A product of its environment: the epaulette shark (Hemiscyllium ocellatum) exhibits physiological tolerance to elevated environmental CO2

Ocean acidification is predicted to affect the performance of marine species, but little is known about the effects on sharks. We found that long-term exposure to elevated CO2 did not affect the epaulette shark, possibly because it experiences fluctuating environmental conditions in its shallow coral reef habitat.


Introduction
Anthropogenic CO 2 emissions have caused an increase in atmospheric CO 2 by almost 40% over the past 250 years (IPCC, 2013). The resulting rise from pre-industrialization levels (~280 ppm) to 400 ppm in 2014 has occurred at a rate unprecedented for the past 800 000-1 000 000 years (Raven et al., 2005;Doney and Schimel, