interaction between diving physiology and behaviour of 13 14 138 Steller sea lions. 15 16 17 18 139 Experimental set-up 19 20 21 140 While the experimental manipulations conducted over the last decade have become 22 23 24 141 more complex, the general experimental set-up has changed little from its initial 25 26 142 conception (Hastie et al. 2006b). When not undertaking studies, the sea lions stay in 27 28 29 143 flow-through holding pens with adjacent haul out space at a local marina. They are 30 31 144 trained each day by professional husbandry staff from the Vancouver Aquarium 32 33 34 145 using positive reinforcement. The sea lions are usually maintained on an ad libitum 35 36 146 diet, composed primarily of herring (Clupea pallasii), and supplemented with 37 38 39 147 vitamins and other prey species. All husbandry and experimental protocols are 40 41 148 approved by Animal Care Committees of the University of British Columbia and the 42 43 44 149 Vancouver Aquarium. It is important to note that all research conducted at the Open 45 46 150 Water Research Station is done voluntarily by the sea lions, and they can end a dive 47 48 49 151 trial at any point they choose. 50 51 52 152 The sea lions are weighed each morning on a platform scale prior to their first meal. 53 54 55 153 When working in the open ocean they are outfitted with a custom, tight-fitting 56 57 154 harness that makes the animal more visible underwater. The harness holds a VHF 58 59 60 155 transmitter in case the animal wanders from the study area, and also permits the 61 62 63 Diving metabolism of Steller sea lions 9 64 65 1 2 3 4 156 temporary attachment of additional experimental equipment, including time depth 5 6 7 157 recorders, accelerometers, heart rate monitors, etc. 8 9 10 158 The actual experimental trials are undertaken in nearby, deep-water fjords that 11 12 13 159 provide the necessary water depth and isolation. Sea lions are transported to these 14 15 160 study sites on board a modified transport boat. A secondary research boat carries 16 17 18 161 scientific equipment, personnel, and a research platform to the selected dive site 19 20 162 (Fig. 1 and 2). The 6.0 x 3.1 m platform contains a 150 L floating respirometry dome 21 22 23 163 in the centre that is attached to flow generators and O2 and CO2 analysers that 24 25 164 continuously feed data into a laptop computer. Below the respirometry dome is a 26 27 28 165 nylon enclosure with a trap door that assists to behaviourally contain (but not 29 30 166 forcibly confine) the sea lions for extended periods at the surface. Lengths of PVC 31 32 33 167 feeding tubes are fitted together and lowered off the side of the platform to desired 34 35 168 depths, usually between 10 and 50 m, to make subsurface feeding stations. Water 36 37 38 169 pumps deliver 20 g pieces of fish to depth at selected rates, thereby controlling the 39 40 170 depth (and durations if required) of the sea lions’ dives. Prior to 2007 only a single 41 42 43 171 feeding station was used, but later studies used two tubes (~6 m apart) with 44 45 172 alternating food delivery to stimulate swimming at depth, as well as enable tests for 46 47 48 173 the effects of choices of depth and/or prey field quality (number of fish per minute) 49 50 174 on behaviour and physiology. The set-up allows the trained sea lions to perform 51 52 53 175 dives similar to their wild counterparts; Steller sea lions in the wild generally dive 54 55 176 for less than two minutes and 90% of their dives are shallower than 50 m (Loughlin 56 57 58 177 et al. 2003; Loughlin et al. 1987). 59 60 61 62 63 Diving metabolism of Steller sea lions 10 64 65 1 2 3 4 178 As detailed later, various experimental protocols can be employed to alter the 5 6 7 179 characteristics of a dive depending on the specific scientific goals. For example, dive 8 9 180 durations can be either determined by the sea lions or controlled by the researchers 10 11 12 181 (via either a light cue at the end of the feeding tubes or cessation of fish delivery). 13 14 182 Similarly, inter-dive surface intervals can be determined by the sea lion or 15 16 17 183 controlled by the researchers (using training cues and food reinforcement). Video 18 19 184 cameras at depth and at the surface allow staff to observe the behaviour of the sea 20 21 22 185 lions throughout the trials. 23 24 25 186 The logistics of this experimental set-up (e.g., the high level of training, the time to 26 27 28 187 run a single trial) inherently limits the number of individual sea lions that can be 29 30 188 used in any set of experiments. This, in turn, limits statistical power, including the 31 32 33 189 ability to make inferences about the population as a whole. This can be partly 34 35 190 overcome by appropriate experimental design and use of repeated-measures and 36 37 38 191 mixed-effect model statistics (Zuur et al. 2009). However, the fact that many of the 39 40 192 studies use the same group of individuals adds to our ability to compare results 41 42 43 193 among studies, and therefore derive conclusions regarding the physiological and 44 45 194 behavioural effects of different experimental conditions. 46 47 48 49 195 Finally, despite its ground-breaking design, we recognize the inherent limitations of 50 51 196 the experimental set-up. As these are trained sea lions kept under human care, their 52 53 54 197 behaviour and life history are not identical to their wild counterparts, and certain 55 56 198 aspects of their physiology may also differ. For example, the study animals are free 57 58 59 199 of disease and injury, are non-breeding, and while they are accustomed to regular 60 61 62 63 Diving metabolism of Steller sea lions 11 64 65 1 2 3 4 200 diving and swimming in the open ocean it is not on the same schedule as wild sea 5 6 7 201 lions (although see Gerlinsky et al. 2013). It is also worth noting that, at the end of 8 9 202 the day, our sea lions do not have to forage to survive. While the open water design 10 11 12 203 differs from the “elongated pool” protocol (e.g., Sparling and Fedak 2004) by 13 14 204 incorporating the effects of depth and pressure, it is limited in the amount of activity 15 16 17 205 (number of dives) the animals will perform while still reliably collecting 18 19 206 respirometry data (i.e., returning to the dome). While using trained animals under 20 21 22 207 controlled conditions permits testing a range of specific physiological, behavioural, 23 24 208 and environmental effects, this set-up cannot reproduce completely “natural” 25 26 27 209 behaviours that can be observed through measuring the energy expenditure of wild 28 29 210 pinnipeds (Kooyman et al. 1980; Iverson et al. 2010). As with all manipulative 30 31 32 211 experiments, studies are designed and interpreted with these considerations in 33 34 212 mind. 35 36 37 38 213 Calculating the cost of diving 39 40 41 214 On first consideration, it would not seem overly complicated to calculate an animal’s 42 43 44 215 metabolism while diving given accurate measures of O2 consumption and CO2 45 46 216 production. However, one difficulty is that, unlike terrestrial mammals, the gas 47 48 49 217 exchange related to physical activity is temporally removed from the measurement 50 51 218 of that consumption. This is because, in diving mammals, the actual O2 consumption 52 53 54 219 occurs at depth away from the respirometry dome, and can only be replenished 55 56 220 (and CO2 off-loaded) after the dive has been completed. 57 58 59 60 61 62 63 Diving metabolism of Steller sea lions 12 64 65 1 2 3 4 221 The most common measure of metabolism while submerged is diving metabolic rate 5 6 7 222 (DMR). For single dives, DMR can be quantified as the excess (above resting levels) 8 9 223 volume of O2 consumed (VO2) and CO2 expelled (VCO2) that occurs post-dive, which 10 11 12 224 is measured until the point when the animal has returned to its resting state. The 13 14 225 definition of “resting state” is, in itself, open to interpretation. For practical and 15 16 17 226 scientific reasons, we use the stable rate of oxygen consumption (V O2) while the sea 18 19 227 lions are resting, inactive at the water’s surface prior to any diving activity, and with 20 21 22 228 minimal food reinforcement (to minimize the potential effect of the heat increment 23 24 229 of feeding; HIF) as our “baseline” measure. This may or may not coincide with 25 26 27 230 classically defined resting metabolic rate, and so we adopted the nomenclature of 28 29 231 “metabolic rate at the surface” (MRs, Fig. 3A) (Fahlman et al. 2008b). 30 31 32 33 232 This additional post-dive V O2 and V CO2 above MRs levels are thought to represent 34 35 233 the aerobic metabolism during the dive. The VO2 consumed during the submerged 36 37 38 234 portion of the dive can therefore be estimated by subtracting the post-dive increase 39 40 235 in V O 2 from the MRs. The total energetic cost of a dive can then be calculated as the 41 42 43 236 total additional oxygen consumed, while DMR can be calculated by dividing the total 44 45 237 cost by the submergence time (Fig. 3A). 46 47 48 49 238 It is vitally important that the animal has sufficient time to completely recover 50 51 239 between dives, or the true DMR will be underestimated by failing to capture all post- 52 53 54 240 dive excess V O 2. In theory, pre-dive MRs and post-dive MRs should be identical. 55 56 241 However, in a subset of our studies, we found the pre-dive MRs was elevated in 57 58 59 242 relation to post-dive MRs. This may be due to natural physiological variation, or it 60 61 62 63 Diving metabolism of Steller sea lions 13 64 65 1 2 3 4 243 may be due to behavioural and physiological anticipation of diving causing an 5 6 7 244 elevation in pre-dive MRs. 8 9 10 245 The importance of an animal returning to steady state is a vital consideration when 11 12 13 246 measuring DMR of sea lions performing dive bouts, as they often do in the wild. Dive 14 15 247 bouts consist of multiple dives interspersed with relatively brief inter-dive surface 16 17 18 248 intervals, followed by an extended post-dive surface recovery period at the end of 19 20 249 the bout. The inter-dive surface intervals are too short for complete gas exchange 21 22 23 250 (i.e., animals do not return to resting state and carry over some level of oxygen debt). 24 25 251 As a result, the V O 2 must be determined over the entire dive bout (Boutilier et al. 26 27 28 252 2001). In fact, a dive bout (in contrast to single dives) could be operationally 29 30 253 defined as a series of dives with incomplete physiological recovery in between. 31 32 33 254 Hence, DMR for dive bouts is calculated from the total additional V O 2 during both 34 35 255 the brief inter-dive surface intervals and the final post-dive recovery, divided by the 36 37 38 256 total subsurface time. As each dive in the bout is not physiologically independent, 39 40 257 only a single estimate of DMR, calculated across all dives in the bout, can be 41 42 43 258 obtained (thereby inherently limiting statistical power). 44 45 46 259 While DMR may seem a logical way to measure the cost of diving, it makes the 47 48 49 260 assumption that the dive and the time at the surface (both inter-dive surface 50 51 261 intervals and post-dive recovery periods) are independent events. In other words, it 52 53 54 262 assumes that the post-dive metabolic rate is simply the sum of the direct cost of the 55 56 263 submerged portion of the dive cycle and resting surface rate. Evidence suggests that 57 58 59 264 this is not physiologically accurate (Fedak 1988; Kooyman and Ponganis 1998; Reed 60 61 62 63 Diving metabolism of Steller sea lions 14 64 65 1 2 3 4 265 et al. 1994). This has led to the calculation of an alternate measure, often referred to 5 6 7 266 as “average metabolic rate” (AMR). For AMR, the total volume of oxygen consumed 8 9 267 is averaged over the entire dive cycle (submergence and inter-dive and post-dive 10 11 12 268 surface times). Unlike DMR calculations, it does not attempt to separate the “surface 13 14 269 resting” and “diving” portions of oxygen consumption measured during the post- 15 16 17 270 dive surface intervals. Rather, AMR entails integrating the total volume of oxygen 18 19 271 consumed and converting it to a rate by dividing over the length of the dive cycle. 20 21 22 272 The dive cycle is the time from the start of the first dive to the end of the recovery 23 24 273 period (when rates of oxygen consumption return to baseline levels; Fig 3B). AMR 25 26 27 274 can be calculated for both single dives and dive bouts although, as with DMR, only a 28 29 275 single value can be calculated for a single dive bout. 30 31 32 33 276 To clarify, DMR and AMR are (in our usage) alternate means of calculating the rate 34 35 277 of oxygen consumption during either single dives or a mean value over a dive bout 36 37 38 278 consisting of series of submergences. There are pros and cons to estimating the 39 40 279 energetic cost of diving as either DMR or AMR. DMR is theoretically a more “direct” 41 42 43 280 cost of the actual submergence time, but makes the questionable assumption that 44 45 281 post-dive rates of oxygen consumption are simply the sum of the exertion of the 46 47 48 282 previous activity and the costs of resting at the surface. DMR is also more dependent 49 50 283 on the measure of MRs; if MRs is elevated, DMR will be underestimated. In 51 52 53 284 comparison, AMR does not make the assumption of physiological independent 54 55 285 between post-dive MRs and the costs of previous physical exercise. It is not as 56 57 58 286 susceptible to errors induced by elevated pre-dive MRs as it is calculated on the 59 60 287 total observed rates of oxygen consumption. However, it is highly susceptible to 61 62 63 Diving metabolism of Steller sea lions 15 64 65 1 2 3 4 288 errors induced by inaccurate estimates of recovery time. As AMR is based on the 5 6 7 289 total oxygen consumption over the entire dive cycle, miscalculations in the recovery 8 9 290 period will proportionally affect the measure (e.g., incorrectly long recovery times 10 11 12 291 will proportionally decrease the apparent AMR by increasing the denominator). 13 14 15 292 It is also important to note that the estimated recovery time is a useful physiological 16 17 18 293 measure in its own right. As later discussed, it can provide valuable information on 19 20 294 post-dive gas exchange rates and can be used to examine the effect of diving 21 22 23 295 duration on foraging efficiency over a complete dive cycle. Finally, when comparing 24 25 296 published studies, it is important to note the specific formula used to calculate 26 27 28 297 diving metabolism, as the terms DMR and AMR have been variously used to denote a 29 30 298 variety of calculations. For clarity in this review, DMR will always refer to the rate of 31 32 33 299 oxygen consumption calculated for the submerged portion of the dive and AMR will 34 35 300 refer to the average metabolic rate over the entire dive cycle. 36 37 38 39 301 Evidence for hypometabolism while submerged 40 41 42 302 One of the first studies conducted at the Open Water Research Station using two 43 44 45 303 Steller sea lions appeared to confirm the general marine mammal model that 46 47 304 metabolism was depressed during diving (Hastie et al. 2007). This initial study 48 49 50 305 employed a relatively straightforward set-up: the animal undertook single dives, of 51 52 306 a controlled (but variable) duration (60-200 sec), to a submerged target positioned 53 54 55 307 at depth (between 5-30 m). Each trial involved single dives followed by a post-dive 56 57 308 recovery. The results indicated that DMR was only 55-57% of MRs, and neither dive 58 59 60 61 62 63 Diving metabolism of Steller sea lions 16 64 65 1 2 3 4 309 duration nor depth was a significant predictor of DMR in the resulting statistical 5 6 7 310 model. 8 9 10 311 These results provided the first estimates of DMR for Steller sea lions and showed 11 12 13 312 that this species can exhibit a marked decrease in oxygen consumption while 14 15 313 submerged relative to surface rates. These results were similar to those reported for 16 17 18 314 California sea lions (Hurley and Costa 2001), and would appear to support 19 20 315 Scholander’s (1940) original hypothesis that marine mammals reduce their 21 22 23 316 metabolic rate during diving to help increase foraging efficiency. 24 25 26 317 However, there were suggestions that our early results (Hastie et al. 2007) did not 27 28 29 318 accurately reflect the metabolism of sea lions while diving. One concern was that the 30 31 319 sea lions, despite an extensive preliminary training period, were not completely 32 33 34 320 comfortable with the experimental protocols. Average pre-dive MRs (~11 ml O2 kg-1 35 36 321 min-1) was slightly higher compared to subsequent studies (and measured recovery 37 38 39 322 periods slightly shorter), potentially leading to underestimates of the costs of the 40 41 323 dives (see earlier discussion on potential errors in calculating DMR). On a related 42 43 44 324 note, there was concern that a lack of familiarity with the experimental protocols — 45 46 325 specifically that the animals did not control dive duration — may have induced a 47 48 49 326 “maximum” dive response, similar to (although less dramatic than) the effect 50 51 327 reported for early “forced” diving experiments (Jobsis et al. 2001). 52 53 54 55 328 A more general concern related to the fact that metabolism was not measured under 56 57 329 “realistic” conditions. The dives were relatively short, the sea lions performed only 58 59 60 330 single dives and not dive bouts, and their activity at depth was minimal. As a result, 61 62 63 Diving metabolism of Steller sea lions 17 64 65 1 2 3 4 331 the effect of predictability and control on physiological response, dive duration, 5 6 7 332 activity level, and the differences between single dives and dive bouts were explored 8 9 333 in subsequent studies. 10 11 12 13 334 The effect of dive duration and dive bouts 14 15 16 335 In our first study, the Steller sea lions did not demonstrate a relationship between 17 18 19 336 DMR and dive duration (Hastie et al. 2007); however, the dive durations were only 3 20 21 337 min. In comparison, California sea lions showed larger decreases in DMR with dive 22 23 24 338 durations of up to 7 min (Hurley and Costa 2001). 25 26 27 339 A subsequent study (Fahlman et al. 2008b) used three sea lions (two which were the 28 29 30 340 same as the previous study) in dive trials that consisted of a series of repeated dives 31 32 341 (2–15 dives) to a single feeding station at depths of 10-50 m. For dives where the 33 34 35 342 sea lions chose both dive and inter-dive surface interval durations, DMR decreased 36 37 343 exponentially with increasing dive duration. For dives longer than 1 min, DMR 38 39 40 344 reached levels ~8% lower than MRs. 41 42 43 345 A follow-up study also investigated the potential metabolic differences between 44 45 46 346 single dives and dive bouts in a trial that employed dive bouts composed of a set of 47 48 347 up to 5 individual dives (Hastie et al. 2006a). Three sea lions dove to depths (5-30 49 50 51 348 m) with controlled dive times (40-200 sec) similar to the earlier study that only 52 53 349 measured metabolism over single dives (Hastie et al. 2007). However, in these trials, 54 55 56 350 the inter-dive surface intervals between individual dives in the bout were limited 57 58 351 (7-25 sec) in order to simulate more natural dive bouts. Variation in dive duration 59 60 61 62 63 Diving metabolism of Steller sea lions 18 64 65 1 2 3 4 352 and inter-dive surface intervals meant that the proportion of the dive cycle spent 5 6 7 353 submerged ranged between 23% and 88% (average = 75%). While the study 8 9 354 determined that diving metabolic rate was affected by dive activity (discussed later), 10 11 12 355 it also determined that DMR decreased curvilinearly with dive depth once activity 13 14 356 was taken into account. This relationship was not observed in the previous study 15 16 17 357 that only employed single dives (Hastie et al. 2007). 18 19 20 358 It should not be surprising that single dives and dive bouts have different cost 21 22 23 359 characteristics, given they have different gas exchange dynamics. Inter-dive surface 24 25 360 intervals during a dive bout represent “recovery” time in which sea lions can off- 26 27 28 361 load CO2 and other metabolites from exercising tissues, and replenish on-board O2 29 30 362 stores. Oxygen management during extended dives is complicated, involving 31 32 33 363 peripheral, arterial, venous, and pulmonary circulatory adjustments (see McDonald 34 35 364 and Ponganis 2013). It is unclear the degree to which diving mammals normally 36 37 38 365 replenish their O2 stores between dives in a bout, although a law of diminishing 39 40 366 returns for oxygen uptake across a decreasing partial pressure gradient would 41 42 43 367 suggest that it might be optimal for a sea lion to maintain some level of constant 44 45 368 oxygen debt through a dive bout (Boutilier et al. 2001). Dive bouts, by definition, are 46 47 48 369 characterized by inter-dive surface intervals that limit the extent of gas exchange 49 50 370 and theoretically affect both dive behaviour and metabolic costs of subsequent dives. 51 52 53 371 Therefore, the next set of trials specifically examined the effect of differing surface 54 55 372 intervals on subsequent dive durations and their associated costs. 56 57 58 59 60 61 62 63 Diving metabolism of Steller sea lions 19 64 65 1 2 3 4 373 While these initial studies (Hastie et al. 2007, 2006a) highlighted the differences in 5 6 7 374 cost between single dives and more natural dive bouts, a critical detail of the 8 9 375 methodology was that both the dive durations and inter-dive surface intervals (for 10 11 12 376 dive bouts) were controlled by the researchers and not the animals. A lack of control 13 14 377 over dive durations may mean that the animals were responding to an uncertainty 15 16 17 378 in submergence times by altering their physiological response compared to “natural” 18 19 379 diving in order to maximize their potential submergence time. Hence, in the 20 21 22 380 following study, the sea lions were allowed to control their dive duration. 23 24 25 381 The dive trials with the three sea lions in the subsequent study (Fahlman et al. 26 27 28 382 2008b) consisted of either a single dive or a bout of repeated dives (2–15 dives) to a 29 30 383 single feeding station at depths of 10-50 m. Two different diving protocols were 31 32 33 384 used: ‘spontaneous’ dives where the trainers exerted no control over dive and inter- 34 35 385 dive surface interval durations and ‘conditioned’ dives where the sea lions 36 37 38 386 controlled the dive duration but were held at the surface between dives until the 39 40 387 instantaneous rate of oxygen consumption returned to baseline levels. The 41 42 43 388 conditioned dive bouts had imposed inter-dive surface intervals (1-8 min) such that 44 45 389 they were, effectively, more similar to a series of single dives with near-complete 46 47 48 390 gas exchange recovery between dives. In contrast, the inter-dive surface intervals 49 50 391 (5-60 sec) were much shorter for the spontaneous dives such that they were 51 52 53 392 equivalent to dive bouts in previous studies. 54 55 56 393 Overall, average DMR for all animals was 1.65±0.66 L O2 min-1, which was lower 57 58 59 394 compared to the previous study (Hastie et al. 2007), but still higher than the average 60 61 62 63 Diving metabolism of Steller sea lions 20 64 65 1 2 3 4 395 MRs of 1.45±0.44 L O2 min-1. The most striking result of this study was that DMR 5 6 7 396 was significantly higher for spontaneous dive events as compared to conditioned 8 9 397 dives (1.74±0.70 vs. 1.57±0.40 L O2 min-1). Part of this difference might attributable 10 11 12 398 to differences in dive duration. The average dive durations during the conditioned 13 14 399 dives was significantly longer than during spontaneous dives (2.28±1.53 min vs. 15 16 17 400 0.97±0.83 min). However, DMR for spontaneous dives decreased exponentially with 18 19 401 increasing dive duration. For dives longer than 1 min, DMR for spontaneous dives 20 21 22 402 was lower than for conditioned dives and, as previously noted, reached levels ~8% 23 24 403 lower than MRs. 25 26 27 28 404 Given the hypothesis that increasing levels of O2 debt are incurred during dive bouts, 29 30 405 this result also suggests that sea lions might alter their rate of oxygen consumption 31 32 33 406 based upon their physiological state (i.e., remaining levels of oxygen stores). This 34 35 407 contention is supported by the observation that the first dive of a series of 36 37 38 408 spontaneous dives was longest and had the lowest apparent V O 2, while the last dive, 39 40 409 which was followed by a long recovery period, had the highest V O 2. These results 41 42 43 410 indicate that the sea lions developed an O2 debt during the first dive, which was not 44 45 411 restored until the sea lion spent considerable time at the surface at the end of the 46 47 48 412 bout. This also demonstrates why DMR cannot be accurately estimated for each 49 50 413 separate dive during a bout. 51 52 53 54 414 Effect of activity on diving metabolism 55 56 57 415 One suggested explanation for the measured hypometabolism in both the Steller sea 58 59 60 416 lions in our first diving study (Hastie et al. 2007) and in a similar study using 61 62 63 Diving metabolism of Steller sea lions 21 64 65 1 2 3 4 417 California sea lions (Hurley and Costa 2001) was that the animals were trained to 5 6 7 418 remain stationary at depth. Given the assumption that active swimming is more 8 9 419 metabolically expensive than remaining at depth (see Diving behaviour), this would 10 11 12 420 suggest that the observed hypometabolism was facilitated by a lack of energy 13 14 421 expenditure due to movement. However, a later study that examined the 15 16 17 422 relationship between activity and diving metabolism extrapolated that — for the 18 19 423 same level of activity — the metabolic rate during diving may be as much as 29% 20 21 22 424 lower than when at the surface between dives (Fahlman et al. 2013). 23 24 25 425 The effect of activity on metabolism while submerged was specifically examined in 26 27 28 426 the previously mentioned study that first incorporated more realistic bouts of 5 29 30 427 dives. The results indicated that DMR was affected by both dive depth and type of 31 32 33 428 activity during the dive (Hastie et al. 2006a). The fact that both factors had a 34 35 429 significant effect on metabolic rate may seem obvious given the presumed cost of 36 37 38 430 active swimming and the fact that total travel time and distance (ascent and 39 40 431 descent) change inherently with dive depth and the number of individual dives in a 41 42 43 432 bout; but the actual results were surprising. As predicted, DMR increased 44 45 433 curvilinearly with increasing total swimming distance, which ranged from 10-300 m 46 47 48 434 during a bout. However, DMR also decreased curvilinearly with maximum dive 49 50 435 depth once travel distance was accounted for, although the physiological cause for 51 52 53 436 this response is unclear (possibly triggered by pressure increases?; Andersen 1966). 54 55 56 437 Overall, distance travelled and dive depth appeared to have significant, but opposing 57 58 59 438 effects on DMR. To put these mathematical relationships in perspective, for dive 60 61 62 63 Diving metabolism of Steller sea lions 22 64 65 1 2 3 4 439 bouts that consisted of a total of 600 m of transit distance, DMR (averaged over all 5 6 7 440 dives in the dive bout) was predicted to be 3.37 L O2 min-1 at 10 m and 2.44 L O2 8 9 441 min-1 at 50 m. Although MRs were not specifically reported, comparison to an 10 11 12 442 average MRs of 1.45±0.44 L O2 min-1 for the same 3 (slightly older and heavier) sea 13 14 443 lions reported in subsequent study (Fahlman et al. 2008b) suggests that DMR during 15 16 17 444 more realistic, deep dive bouts was up to 68% greater than when resting at the 18 19 445 surface. These results provided the first indication that the behaviour of the animals 20 21 22 446 while diving greatly affects the calculated costs of diving. 23 24 25 447 Differences in dive behaviour might also partly explain why the overall metabolic 26 27 28 448 rate was significantly lower for conditioned dives than spontaneous dives over the 29 30 449 entire dive bout in the later study (Fahlman et al. 2008b). On average, maximum 31 32 33 450 dive depth was shallower during the spontaneous dive trials (11.5±6.3 m) than 34 35 451 during the conditioned ones (20.1 ±14.3 m). As underwater activity is generally 36 37 38 452 higher during shallow dives (as a proportion of total dive time; Fahlman et al. 2013), 39 40 453 particularly in the relative amount of higher cost transiting behaviour (Goundie et al. 41 42 43 454 2015b; discussed later; Fahlman et al. 2008c), the slightly higher average metabolic 44 45 455 rate for spontaneous dives might be partly due to differences in the costs associated 46 47 48 456 with activity. 49 50 51 457 Cardiac responses and activity levels 52 53 54 55 458 The results of our earlier diving studies highlight that estimating “the” cost of diving 56 57 459 is complicated by various interconnecting characteristics of the dive: activity level, 58 59 60 460 dive depth, single dive vs. dive bout, etc. However, the relationships are made more 61 62 63 Diving metabolism of Steller sea lions 23 64 65 1 2 3 4 461 complex by the fact that marine mammals have the ability to make a number of 5 6 7 462 physiological adjustments during diving (reviewed in a number of sources, including 8 9 463 Kooyman 2002; Davis 2014; Butler and Jones 1997). To begin to clarify the role of 10 11 12 464 internal physiological changes on changes in the cost of diving, we measured V O 2 in 13 14 465 tandem with direct measures of both the level of physical activity and a quantified 15 16 17 466 metric of the extent of the dive response (Hindle et al. 2010b). This facilitated 18 19 467 understanding the effect of these variables on total costs, and also enabled us to 20 21 22 468 examine the temporal pattern of the onset of the dive response. 23 24 25 469 Specifically, the dive response study compared the extent of bradycardia (decrease 26 27 28 470 in heart rate, fH) during shallow and deep dives, and its relationship to V O 2 and level 29 30 471 of activity (Hindle et al. 2010b). In terrestrial mammals, fH increases with exercise, 31 32 33 472 as a means of delivering oxygen to highly active locomotor muscles. Hence, there is a 34 35 473 reasonable relationship between physical activity, fH, and V O2. However, fH is 36 37 38 474 predicted to be disassociated with energy expenditure and activity in marine 39 40 475 mammals (Fedak 1988). As noted previously, bradycardia and vasoconstriction 41 42 43 476 serve to limit blood flow to locomotor muscles, partly to limit blood oxygen 44 45 477 depletion and partly to assist the release of oxygen from myoglobin stores (Davis 46 47 48 478 2014). However, it is unclear how fH changes during the course of different types of 49 50 479 dives, or how maximum bradycardia relates to dive characteristics (depth, duration) 51 52 53 480 or energy expenditure (despite suggestions that maximum bradycardia is unrelated 54 55 481 to maximum dive duration across pinniped species; Mottishaw et al. 1999). The 56 57 58 482 relationship between fH, and V O2 is also likely complicated by the fact that fH in 59 60 483 marine mammals is at least partly under voluntary control. 61 62 63 Diving metabolism of Steller sea lions 24 64 65 1 2 3 4 484 Hindle et al. (2010b) monitored fH in diving sea lions via two subcutaneous 5 6 7 485 electrodes while simultaneously measuring acceleration in 3 dimensions via a data 8 9 486 logger. The latter could be converted into a measure of total body movement, known 10 11 12 487 as overall dynamic body acceleration (ODBA; Wilson et al. 2006), which is used as a 13 14 488 proxy of physical activity (see a more detailed discussion in Predicting rates of 15 16 17 489 oxygen consumption). Similar to previous studies, three sea lions undertook single 18 19 490 short dives, single long dives, or bouts of 4 dives to either 10 m or 40 m. To better 20 21 22 491 mimic natural diving, a pair of feeding stations (with alternating timing of fish 23 24 492 delivery) was positioned about 6 m apart to promote horizontal movement at depth. 25 26 27 493 The sea lions controlled both the inter-dive surface intervals and the dive durations; 28 29 494 longer dive times were encouraged during the single dives by increased food 30 31 32 495 delivery rates. 33 34 35 496 Based on decreases in DMR with increasing dive duration reported in previous 36 37 38 497 studies, the dive response (i.e., degree of bradycardia) was expected to be stronger 39 40 498 during extended dives—particularly when diving to deeper depths and for longer 41 42 43 499 durations. Minimum fH (our primary measure of bradycardia) was lower during 44 45 500 dive bouts than single dives at either depth. Similarly, dive duration correlated 46 47 48 501 negatively with minimum instantaneous fH at both trial depths, but only for dive 49 50 502 bouts (a similar trend was observed in single dives, but this was not statistically 51 52 53 503 significant). In other words, minimum fH was lower for longer dive bouts at both 10 54 55 504 m and 40 m. This result highlights the flexibility in the dive response within 56 57 58 505 individual animals. 59 60 61 62 63 Diving metabolism of Steller sea lions 25 64 65 1 2 3 4 506 Another notable result of the Hindle et al. (2010b) study was that minimum fH 5 6 7 507 appeared to be a more accurate measure for quantifying the extent of bradycardia 8 9 508 than mean fH. For example, mean fH only showed a non-significant trend in 10 11 12 509 differences between single dives and dive bouts, and demonstrated no relationship 13 14 510 to dive duration. Minimum fH may be a stronger indicator of the extent of the dive 15 16 17 511 response than mean fH because it not only (by definition) indicates maximum 18 19 512 bradycardia, but is unaffected by the fact that fH is not constant during a dive cycle. 20 21 22 513 Mean values can be greatly affected by the rate of onset and release of bradycardia 23 24 514 and by changes in fH during the dive, such as pre-surface tachycardia. For example, 25 26 27 515 instantaneous fH dropped an average of 64% from pre-dive baseline within the first 28 29 516 few seconds of the 10 m dives, reaching a minimum about 29 s post-submergence, 30 31 32 517 and remained consistently reduced during these dives. As a result, mean fH 33 34 518 decreased with increasing dive duration. In contrast, the onset of bradycardia was 35 36 37 519 much more gradual during the deeper, longer 40 m dives, reaching a minimum after 38 39 520 45 seconds. Part of the difference in bradycardia onset time may be attributable to 40 41 42 521 the differences in the time it takes to reach depths of 10 or 40 m (~9 vs. 30 sec). As a 43 44 522 result, mean fH during deeper dives was actually higher than during 10 m dives. In 45 46 47 523 addition, transient increases in fH were observed periodically throughout 48 49 524 submergence at 40 m, effectively increasing mean fH but not affecting minimum 50 51 52 525 values. Further, ascent tachycardia (a term describing the release of bradycardia 53 54 526 which occurs prior to surfacing) occurred for both 10 and 40 m dives, but the 55 56 57 527 deeper (longer) dives had a longer period of ascent tachycardia, which increased 58 59 528 mean fH. 60 61 62 63 Diving metabolism of Steller sea lions 26 64 65 1 2 3 4 529 While the Hindle et al. (2010b) study demonstrated a relationship between the 5 6 7 530 extent of bradycardia and dive duration, it is not the same as saying there is a 8 9 531 relationship between cardiovascular function and physical activity (Fedak 1988). If 10 11 12 532 locomotor muscle remains at least partially perfused during diving, a correlation 13 14 533 should occur between underwater locomotion and heart rate during diving (see 15 16 17 534 further discussion in Davis 2014). In contrast, measures of exercise output (e.g., 18 19 535 flipper stroke frequency) and fH should be independent over the dive period for 20 21 22 536 species in which there is likely minimal blood flow to muscle during diving (e.g., 23 24 537 emperor penguins, Aptenodytes forsteri, Meir et al. 2008). As an added complication, 25 26 27 538 periods of elevated activity may alter peripheral reperfusion of muscles (Williams et 28 29 539 al. 2015), resulting in intermittent periods of increased blood flow during activity. 30 31 32 33 540 Hindle et al. (2010b) found that changes in fH during the dive and the differences in 34 35 541 bradycardia patterns observed between different types of dives were not reflected 36 37 38 542 in the apparent levels of physical activity, as measured by the ODBA accelerometry 39 40 543 values. Activity was high in the first 2 s of diving, reflecting initial orientation and 41 42 43 544 thrust to depth (see Fahlman et al. 2008a), followed by a pronounced decline of 44 45 545 ~85% for dives at both depths. This decline was prolonged in the 40 m dives 46 47 48 546 (reflecting a longer transit time to depth), and was followed by a considerable rise 49 50 547 ~20 s later corresponding to the start of the dive's bottom phase. Activity then 51 52 53 548 fluctuated throughout the remaining underwater time, a pattern that was observed 54 55 549 over the majority of the time course of 10 m dives. 56 57 58 59 60 61 62 63 Diving metabolism of Steller sea lions 27 64 65 1 2 3 4 550 Contrary to the temporal pattern of fH, and aside from a minor elevation in the final 5 6 7 551 3 s before surfacing, there was no clear elevation in ODBA during the ascent 8 9 552 (although see Fahlman et al. 2008c). The overall result was a significant positive 10 11 12 553 linear relationship between ODBA and mean diving fH for dives to 10 m, but not for 13 14 554 dives to 40 m. Further, for both diving scenarios, surface fH correlated with dive 15 16 17 555 activity, suggesting that some underwater locomotor costs were deferred to the 18 19 556 post-dive surface interval. This further supports the speculation that locomotor 20 21 22 557 muscles of Steller sea lions become hypoxic during diving, regardless of dive depth. 23 24 558 The likely mechanism for this is a limitation of perfusion to muscle vascular beds, 25 26 27 559 presumably occurring to a lesser degree in shallow dives. 28 29 30 560 Overall, the results of the Hindle et al. (2010b) study confirm that the processes of 31 32 33 561 physical activity, oxygen utilization, and cardiac output are more independent in 34 35 562 exercising diving mammals than their terrestrial counterparts. The predictive 36 37 38 563 power of the relationship between fH and oxygen consumption over an entire dive is 39 40 564 discussed later (see Predicting rates of oxygen consumption). 41 42 43 44 565 Physiological constraints to diving 45 46 47 566 The presumed goal of the dive response is to maximize time spent being active 48 49 50 567 while submerged. The results from previous experiments confirm the ability of 51 52 568 Steller sea lions to use variable physiological adjustments to maximize both foraging 53 54 55 569 activity and time at depth. While marine mammals can and do also rely on anaerobic 56 57 570 metabolism to extend submergence time, an additional assumption is that an animal 58 59 60 571 which manages to fulfill its energetic demands through aerobic metabolism will be 61 62 63 Diving metabolism of Steller sea lions 28 64 65 1 2 3 4 572 more “efficient” than one that relies on anaerobic metabolism. This is related to the 5 6 7 573 additional costs associated with production of metabolic byproducts, and the time 8 9 574 required for their post-dive clearance (Kooyman and Ponganis 1998). Hence, the 10 11 12 575 aerobic dive limit (ADL) has become a standard comparative metric of diving 13 14 576 capacity among marine mammals (Costa et al. 2001). This attribute is rarely 15 16 17 577 determined directly (via appearance of blood lactate) and so researchers rely upon 18 19 578 the calculated aerobic dive limit (cADL) to approximate the actual ADL. The cADL is 20 21 22 579 quantified from total body oxygen stores (TBO; the oxygen reserve) and diving 23 24 580 metabolic rate (i.e., the rate at which the reserves are consumed) (Butler 2006). 25 26 27 28 581 In the wild, adult female Steller sea lions largely exhibit a pattern of relatively brief 29 30 582 (averaging 1.9–2.4 min), shallow (averaging 21–53 m), and frequent dives (Merrick 31 32 33 583 et al. 1994; Loughlin et al. 1998). In fact, 92% of dives in winter and 98% of dives in 34 35 584 summer made by adult females are <4 min (Merrick and Loughlin 1997). An 36 37 38 585 important question is how much of this diving pattern represents responses to 39 40 586 extrinsic factors (e.g., fish distribution, water depth) and how much is due to 41 42 43 587 intrinsic factors (e.g., physiological constraints). 44 45 46 588 Surprisingly, the cADL of otariids has rarely been estimated, primarily because of 47 48 49 589 the difficulties associated with obtaining measures of DMR. The previously 50 51 590 described natural variation in diving metabolic rate under diverse diving regimes in 52 53 54 591 combination with differences in the method of calculating metabolic expenditure 55 56 592 combine to make estimates of cADL less clear than standard definitions might 57 58 59 593 indicate. 60 61 62 63 Diving metabolism of Steller sea lions 29 64 65 1 2 3 4 594 We estimated cADL in four Steller sea lions by combining estimates of total body 5 6 7 595 oxygen (TBO) stores with measures of DMR while diving to 10 or 40 m (Gerlinsky et 8 9 596 al. 2013). We started by calculating TBO from direct measures of blood oxygen 10 11 12 597 stores and lean tissue mass, and estimates of lung volume and myoglobin 13 14 598 concentrations—and found our estimated TBO (35.9 ml O2 kg-1) was slightly lower 15 16 17 599 than previously reported for young Steller sea lions and other otariids (Lenfant et al. 18 19 600 1970; Richmond et al. 2006). As with previous studies, pre-dive metabolism (1.78 L 20 21 -1 22 601 O2 min for animals averaging 193 kg) was lower than metabolic rate during a dive. 23 24 602 Mean DMR for single dives was 2.88 L O2 min-1 but also depended significantly on 25 26 -1 27 603 dive duration. A ‘‘minimum’’ DMR was estimated as 2.68 L O2 min for dives >4.5 28 29 604 min, as DMR for these dives no longer depended on dive duration. Mean AMR was 30 31 -1 -1 32 605 2.24 L O2 min for single dives and 2.44 L O2 min for a bout of four consecutive 33 34 606 dives. It should be noted that these values are significantly higher than reported for 35 36 37 607 earlier studies (1.20-1.74 L O2 min-1, Fahlman et al. 2008b; Hastie et al. 2006a), and 38 39 608 therefore will result in a proportionally lower cADL. The source of these differences 40 41 42 609 are unclear – while the animals were larger on average than in previous studies, 43 44 610 some of the difference was likely due to a higher level of bottom foraging activity 45 46 47 611 from the use of dual feeding stations, and may therefore more accurately reflect 48 49 612 estimates of ADL in free-ranging sea lion. 50 51 52 53 613 The cADL for a single dive was 3.0 min based on the AMR and 2.5 min based on the 54 55 614 minimal DMR. Actual dive durations for single dives were conspicuously longer (4.4 56 57 58 615 min on average) than the cADL. However, the dive durations of the sea lions when 59 60 616 performing consecutive dives in a bout became progressively shorter and were, on 61 62 63 Diving metabolism of Steller sea lions 30 64 65 1 2 3 4 617 average, ~2.2 min each — slightly less than their cADL, and more similar to the 5 6 7 618 behaviour of wild animals. Undertaking shortened dives during bouts may reflect 8 9 619 the incurred oxygen debt maintained by sea lions during such diving (Fahlman et al. 10 11 12 620 2008b). Our sea lions spent short (average 18 sec) surface intervals between their 13 14 621 dives, which was likely insufficient to fully replenish their oxygen stores—indicating 15 16 17 622 a delayed payoff of oxygen debt. Hence, a consecutive bout of diving (akin to what 18 19 623 animals typically perform in the wild) may be more constrained by aerobic limits 20 21 22 624 than a single dive, and the average duration of dives in a bout may be correspond 23 24 625 more closely to the ADL. In other words, if there is no immediate need to fully 25 26 27 626 balance oxygen stores or process accumulated lactate, single dives may be 28 29 627 ‘‘uncoupled’’ from aerobic limits (Horning 2012). 30 31 32 33 628 The importance traditionally placed on ADL as an ecological and physiological 34 35 629 meaningful measure of diving capacity is a bit misleading. In most mammals, high 36 37 38 630 partial pressures of blood carbon dioxide (PCO2) rather than low blood oxygen 39 40 631 pressures (PO2) plays a central role in the control of ventilation (Phillipson et al. 41 42 43 632 1981; Schagatay 2010). There is no reason to expect that the same general 44 45 633 mechanism does not control diving in marine mammals, with high PCO2 ultimately 46 47 48 634 driving a return to the surface to breathe. However, it would seem advantageous for 49 50 635 marine mammals to defer surfacing for gas exchange as long as possible (although 51 52 53 636 see discussion in Foraging strategies on the effect of increased recovery times). 54 55 637 Hence, apnea is generally considered to be one of the critical characteristics of the 56 57 58 638 classical dive response. At first glance, listing the need for a mammal to not breathe 59 60 639 while submerged may seem needlessly obvious. However, in the case of diving 61 62 63 Diving metabolism of Steller sea lions 31 64 65 1 2 3 4 640 mammals, the apneic adaptation refers to a dampening of the breathing response in 5 6 7 641 conditions that would induce respiratory drive in most mammals. 8 9 10 642 The blood PO2 of marine mammals can reach levels below that typically exhibited by 11 12 13 643 terrestrial counterparts (Meir et al. 2009). At the same time, the blood of marine 14 15 644 mammals can carry more CO2 as carboxyhemoglobin than terrestrial mammals (Tift 16 17 18 645 et al. 2014), and they may be more tolerant of higher PCO2 levels and better able to 19 20 646 buffer its effects (Castellini 1991; Lenfant et al. 1970). While a number of studies 21 22 23 647 conducted in tanks suggest that breath-hold duration of marine mammals is affected 24 25 648 by high blood PCO2 levels (hypercapnia) (Pasche 1976; Gallivan 1980), no work had 26 27 28 649 been conducted on animals diving freely to realistic depths. While there is some 29 30 650 evidence that surface interval durations are managed to allow adequate removal of 31 32 33 651 CO2 (Boutilier et al. 2001; Fahlman et al. 2008b), it is unclear what role PO2 plays in 34 35 652 regulating dive duration, particularly during dive bouts. 36 37 38 39 653 To assess the influence of PCO2 and PO2 on physiological control of dive behaviour, 40 41 654 we tested how increasing levels of inspired CO2 (hypercarbia) and decreasing 42 43 44 655 inspired O2 (hypoxia) affected the diving metabolic rate, submergence times, and 45 46 656 dive recovery times (time to replenish O2 stores and eliminate CO2) in our freely 47 48 49 657 diving Steller sea lions by altering the gas composition within the respirometry 50 51 658 dome (Gerlinsky et al. 2014a). While hypercarbia (as low as 2% CO2) increased 52 53 54 659 breathing frequency, it did not affect metabolic rate, or the duration of dives or 55 56 660 surface intervals (tested up to 3% CO2). Changes in breathing rates indicated 57 58 59 661 respiratory drive was altered by hypercarbia at rest, but also suggested that blood 60 61 62 63 Diving metabolism of Steller sea lions 32 64 65 1 2 3 4 662 CO2 levels remained below the threshold that would alter normal dive behaviour. 5 6 7 8 663 Regardless of the ambient air conditions (whether breathing ambient air, 9 10 664 hypercarbia, or hypoxia), it took the sea lions longer to remove accumulated CO2 11 12 13 665 than it did for them to replenish their O2 stores following dives (as seen in other 14 15 666 mammals). This difference between O2 and CO2 recovery times grew with increasing 16 17 18 667 dive durations, increasing hypercarbia, and was greater for bout dives, suggesting 19 20 668 there could be a build-up of CO2 load with repeated dives (despite any 21 22 23 669 compensatory changes in ventilation rate). Although we saw no evidence of CO2 24 25 670 limiting dive behaviour within our testing conditions, the longer time required to 26 27 28 671 remove CO2 may eventually exhibit control over the overall time they can spend in 29 30 672 apnea and thus regulate overall foraging duration. 31 32 33 34 673 Foraging strategies: interacting effects of physiology, prey, and nutritional status 35 36 37 674 Of course, sea lions are not slaves to their physiology. Motivation and cost-benefit 38 39 40 675 considerations are important in shaping behaviour. This is the basis of an entire 41 42 676 area of study — optimal foraging theory — which has its own subset of theories 43 44 45 677 regarding how a breath-hold diver should optimize its subsurface foraging 46 47 678 behaviour based on prey patch characteristics and physiological limits (see 48 49 50 679 Thompson and Fedak 2001; Carbone and Houston 1996). To address these theories, 51 52 680 we conducted a study to quantify how sea lions respond to changes in prey 53 54 55 681 availability, and how this affects their foraging efficiency by measuring DMR, dive 56 57 682 durations, and food intake of animals diving on prey patches of varying depths (10 58 59 -1 60 683 and 40 m) and densities (delivered at 4 and 12 fish min ) (Goundie et al. 2015a). 61 62 63 Diving metabolism of Steller sea lions 33 64 65 1 2 3 4 684 The rate of energy expenditure of the four sea lions did not change while diving in 5 6 7 685 any of the simulated foraging conditions, but the proportion of time spent 8 9 686 consuming prey increased with prey patch density due to alterations in diving 10 11 12 687 patterns. While it may seem an obvious choice to prolong dive duration to remain on 13 14 688 a higher quality forage patch, there is a consequence to this strategy in the form of 15 16 17 689 extended post-dive recovery times. However, for the sea lions, the increased time at 18 19 690 depth more than offset the subsequent increases in surface recovery time. At both 20 21 22 691 depths, sea lions spent a greater proportion of the dive bout foraging in the high- 23 24 692 density prey patches (45% vs. 28%), leading to higher rates of energy gain and 25 26 27 693 foraging efficiency (cost:benefit ratio). Conversely, in low-density prey patches, the 28 29 694 animals spent a smaller proportion of the dive bout actively feeding, and 30 31 32 695 consequently had lower rates of energy gain and foraging efficiency. In other words, 33 34 696 by extending the dive duration in a high-density patch, the increase in overall 35 36 37 697 foraging efficiency (taking into account the increased recovery times) was still 38 39 698 significantly greater (~5:1) than that due to differences in prey availability alone 40 41 42 699 (3:1). Our results indicate that sea lions foraging in areas with reduced prey 43 44 700 availability forage less efficiently, and therefore would have greater difficulty 45 46 47 701 fulfilling their daily energy requirements than might be predicted from estimates of 48 49 702 prey density alone. 50 51 52 53 703 Understanding the interactions between foraging behaviour and aerobic capacity is 54 55 704 further complicated by the fact that neither the physiology (e.g., metabolism) nor 56 57 58 705 the oxygen stores of sea lions remains constant, resulting in variable diving abilities. 59 60 706 For example, total oxygen stores were only slightly higher during the winter than 61 62 63 Diving metabolism of Steller sea lions 34 64 65 1 2 3 4 707 the summer or spring in Steller sea lions. However, given the seasonal differences in 5 6 7 708 body mass, this meant that mass-specific blood volume of Steller sea lions is 8 9 709 significantly higher in the winter than in summer (Gerlinsky et al. 2013). Of course, 10 11 12 710 any form of mass-scaling is fraught with difficulties and differences of opinion. 13 14 711 However, in this case the differences are physiologically relevant for their effect on 15 16 17 712 cADL given that DMR also changes with body mass. 18 19 20 713 The findings of Gerlinsky et al. (2013) highlight that sea lion physiology is markedly 21 22 23 714 seasonal; their net aerobic diving capacity is the product of simultaneous changes in 24 25 715 blood volume, body mass (including muscle oxygen stores) and resting metabolism 26 27 28 716 (which may itself affect DMR). These changes are even greater during periods of 29 30 717 nutritional stress, which are characterized by rapid changes in body mass (with a 31 32 33 718 seasonally varying portion from core tissues; Rosen 2009) and either increases or 34 35 719 decreases in resting and diving metabolism (depending on season and type of 36 37 38 720 nutritional challenge; Rosen and Trites 2002; Svärd et al. 2009). 39 40 41 721 Given the aforementioned considerations, we conducted a set of studies to 42 43 44 722 specifically examine whether episodes of nutritional stress affect the sea lions’ 45 46 723 capacity to successfully forage. Animals under some level of nutritional challenge 47 48 49 724 often exhibit depressed resting metabolic rates in air (Guppy and Withers 1999). 50 51 725 Studies with non-diving Steller sea lions have demonstrated that this response is 52 53 54 726 contingent upon the type of nutritional challenge and season (Rosen and Trites 55 56 727 2002). If altered metabolic rate at rest parallels changes during diving, this would 57 58 59 728 result in proportional (but inverse) alterations in ADL. At the same time, decreasing 60 61 62 63 Diving metabolism of Steller sea lions 35 64 65 1 2 3 4 729 core body mass due to energy deficits would theoretically decrease both metabolic 5 6 7 730 requirements and myoglobin oxygen stores, each with opposite effects on ADL. Also, 8 9 731 reductions in the subcutaneous blubber layer could increase thermoregulatory costs 10 11 12 732 during diving (Fahlman et al. 2005; Rosen et al. 2007). In theory, any increase in 13 14 733 diving metabolism or decrease in oxygen stores would reduce ADL and impede 15 16 17 734 foraging capacity; however, the actual net effect of episodes of nutritional stress on 18 19 735 diving sea lions is difficult to predict. 20 21 22 23 736 To address the potential effects of nutritional status and body condition on diving 24 25 737 abilities, we first examined the effect of an acute period of nutritional stress on 26 27 28 738 diving behaviour and costs (Svärd et al. 2009). Specifically, we measured changes in 29 30 739 metabolism while resting at the water surface and while diving, before and after 31 32 33 740 three 9-10 d fasting periods; two trials were conducted in summer and one in 34 35 741 winter. MRs was greater in the winter, both before and after the fasts, despite no 36 37 38 742 seasonal differences in initial body mass. Over the course of the fasts, mass- 39 40 743 corrected MRs decreased significantly during both seasons, but more during the 41 42 43 744 summer fasts (–16.4 ± 4.7%) than the winter (–8.0 ± 9.0%). Perhaps as a result of 44 45 745 these metabolic differences, the sea lions experienced a slightly greater degree of 46 47 48 746 mass loss during the winter fasts than the summer (-10.6 ± 0.8% vs. -9.5 ± 0.03%). 49 50 747 In contrast to MRs, mass-corrected DMR (over single dives) increased significantly 51 52 53 748 following the winter fasts (13.5 ± 8.1%), but did not change during summer trials (– 54 55 749 1.1 ± 3.2%). As a result, the ratio between diving and surface metabolism was 56 57 58 750 significantly higher in winter. We hypothesized that the increased DMR during the 59 60 751 winter fasts (both as an absolute cost and relative to MRs) was attributable to an 61 62 63 Diving metabolism of Steller sea lions 36 64 65 1 2 3 4 752 increased thermal challenge via convective heat loss while diving. This suggests that 5 6 7 753 Steller sea lions are more sensitive to changes in body condition due to acute food 8 9 754 shortages in the winter compared with the summer. 10 11 12 13 755 Interestingly, the metabolism of the sea lions at the surface in-between individual 14 15 756 dives in a bout was constant prior to the food restriction period, but increased 16 17 18 757 significantly after the first dive in a bout when sea lions were fasted, regardless of 19 20 758 season (Svärd et al. 2009). If this were an effect of the heat increment of feeding 21 22 23 759 (HIF), it implies that immediate processing of ingested energy was of higher priority 24 25 760 than optimizing diving ability (which is adversely affected by increases in 26 27 28 761 metabolism due to digestion) in nutritionally challenged sea lions. Inhibition of 29 30 762 digestion until completion of foraging dives under “nutritionally normal” conditions 31 32 33 763 has been seen in other diving vertebrates and may be a crucial mechanism to 34 35 764 conserve food or alter diving ability (Sparling et al. 2007a; Rosen et al. 2015), as 36 37 38 765 discussed below and in Rosen et al. (2007). 39 40 41 766 While Svärd et al. (2009) examined changes in diving metabolism in response to an 42 43 44 767 acute period of nutritional stress, sea lions in the wild may be more typically 45 46 768 exposed to longer periods of partial under-nutrition. Additionally, changes in TBO 47 48 49 769 stores that accompany mass loss must also be measured to fully appreciate any 50 51 770 potential impacts of nutritional status on ADL. Resulting changes in ADL will directly 52 53 54 771 affect overall diving behaviour, costs, and foraging capacity. 55 56 57 772 We addressed this issue by examining the effects of nutritional status on both sides 58 59 60 773 of the ADL equation (Gerlinsky et al. 2014b). We measured DMR, blood O2 stores, 61 62 63 Diving metabolism of Steller sea lions 37 64 65 1 2 3 4 774 body composition, and dive behaviour prior to and during a period of restricted 5 6 7 775 food intake (resulting in a 10% decrease in body mass over 3 weeks). The four sea 8 9 776 lions completed either single long dives or bouts of 4 dives to either 10 or 40 m 10 11 12 777 before and after the period of mass loss. The sea lions controlled both the dive 13 14 778 durations and inter-dive surface intervals. We predicted that total body oxygen 15 16 17 779 (TBO) stores would decrease following a nutritional stress event due to changes in 18 19 780 core body mass (and associated decreases in myoglobin stores) with blood oxygen 20 21 22 781 stores likely being proportionally reduced. We also predicted that DMR would 23 24 782 increase as seen in the previous study (Svärd et al. 2009) rather than mirror the 25 26 27 783 metabolic depression exhibited by fasting sea lions on land (Rosen and Trites 2002). 28 29 784 In combination, these proposed changes should lead to a dramatic decrease in ADL, 30 31 32 785 with a resulting decrease in average dive durations. 33 34 35 786 What we found was quite different from what we had expected. While sea lion lean 36 37 38 787 body mass declined 6% as a result of the nutritional stress event, TBO stores 39 40 788 actually increased 32%. This increase was due to increased blood oxygen stores, 41 42 43 789 driven by a 23% increase in absolute blood volume coupled with an 8% increase in 44 45 790 red blood cell counts. While mass-specific DMR did increase, this was primarily due 46 47 48 791 to decreases in mass rather than changes in absolute metabolism. More importantly, 49 50 792 this increase in DMR was less than the concurrent increase in TBO stores. As a result, 51 52 53 793 the cADL increased by 10% in nutritionally stressed sea lions. 54 55 56 794 Nutritionally stressed sea lions also increased the duration of their single long dives 57 58 59 795 (from 4.6 to 5.2 min), with an associated increase in post-dive recovery times (from 60 61 62 63 Diving metabolism of Steller sea lions 38 64 65 1 2 3 4 796 5.6 to 6.1 min). While this increase in dive duration is thought to have been the 5 6 7 797 result of the longer ADL, the role of increased motivation for food in nutritionally 8 9 798 stressed sea lions cannot be discounted. As demonstrated in earlier studies, longer 10 11 12 799 dive durations in this study also resulted in lower DMR. However, mass-specific 13 14 800 DMR was still higher for a given dive duration in nutritionally stressed sea lions. 15 16 17 18 801 As with single dive cycle duration, the total duration of a bout dive cycle also 19 20 802 increased with nutritional stress. However, the overall increase in duration for a 21 22 23 803 complete dive bout was not due to an increase in submergence time, but rather the 24 25 804 proportion of time the sea lions spent at the surface during a cycle of 4 dives (due to 26 27 28 805 both increased inter-dive surface intervals and post-dive recovery periods). This is 29 30 806 consistent with the proposition that the sea lions were consuming O2 (and 31 32 33 807 producing CO2) at a higher rate when nutritionally stressed for a given dive duration, 34 35 808 and therefore had to spend more time (relative to unstressed animals) replenishing 36 37 38 809 oxygen stores and off-loading CO2 while at the surface. 39 40 41 810 Physiologically induced changes in diving behaviours likewise affect sea lion 42 43 44 811 foraging efficiencies. The increased time spent at the surface for a given subsurface 45 46 812 time affects the proportion of their dive cycle spent actively foraging/ingesting prey. 47 48 49 813 For single dives, the nutritionally stressed animals consumed more fish per minute 50 51 814 of a dive cycle (dive and post-dive recovery duration) and had slightly higher 52 53 54 815 foraging efficiency (~7% higher), despite having higher mass-specific DMRs. 55 56 816 However, for dive bouts, the amount of fish consumed per minute of dive cycle by 57 58 59 817 the nutritionally stressed sea lions was lower and their mass-specific DMR was 60 61 62 63 Diving metabolism of Steller sea lions 39 64 65 1 2 3 4 818 higher, which combined to reduce their foraging efficiency by ~20%. 5 6 7 8 819 Past foraging success can also affect the cost of diving through the effects of HIF, the 9 10 820 increase in metabolism associated with the processing of a meal. An increase in 11 12 13 821 DMR caused by HIF would effectively decrease the ADL (which, as previously noted, 14 15 822 may have explain the results of Svärd et al. 2009). Conversely, if digestion is 16 17 18 823 suspended while foraging due to vasoconstriction that limits blood flow to the 19 20 824 stomach or intestine during diving (Zapol et al. 1979), neither V O 2 nor ADL will be 21 22 23 825 affected by successful foraging. In addition to serving to maintain foraging efficiency 24 25 826 by maintaining the ADL, this could also lead to a temporal separation of diving and 26 27 28 827 digestion (as proposed in phocid seals; Sparling et al. 2007a; Crocker et al. 1997) 29 30 828 that would result in the post-foraging surface intervals being extended, as is often 31 32 33 829 observed in wild pinnipeds. 34 35 36 830 To further examine the nature of the metabolic interactions between digestion and 37 38 39 831 diving, we compared the cost of diving in fasted and pre-fed trained Steller sea lions 40 41 832 foraging under controlled conditions (Rosen et al. 2015). Four sea lions completed a 42 43 44 833 bout of four, 3-minute dives with 1-minute inter-dive surface intervals, after either 45 46 834 fasting overnight (at least 18 hours), or one hour after being fed a 3 kg meal of 47 48 49 835 herring. Pre-dive MRs was higher in the pre-fed animals than the fasted animals, 50 51 836 indicating an effect of digestion on metabolism. However, while DMR was also 52 53 54 837 higher in the pre-fed animals, the sea lions displayed a significant reduction in the 55 56 838 apparent additive effect of digestion during diving. The increase in rate of oxygen 57 58 59 839 consumption during diving was only 54% of the increase observed in pre-dive 60 61 62 63 Diving metabolism of Steller sea lions 40 64 65 1 2 3 4 840 metabolism, suggesting partial deferment of digestion during the active diving phase. 5 6 7 841 Immediately following a dive, there was a rapid, almost complete return to pre-dive 8 9 842 levels of metabolism, suggesting a rapid reinstatement of the digestive process. 10 11 12 13 843 The results of Rosen et al. (2015), in combination with the findings of Svärd et al. 14 15 844 (2009) on the effects of nutritional state on the extent of digestion while diving, 16 17 18 845 suggest that Steller sea lions diving to depth partially deferred digestion while 19 20 846 actively foraging and that the classically held view that digestion and diving are 21 22 23 847 incompatible processes may be much more variable and adaptable to specific diving 24 25 848 conditions and behaviours than previously thought. Thus, when nutritionally 26 27 28 849 compromised, ADL may be even shorter as sea lions may prioritize digestion as soon 29 30 850 as prey is ingested. 31 32 33 34 851 Predicting rates of oxygen consumption 35 36 37 852 While measures of rates of oxygen consumption and carbon dioxide production are 38 39 40 853 usually considered to represent the “gold standard” for estimating rates of energy 41 42 854 expenditure during diving, respirometry is largely a logistically unfeasible 43 44 45 855 methodology in studies of wild animals. As noted previously, a rare exception is the 46 47 856 ability to transport seals to isolated ice holes, although this may, in itself, set up an 48 49 50 857 artificial diving situation (Castellini et al. 1992). In lieu of direct measures of oxygen 51 52 858 consumption, scientists have relied on alternate indirect measures to predict energy 53 54 55 859 expenditure. All of these proxy measures require calibration experiments to 56 57 860 produce predictive mathematic equations, and ideally to determine the accuracy 58 59 60 861 and precision of these techniques. Some, such as the doubly labelled water turnover 61 62 63 Diving metabolism of Steller sea lions 41 64 65 1 2 3 4 862 method, have been well-described, but only provide a single value of energy 5 6 7 863 expenditure over extended timeframes rather than specific costs of diving, and are 8 9 864 therefore not discussed here (for validation studies in otariids see Butler et al. 2004; 10 11 12 865 Dalton et al. 2014). Our laboratory has been actively investigating the efficacy of 13 14 866 three potential proxies for estimating energy expenditure during diving: heart rate, 15 16 17 867 body acceleration, and behavioural budgets. 18 19 20 868 Heart rate: 21 22 23 24 869 In terrestrial mammals, increases in V O 2 from activities such as exercise induce a 25 26 870 proportional increase in heart rate (fH) (Hiilloskorpi et al. 2003). Fick’s Equation 27 28 29 871 (Fick 1870) formulized the relationship between V O 2, fH, stroke volume, blood 30 31 872 oxygen content, and tissue oxygen extraction. It is not feasible to measure all of the 32 33 34 873 physiological variables in Fick’s equation on freely diving animals (Ponganis et al. 35 36 874 1991; Ponganis et al. 1990). Consequently, application of this method on marine 37 38 39 875 mammals assumes that an increase in fH is the primary response to increased 40 41 876 oxygen demand, and that the remaining parameters of Fick’s equation either vary 42 43 44 877 proportionally to fH or remain constant (although see Miedler et al. 2015). 45 46 47 878 Given the apparent relationship between fH and whole-body O2 use in air, and the 48 49 50 879 fact that fH is relatively easy to obtain via attached telemetry devices from a free- 51 52 880 diving animal (at least compared to other circulatory variables; Ponganis 2007), 53 54 55 881 many researchers have made a case for using fH as an indirect measure of free- 56 57 882 ranging energy expenditure in marine mammals (e.g., Green et al. 2009; Butler et al. 58 59 60 883 2004). In captive swimming (non-diving) California sea lions, fH overestimated rates 61 62 63 Diving metabolism of Steller sea lions 42 64 65 1 2 3 4 884 of oxygen consumption by an average of only 2.7%, although error estimates ranged 5 6 7 885 widely from –28% to +23% (Boyd et al. 1995). 8 9 10 886 Our initial investigations with Steller sea lions also began by simultaneously 11 12 13 887 monitoring V O 2 and fH in four non-diving juvenile animals while resting in air, 14 15 888 resting in water, or while swimming in a 5800 L metabolic flume tank (1.8 m max 16 17 18 889 depth; McPhee et al. 2003). The study was limited by several logistical constraints, 19 20 890 particularly for the trials in the flume tank. The activity of the animals was limited 21 22 23 891 by the size and depth of the tank (3.2 m X 1.8 m X 1.0 m), although changes in the 24 25 892 internal water current were used to promote a range of V O 2 and fH from the animals. 26 27 28 893 Additionally, due to the necessity of attaching the surface electrodes to the fur under 29 30 894 gas anesthesia, multiple measurements (up to 18) were conducted in the same 31 32 33 895 session (although the individual periods of data collection were separated to try to 34 35 896 maintain a level of statistical independence). 36 37 38 39 897 Despite these limitations, the preliminary tests seemed to confirm the hypothesis 40 41 898 that fH accurately predicted V O 2, at least in non-diving pinnipeds. Combining all of 42 43 44 899 the data obtained in both air and water environments demonstrated that fH could 45 46 900 account a large amount of the variance (~70%) in mass-specific V O 2. The resulting 47 48 49 901 predictive equation was different from that of other marine mammal species, 50 51 902 although it was most similar to other otariids (e.g., Butler et al. 1992; Boyd et al. 52 53 54 903 1995; Williams et al. 1991). While this initial study demonstrated the validity of the 55 56 904 basic principal, it did not test the efficacy of the technique under realistic conditions 57 58 59 905 for a diving marine mammal. As discussed earlier, the relationship between exercise 60 61 62 63 Diving metabolism of Steller sea lions 43 64 65 1 2 3 4 906 and fH in diving marine mammals is different than their terrestrial counterparts. 5 6 7 907 Diving is characterized by variable periods of bradycardia and tachycardia (which 8 9 908 were not observed in the swimming sea lions) that are temporally independent of 10 11 12 909 physical work and associated aerobic metabolism (Williams et al. 2015). 13 14 15 910 A follow-up study examined the relationship between V O 2 and fH in both diving and 16 17 18 911 non-diving sea lions (Young et al. 2011b). Data from non-diving animals that were 19 20 912 tested in either the aforementioned flume tank or while resting at the water’s 21 22 23 913 surface prior to diving in the open ocean provided a single predictive equation with 24 25 914 a relatively low standard error of the estimate. Curiously, the equation predicting 26 27 28 915 metabolism while resting in water (either in the flume tank or at the water’s surface 29 30 916 in the open ocean) was different than the one resulting from data gathered from 31 32 33 917 post-absorptive animals resting in ambient air. Mathematically, the resulting 34 35 918 regression equations indicated that changes in fH while in air were associated with 36 37 38 919 much greater changes in V O 2 than when in water, although the reason for this 39 40 920 difference is unknown. 41 42 43 44 921 The ability of fH to predict V O2 in diving sea lions was less clear. When examined 45 46 922 over the dive portion only (i.e., DMR), fH could not be used to predict V O2 for either 47 48 49 923 single dives or dive bouts. This would be expected, given the previously discussed 50 51 924 changes in fH during a dive (i.e., intermittent bradycardia and pre-surface 52 53 54 925 tachycardia; Hindle et al. 2010b), as well as potential changes in stroke volume and 55 56 926 the level of pulmonary shunting. However, fH could predict AMR calculated over the 57 58 59 927 entire dive cycle, which is more consistent with the concept that the dive cycle 60 61 62 63 Diving metabolism of Steller sea lions 44 64 65 1 2 3 4 928 (submergence and subsequent surface periods) is a distinct physiological unit 5 6 7 929 (Kramer 1988; Butler and Jones 1997; Green et al. 2003). 8 9 10 930 However, when the AMR dive data was examined more closely, two statistically 11 12 13 931 distinct predictive equations could be produced for single long dives and bouts of 4 14 15 932 consecutive dives. Further, the equation predicting AMR from fH for single long 16 17 18 933 dives was not statistically distinct from the regression predicting V O 2 resting at the 19 20 934 surface, and hence could be combined into a single predictive equation. This 21 22 23 935 suggests that single extended dives were more physiologically similar (in the 24 25 936 relationship between cardiac function and oxygen consumption) to that of animals 26 27 28 937 resting at the water’s surface than those undertaking a bout of multiple dives. This 29 30 938 result may be because, unlike in dive bouts, there is no cumulative oxygen debt in 31 32 33 939 between single dives so that the relationship between fH and V O 2 remains more 34 35 940 similar to that observed in non-diving activities. The separate equation required to 36 37 38 941 predict V O 2 from fH during dive bouts not only had a low predictive error but was 39 40 942 also quite robust. The fH:V O 2 relationship was not significantly affected by dive 41 42 43 943 duration, dive depth, or water temperature (under the conditions tested). 44 45 46 944 The results of the Young et al. (2011b) study reinforced the concept that different 47 48 49 945 types of dives are physiologically distinct. On a practical level, the results 50 51 946 demonstrate the importance of applying the appropriate equation for the correct 52 53 54 947 type of diving behaviour when predicting V O 2 from fH. For example, incorrectly 55 56 948 applying the predictive equation for animals resting in water to estimate V O 2 of sea 57 58 59 949 lions performing dive bouts would overestimate V O 2 by ~25%. Fortunately, in 60 61 62 63 Diving metabolism of Steller sea lions 45 64 65 1 2 3 4 950 studies with wild sea lions, dive types (single dives vs. bouts) can be identified 5 6 7 951 through simultaneous collection of data from time-depth recorders (TDRs). 8 9 10 952 Digestion is another factor that might affect the relationship between V O 2 and fH. As 11 12 13 953 previously noted, the digestion of food causes an increase in rates of oxygen 14 15 954 consumption and the heat increment of feeding (HIF) in Steller sea lions on land has 16 17 18 955 been shown to double V O 2 (Rosen and Trites 1997). Previous studies have indicated 19 20 956 that digestion is partly suppressed during diving (Rosen et al. 2015), although HIF 21 22 23 957 during diving may be more pronounced during periods of nutritional stress (Svärd 24 25 958 et al. 2009). It is unknown whether this increase in V O 2 associated with digestion is 26 27 28 959 accompanied by a parallel increase in fH, and what effect this might have on the 29 30 960 predictive strength of heart rate. 31 32 33 34 961 Preliminary investigation into this question was undertaken as part of the original 35 36 962 fH:V O 2 calibration study (McPhee et al. 2003). The fH and V O 2 for one of the male 37 38 39 963 sea lions was monitored for 3 hr following ingestion of a bulk meal of either 6 or 12 40 41 964 kg of herring. As expected, V O 2 increased over the measurement period, but heart 42 43 44 965 rate displayed no apparent parallel increase. 45 46 47 966 A more detailed study monitored the fH and V O 2 of four sea lions for 4-4.5 hr after 48 49 50 967 being fed 4 kg or 6 kg meals of herring in the previously described metabolic tank or 51 52 968 dry metabolic chamber (Young et al. 2011a). The results seemed to indicate 53 54 55 969 differences in the fH:V O 2 relationship depending on whether they were tested on 56 57 970 land or in the water. When sea lions were tested on land, the fH:V O 2 relationship 58 59 60 971 was not impacted by meal size or whether they were fed or not, enabling a single 61 62 63 Diving metabolism of Steller sea lions 46 64 65 1 2 3 4 972 predictive equation. The picture was slightly different for sea lions tested while in 5 6 7 973 water (but not diving). Although fH could be used to predict V O 2 after consuming a 4 8 9 974 kg meal, the relationship was different than that for when the same animals were 10 11 12 975 fasted. Furthermore, fH could not be used to predict V O 2 when the sea lions had 13 14 976 consumed a 6 kg meal. 15 16 17 18 977 It is unclear what caused the apparent collapse of the relationship after consuming 19 20 978 the 6 kg meal. We hypothesized that the larger meal would produce a larger 21 22 23 979 observed increase in V O 2 that was not accompanied by a comparative increase in fH, 24 25 980 but no such difference in scope of V O 2 with meal size was observed (although the 26 27 28 981 effect was longer). Overall, the results indicated that meal size and submergence in 29 30 982 water changed the relationship between V O 2 and fH. It is unclear the degree to 31 32 33 983 which these differences are associated with circulatory changes that sea lions may 34 35 984 undergo in preparation for diving that at least partly alter the course of digestion (as 36 37 38 985 discussed earlier in relation to Rosen et al. 2015; Svärd et al. 2009). 39 40 41 986 Overall, the ability of heart rate to accurately predict rates of energy expenditure in 42 43 44 987 wild sea lions is complicated beyond the associated technical concerns. The 45 46 988 relationships between activity, metabolic expenditure, and heart rate during apneic 47 48 49 989 exercise are made problematic by the cardiovascular adjustments associated with 50 51 990 the dive response. Hence, while reasonable predictive equations can be formulated 52 53 54 991 for sea lions on land, at the water surface, or actively diving, substantial errors can 55 56 992 be accrued by improper application of the different equations. Further, there is 57 58 59 993 evidence that digestion can significantly disrupt these relationships. Collectively, 60 61 62 63 Diving metabolism of Steller sea lions 47 64 65 1 2 3 4 994 this makes effective application of this technique to estimate energy expenditure in 5 6 7 995 wild sea lions extremely difficult. 8 9 10 996 Physical activity: 11 12 13 14 997 Many of the problems associated with proxies (such as heart rate or doubly labeled 15 16 998 water) that might be used to predict rates of energy expenditure are logistical. 17 18 19 999 Methods that require substantial data processing, serial timed captures of individual 20 21 1000 animals, and/or attachment of a significant mass of sensitive electronics present 22 23 24 1001 difficulties when dealing with wild marine mammals. 25 26 27 1002 Using measures of body acceleration would, in theory, avoid some of these problems 28 29 30 1003 (Halsey et al. 2011b). It requires a single attachment of a relatively small, 31 32 1004 inexpensive biologger (often encapsulated into existing electronics such as time- 33 34 35 1005 depth recorders) which can collect data at a high frequency that can be readily 36 37 1006 converted into a single index per unit time (e.g., per dive). Use of body acceleration 38 39 40 1007 as a proxy for metabolic rate is based on the Newtonian theory that an animal exerts 41 42 1008 force to achieve motion, and that the force required scales to levels of energy 43 44 45 1009 expenditure (Gleiss et al. 2011). A common metric for remotely measuring activity 46 47 1010 levels is overall dynamic body acceleration (ODBA; Wilson et al. 2006). ODBA uses 48 49 50 1011 data from 3-dimensional accelerometers to calculate dynamic movement (i.e., 51 52 1012 movement not due to gravity) in space and time, and has been found to be closely 53 54 55 1013 related to measures of V O 2 in a number of diving vertebrates (Enstipp et al. 2011; 56 57 1014 Halsey et al. 2011a), although this success is not far from universal (Dalton et al. 58 59 60 1015 2014; Halsey et al. 2011b). The equation used to predict V O 2 from measures of 61 62 63 Diving metabolism of Steller sea lions 48 64 65 1 2 3 4 1016 ODBA — derived from controlled calibration studies — is an expression of the basic 5 6 7 1017 cost of metabolism (represented by the equation’s intercept) and an incremental, 8 9 1018 linear increase in metabolism with increased work attributable to physical 10 11 12 1019 movement. 13 14 15 1020 Initial tests suggested that ODBA worked well for predicting V O 2 for diving Steller 16 17 18 1021 sea lions. An analysis of 141 independent dives (between 1 and 12 dives with full 19 20 1022 recovery during each trial, 1 trial per day) from three sea lions diving to depths 21 22 23 1023 between 10 and 50 m reported that ODBA alone was able to predict metabolic rate 24 25 1024 to within 7% of the measured value (Fahlman et al. 2008c). While this study also 26 27 28 1025 suggested that the predictive relationship was not different whether the animals 29 30 1026 were diving or resting at the surface, a follow-up study including additional data (n 31 32 33 1027 = 266) found that the relationship differed between when the sea lion was actively 34 35 1028 diving and when they were resting at the surface (Fahlman et al. 2013). This study 36 37 38 1029 also suggested that there were no effects of a 9-10 day fasting period on the 39 40 1030 relationship between ODBA and V O 2, despite a 7% decrease in DMR. 41 42 43 44 1031 However, a subsequent study by Volpov et al. (2015) using four sea lions 45 46 1032 undergoing a set of controlled dives was less supportive of the use of ODBA as a 47 48 49 1033 proxy of V O 2. These trials incorporated a second feeding station at depth, longer 50 51 1034 dive durations, and more dives per bout in an effort to assess if ODBA could predict 52 53 54 1035 V O 2 with increased levels of activity and oxygen depletion. The sea lions executed 55 56 1036 three dive types—single dives, bouts of multiple long dives with 4–6 dives per bout, 57 58 59 1037 or bouts of multiple short dives with 10–12 dives per bout—to depths of 40 m, 60 61 62 63 Diving metabolism of Steller sea lions 49 64 65 1 2 3 4 1038 resulting in a range of activity and oxygen consumption levels. 5 6 7 8 1039 The Volpov et al. (2015) study found that there was no statistical relationship 9 10 1040 between ODBA and V O 2 when sea lions were resting at the surface in contrast to the 11 12 13 1041 earlier studies (Fahlman et al. 2013; Fahlman et al. 2008c). Similar to these earlier 14 15 1042 studies, ODBA could predict AMR over the dive cycle when data from all dive types 16 17 18 1043 were combined. Interestingly, dive type (as defined above) was a significant model 19 20 1044 factor –and there were no significant linear relationships between AMR and ODBA 21 22 23 1045 within each dive type when data for each dive type were analyzed separately. 24 25 26 1046 There is a natural temptation when disparate results are presented to try to identify 27 28 29 1047 which study is “correct”. A more useful approach is to try to determine the possible 30 31 1048 reasons for the disparity in results among these studies. Possible suggestions 32 33 34 1049 include variation in statistical approach, including different analysis time scales (e.g., 35 36 1050 see Jeanniard du Dot et al. 2016), or seasonal differences (e.g., see Dalton et al. 37 38 39 1051 2014). While there have been suggestions that the non-linear movements (“twists 40 41 1052 and turns”) that otariids exhibit while diving decrease the capacity for ODBA to 42 43 44 1053 predict V O 2 (see Ware et al. 2016), this hurdle is still only theoretical. In the end, 45 46 1054 while ODBA may eventually prove to be a reasonable way to predict the cost of 47 48 49 1055 diving in wild, free-swimming sea lions, significant questions remain regarding its 50 51 1056 future application and interpretation. 52 53 54 55 1057 Diving behaviour: 56 57 58 1058 An alternate, potentially simpler method to determine energy expenditure of diving 59 60 61 62 63 Diving metabolism of Steller sea lions 50 64 65 1 2 3 4 1059 sea lions using behavioural time budgets was also tested at the Open Water Station. 5 6 7 1060 Traditionally, time budgets have been used to estimate the energy expenditure of 8 9 1061 wild animals by employing single value estimates of energetic costs of defined 10 11 12 1062 activities. For example, the amount of time “diving” would be multiplied by a single 13 14 1063 estimated rate cost, allowing no flexibility for differences in diving behaviours 15 16 17 1064 normally exhibited by animals. This level of estimation might be suitable for many 18 19 1065 ecological questions, but cannot be used to estimate the energetic consequences of 20 21 22 1066 differences in diving behaviours between individuals or changes across time, such 23 24 1067 as those induced by environmental changes. 25 26 27 28 1068 As already noted, previous work in our lab clearly indicated that changes in the 29 30 1069 behaviour and structure of a dive affected its energetic cost (e.g., Fahlman et al. 31 32 33 1070 2008c; Hastie et al. 2006a). To that end, we investigated whether a foraging dive 34 35 1071 could be broken down into its major components — surface time, transiting to and 36 37 38 1072 from depth, and bottom time — to inform a mathematic model that could be used to 39 40 1073 predict total V O 2 using these gross behavioural categories (Goundie et al. 2015b). 41 42 43 1074 This study measured DMR of four Steller sea lions performing 4 types of dives to 10 44 45 1075 and 40 m. The dives differed in the amount of bottom time (including a V-shaped 46 47 48 1076 “bounce” dive) and level of bottom activity such that estimates could be made of the 49 50 1077 separate costs of 4 different dive components: surface time, transiting to and from 51 52 53 1078 depth, resting bottom time, active bottom time. It also allowed for comparisons to be 54 55 1079 made between the costs of single dives and dive bouts. Not surprisingly, the sea 56 57 -1 - 58 1080 lions’ DMR was higher while transiting to and from depth (20.5±13.0 ml O2 min kg 59 60 1081 1) than while swimming and “foraging” at depth (13.5±4.1 ml O2 min-1 kg-1), both of 61 62 63 Diving metabolism of Steller sea lions 51 64 65 1 2 3 4 1082 which were higher than when at the surface (9.2±1.6 ml O2 min-1 kg-1). A resulting 5 6 7 1083 time-energy model that incorporated these individual behavioural cost estimates 8 9 1084 accurately predicted oxygen consumption for both the time that sea lions were 10 11 12 1085 submerged (within 9.5%) and for complete dive cycles (submerged and subsequent 13 14 1086 surface time, within 7.7%). 15 16 17 18 1087 The results also explained why diving costs were higher for dive bouts than for 19 20 1088 single dives that had equal total bottom times. We found that the DMR of a single 21 22 23 1089 dive bout with a 3-minute bottom time (14.4±3.1 ml O2 min-1 kg-1) was significantly 24 25 1090 lower than a bout of 3 dives, each with a 1-minute bottom time (18.0±2.8 ml O2 min- 26 27 28 1091 1 kg-1). The difference in costs directly related back to differences in the behaviour 29 30 1092 during the dive. Specifically, a higher proportion of time during a dive bout is spent 31 32 33 1093 performing the more energetically expensive transiting behaviour compared to an 34 35 1094 equivalent single dive. 36 37 38 39 1095 This difference in costs between single dives and dive bouts also has several impacts 40 41 1096 on overall foraging efficiency. Assuming that bottom time scales to energy intake 42 43 44 1097 opportunities (foraging), then the higher costs of dive bouts would lead to a lower 45 46 1098 overall energetic efficiency compared to single dives. However, the sea lions were 47 48 49 1099 more efficient at replenishing their oxygen stores when undertaking bout dives 50 51 1100 compared to single dives, as determined by total time required to be at the surface 52 53 54 1101 before full apparent metabolic recovery (including inter-dive surface intervals for 55 56 1102 bouts). As a result, the sea lions could therefore spend a greater portion of their total 57 58 59 1103 time foraging while undertaking bout dives. So, although single dives were 60 61 62 63 Diving metabolism of Steller sea lions 52 64 65 1 2 3 4 1104 energetically less costly than dive bouts, dive bouts were more efficient in terms of 5 6 7 1105 long-term foraging gain. 8 9 10 1106 Insight to date: 11 12 13 14 1107 To date, we have examined numerous methods for accurately measuring the energy 15 16 1108 expenditure of Steller sea lions in the wild. Some, such as heart rate, demonstrate 17 18 19 1109 high statistical relationships, but may be limited by logistical issues. Body 20 21 1110 acceleration has shown promise in a number of terrestrial species, but its predictive 22 23 24 1111 abilities for air-breathing diving vertebrates remains contested. We have also 25 26 1112 examined the use of simpler metrics, such as time-energy budgets, that seem 27 28 29 1113 statistically robust and are relatively simple to apply. Although no single measure 30 31 1114 has emerged as a superior method of estimating energy expenditure in wild marine 32 33 34 1115 mammals, our studies have made it clear that appropriate calibration trials, across a 35 36 1116 range of realistic behaviours, are essential before any such techniques can be 37 38 39 1117 employed in the field. 40 41 42 1118 Past progress and future research 43 44 45 46 1119 The research program with the trained Steller sea lions at the Open Water Research 47 48 1120 Station was designed to answer what we originally, naively conceived to be a simple 49 50 51 1121 question: “What is the diving metabolic rate of Steller sea lions?” It quickly became 52 53 1122 apparent that this question was unsophisticated. The more pertinent, and far more 54 55 56 1123 interesting, question became “What is the diving metabolic rate of Steller sea lions 57 58 1124 under different circumstances, and what are the energetic and ecological 59 60 61 62 63 Diving metabolism of Steller sea lions 53 64 65 1 2 3 4 1125 consequences?” This more complex view, in turn, affected the pursuit of our 5 broad 5 6 7 1126 program objectives: 8 9 10 1127 1. Measure the energetic cost of diving. 11 12 13 1128 2. Investigate the relationship between individual physiology and diving 14 15 1129 capacity. 16 17 18 1130 3. Investigate the physiological mechanisms that underpin dive behaviour. 19 20 1131 4. Test specific optimal foraging theories for diving mammals. 21 22 23 1132 5. Develop and test proxies for energy expenditure that could be used to 24 25 1133 measure the energy expenditure of diving in wild sea lions. 26 27 28 29 1134 Over the past decade, we made extensive progress towards these goals, as 30 31 1135 illustrated in Figure 4. Conducting studies with trained animals under a carefully 32 33 34 1136 controlled set of environmental and physiological conditions allowed us to quantify 35 36 1137 the effect of specific, known, controlled changes on dive behaviour and energetics. 37 38 39 40 1138 We have realized that there is not a single “cost to diving”, but rather that it varies 41 42 1139 with a number of behavioural (i.e., activity level, dive profile) and physiological (i.e., 43 44 45 1140 dive response, nutritional status) parameters. While metabolic rate while diving 46 47 1141 may be lower than that at the surface under a set of specific conditions, these do not 48 49 50 1142 reflect natural circumstances. When undertaking more natural diving behaviour, the 51 52 1143 cost of diving and foraging is affected by the type of diving (dive bouts or single 53 54 55 1144 dives), the level of underwater activity, the dive duration and depth, and the 56 57 1145 nutritional status and physical condition of the animal. These factors also affect the 58 59 60 1146 aerobic diving capacity of individual animals, although behavioural motivation 61 62 63 Diving metabolism of Steller sea lions 54 64 65 1 2 3 4 1147 (perhaps tied to nutritional status and season) may blunt the influence (although 5 6 7 1148 not the physiological consequences) of the aerobic dive limit on actual dive 8 9 1149 behaviour. 10 11 12 13 1150 Of course, the usual motivation for diving is to undertake foraging. Our studies have 14 15 1151 quantified the trade-offs that sea lions must make when balancing their time at 16 17 18 1152 depth acquiring prey and at the surface acquiring oxygen (and, perhaps more 19 20 1153 importantly, offloading CO2 and other metabolic byproducts). These have also 21 22 23 1154 demonstrated the potential energetic effects of changes in their environment, such 24 25 1155 as changes in the distribution (depth) or abundance of their prey. 26 27 28 29 1156 Through such controlled manipulations we have been able to test specific 30 31 1157 hypotheses, such as those modelling the optimal foraging and diving of pinnipeds 32 33 34 1158 and other marine mammals. Obviously, we have not tested all possible variables. 35 36 1159 However, our research program has revealed a consistent theme — that dive 37 38 39 1160 behaviour and physiology can vary considerably. While variability between 40 41 1161 individuals has often been discussed, these data reveal the wide extent of within- 42 43 44 1162 individual variability. Taken together, they provide the first comprehensive look at 45 46 1163 both physiological and behavioural plasticity in a foraging marine mammal. As the 47 48 49 1164 complexity and interconnectedness of physiology and behaviour become better 50 51 1165 understood, these insights will be critical for understanding individual limits and 52 53 54 1166 the ability of species to cope with changing habitat and altered environmental 55 56 1167 conditions. 57 58 59 60 61 62 63 Diving metabolism of Steller sea lions 55 64 65 1 2 3 4 1168 The breakthroughs we achieved were made possible by eliminating the physical 5 6 7 1169 constraints usually associated with working with trained marine mammals. Our 8 9 1170 experimental set-up allows our trained Steller sea lions to mimic the diving 10 11 12 1171 behaviour of their wild counterparts. That is not to say that the range of 13 14 1172 environmental conditions the trained sea lions are exposed to is identical to that 15 16 17 1173 experienced by animals in the wild. However, this is one of the strengths of the 18 19 1174 experimental paradigm. By controlling which aspects of the sea lion’s biotic or 20 21 22 1175 abiotic environment are varied or held constant, it is easier to define their effect. 23 24 1176 Additionally, such experimental manipulations can shed light on how tightly specific 25 26 27 1177 aspects of behaviour and physiology are ingrained, or the scope of their potential 28 29 1178 expression. Such investigations can further inform future studies about what might 30 31 32 1179 be important factors to consider (such as the effect of disturbance on diving 33 34 1180 behaviour and physiology). 35 36 37 38 1181 In addition to understanding the effects of biotic and abiotic changes on sea lion 39 40 1182 foraging behaviours, our studies also tested a number of theories that examine the 41 42 43 1183 motivations and consequences to an animal for extending dives beyond its aerobic 44 45 1184 dive limit. These have primarily taken the form of measuring the energetic and 46 47 48 1185 behavioural consequences of increased dive duration. A limit to all of these studies, 49 50 1186 however, has been an inability to actually determine ADL, instead having to rely 51 52 53 1187 upon comparison of observed rates of oxygen consumption with estimated body 54 55 1188 oxygen reserves (e.g., Gerlinsky et al. 2014b; Gerlinsky et al. 2013). The next obvious 56 57 58 1189 step is to integrate measures of blood PO2 during dives and post-dive blood lactate 59 60 1190 levels to directly measure ADL. Such measures have only been obtained for a single 61 62 63 Diving metabolism of Steller sea lions 56 64 65 1 2 3 4 1191 Otariid species to date (California sea lion; Ponganis et al. 1997). Similar 5 6 7 1192 experiments with our Steller sea lions would provide valuable insight into their 8 9 1193 physiological capacity. 10 11 12 13 1194 While this review has focussed on our studies of sea lion diving energetics and 14 15 1195 physiology, this unique experimental set-up can be used to conduct other avenues of 16 17 18 1196 scientific inquiry. For example, we have conducted studies examining the ways that 19 20 1197 sea lions compensate for changes in buoyancy (Fahlman et al. 2008a), and shown 21 22 23 1198 the significant effects of ocean currents and swimming depth on surface swimming 24 25 1199 costs and behaviour in Steller sea lions (Hindle et al. 2010a). We have also 26 27 28 1200 conducted studies exploring the possible use of blood microparticles in diving sea 29 30 1201 lions as a biomarker for decompression sickness in marine mammals, such as that 31 32 33 1202 hypothesised to result from sudden changes in diving behaviour induced by 34 35 1203 anthropogenic sound exposure (Fahlman et al. 2016). 36 37 38 39 1204 Our ability to control and monitor the behaviour, energetics, and behaviour of the 40 41 1205 sea lions has also provided opportunities to develop, test, and calibrate methods and 42 43 44 1206 technologies to study wild sea lions. For example, in addition to the previously 45 46 1207 described studies of the efficacy of using heart rate or accelerometry to predict 47 48 49 1208 energy expenditure in sea lions, we have also sought to develop new devices and 50 51 1209 methods to predict energy expenditure (Ware et al. 2016). We have also conducted 52 53 54 1210 studies testing a satellite-linked fluorometer mounted on sea lions to monitor 55 56 1211 zooplankton (Lander et al. 2015), thereby turning sea lions into oceanographic 57 58 59 1212 platforms of opportunity. 60 61 62 63 Diving metabolism of Steller sea lions 57 64 65 1 2 3 4 1213 Ultimately, understanding the physiology, energetics, and behaviour of diving in sea 5 6 7 1214 lions is not just important for matters of comparative physiology or testing optimal 8 9 1215 foraging theories. This information is also central to aspects of conservation ecology. 10 11 12 1216 The interaction between marine mammals and their prey base is becoming of 13 14 1217 increasing importance as potential conflicts between human activities (fishing, 15 16 17 1218 climate change) and populations become central in discussions of resource 18 19 1219 partitioning and species conservation. The cost of diving and foraging has a major 20 21 22 1220 impact on the total prey resources required by marine mammal populations, and the 23 24 1221 potential impact those populations have on prey resources. Conversely, the 25 26 27 1222 behavioural and energetic consequences of changes in an individual sea lion’s biotic 28 29 1223 and abiotic environment are ultimately constrained by their physiological capacity. 30 31 32 1224 Understanding the interactions between these process will not only inform how a 33 34 1225 sea lion might respond to environmental changes, but also their capacity for 35 36 37 1226 compensation and the consequences for failing to adjust to their new conditions. 38 39 40 1227 Acknowledgements 41 42 43 44 1228 The studies we described reflect the tremendous efforts of a large team of 45 46 1229 researchers, graduate students, research assistants, trainers, and veterinary staff. 47 48 49 1230 We would particularly like to acknowledge the contributions of Andreas Fahlman to 50 51 1231 the research program and to this manuscript. We would also like to thank the 3 52 53 54 1232 anonymous reviewers for there suggestions on improving this review. The research 55 56 1233 was funded through a number of sources, including grants provided by the Natural 57 58 59 1234 Sciences and Engineering Research Council (Canada) and from the US National 60 61 62 63 Diving metabolism of Steller sea lions 58 64 65 1 2 3 4 1235 Oceanic and Atmospheric Administration to the North Pacific Universities Marine 5 6 7 1236 Mammal Research Consortium through the North Pacific Marine Science Foundation. 8 9 1237 All studies were carried out under Animal Care Permits issued by the Vancouver 10 11 12 1238 Aquarium and the University of British Columbia. 13 14 15 1239 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 Diving metabolism of Steller sea lions 59 64 65 1 2 3 4 1240 REFERENCES CITED 5 6 7 1241 Andersen HT (1966) Physiological adaptations in diving vertebrates. 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J Comp Physiol B 185 (6):695-708. 5 6 7 1504 doi:DOI:10.1007/s00360-015-0911-y 8 9 1505 Ware C, Trites AW, Rosen DAS, Potvin J (2016) Averaged Propulsive Body 10 11 12 1506 Acceleration (APBA) can be calculated from biologging tags that incorporate 13 14 1507 gyroscopes and accelerometers to estimate swimming speed, hydrodynamic 15 16 17 1508 drag and energy expenditure for Steller sea lions. PloS one 11 (6):e0157326 18 19 1509 Williams TM, Fuiman LA, Kendall T, Berry P, Richter B, Noren SR, Thometz N, 20 21 22 1510 Shattock MJ, Farrell E, Stamper AM (2015) Exercise at depth alters 23 24 1511 bradycardia and incidence of cardiac anomalies in deep-diving marine 25 26 27 1512 mammals. Nature communications 6:6055 28 29 1513 Williams TM, Kooyman GL, Croll DA (1991) The effect of submergence on heart rate 30 31 32 1514 and oxygen consumption of swimming seals and sea lions. J Comp Physiol B 33 34 1515 160:637-644 35 36 37 1516 Wilson RP, White CR, Quintana F, Halsey LG, Liebsch N, Martin GR, Butler PJ (2006) 38 39 1517 Moving towards acceleration for estimates of activity-specific metabolic rate 40 41 42 1518 in free-living animals: the case of the cormorant. J Anim Ecol 75:1081-1090 43 44 1519 Young BL, Rosen DAS, Haulena M, Hindle AG, Trites AW (2011a) Environment and 45 46 47 1520 feeding change the ability of heart rate to predict metabolism in resting 48 49 1521 Steller sea lions (Eumetopias jubatus). J Comp Physiol B 181:105-116 50 51 52 1522 Young BL, Rosen DAS, Hindle AG, Haulena M, Trites AW (2011b) Dive behaviour 53 54 1523 impacts the ability of heart rate to predict oxygen consumption in Steller sea 55 56 57 1524 ions (Eumetopias jubatus) foraging at depth. J Exp Biol 214:2267-2275 58 59 60 61 62 63 Diving metabolism of Steller sea lions 72 64 65 1 2 3 4 1525 Zapol WM, Liggins GC, Schneider RC, Qvist J, Snider MT, Creasy RK, Hochachka PW 5 6 7 1526 (1979) Regional blood flow during simulated diving in the conscious Weddell 8 9 1527 seal. J Appl Physiol 47 (5):968-973 10 11 12 1528 Zuur A, Ieno EN, Walker N, Saveliev AA, Smith GM (2009) Mixed effects models and 13 14 1529 extensions in ecology with R. Springer Science & Business Media, 15 16 17 1530 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 Diving metabolism of Steller sea lions 73 64 65 1 2 3 4 1531 FIGURE CAPTIONS 5 6 7 1532 8 9 1533 Figure 1. Photograph of the experimental set-up for diving trials. Panel A: The sea 10 11 12 1534 lions are trained to dive between a respirometry dome situated in the middle of the 13 14 1535 floating barge (foreground) to the bottom of feeding stations that deliver 20 g pieces 15 16 17 1536 of fish to specific depths. A research boat (seen in the background) carries the 18 19 1537 associated respirometry equipment and support personnel. Sea lions are 20 21 22 1538 transported to the dive trial site on a separate vessel (seen on left). Panel B: When 23 24 1539 performing trials in the open water, the sea lions carry assorted scientific and safety 25 26 27 1540 equipment on a custom-fit harness. Panel C: Aerial view of the Open Water Station 28 29 1541 in Port Moody, BC. showing the laboratory (foreground) and flow-through water 30 31 32 1542 pens for sea lions. When not actively participating in trials, the animals interact daily 33 34 1543 in the adjacent waters with training and research staff. 35 36 37 1544 38 39 1545 Figure 2. Schematic of typical set-up for diving experiments at the Open Water 40 41 42 1546 Station with Steller sea lions. A floating platform holds the respirometry dome, and 43 44 1547 is connected to gas analyzers and flow generators on an accompanying research 45 46 47 1548 vessel. The platform also has a nylon webbed enclosure (not shown) that can be 48 49 1549 used to encourage the sea lions to remain at the surface (but does not physically 50 51 52 1550 restrict them). The sea lions are trained to dive between the respirometry dome and 53 54 1551 the bottom of a pair of PVC tubes placed at desired depths (feeding stations). Pieces 55 56 57 1552 of fish are alternately pumped to the bottom of the two tubes at given rates to 58 59 1553 represent different quality prey patches (which may also be at different depths). 60 61 62 63 Diving metabolism of Steller sea lions 74 64 65 1 2 3 4 1554 Dive durations can be either chosen by the sea lion or controlled by fish delivery or 5 6 7 1555 light signals at depth. 8 9 1556 10 11 12 1557 Figure 3. Schematic representing measurement of diving metabolic rate (DMR) and 13 14 1558 average metabolic rate (AMR) of diving sea lions from recorded changes in 15 16 17 1559 spontaneous oxygen consumption. Panel A represents a theoretical series of 3 dives 18 19 1560 to 40 meters, with submergence indicated by the grey bars. DMR (Panel B) is 20 21 22 1561 measured as the post-dive increase in total oxygen consumption above resting 23 24 1562 (MRs) levels (identified by the striped areas) divided by the actual submergence 25 26 27 1563 time (identified by arrows). AMR (Panel C) is calculated from total oxygen 28 29 1564 consumption (integrated above “zero”) from the start of the dive until the rate of 30 31 32 1565 oxygen consumption reaches the original steady state (MRs), divided by the time 33 34 1566 from the start of the dive until recovery is reached. These measures can be applied 35 36 37 1567 to either single dives (which are followed by complete recovery) or bouts of 38 39 1568 multiple dives. As individual dives within a dive bout are not physiologically 40 41 42 1569 independent (due to incomplete oxygen recovery), only a single value for either 43 44 1570 DMR or AMR can be obtained for a given bout. 45 46 47 1571 48 49 1572 Figure 4. Schematic outlining major study questions and results arising from the 50 51 52 1573 Steller sea lion Open Water research program. 53 54 55 56 57 58 59 60 61 62 63 Diving metabolism of Steller sea lions 75 64 65
Does nutri Diving metabolism can be ~55% of surface rate, Has$e et al. 2007, BUT metabolism is higher under more realis Yes, ADL is affected by Diving metabolism affected by dive dura How does the ADL constrain Does DMR reflect physiological How does dive behaviour affect diving in Steller sea lions? adapta Dives in bouts appear Minimum heart Heart rate is not related Different dive phases constrained by ADL; single dives rate decreases with to physical ac ODBA differs between However, predic Yes, ODBA predicts No, ODBA does not VO2 at surface and predict VO2 within Yes, in non-diving sea lions. Yes, if dives are broken down while diving. different dive types. McPhee et al. 2003 into major phases. Fahlman et al. 2008c Volpov et al. 2015 Young et al. 2011b Goundie et al. 2015a Body accelera Are there reasonable proxies for es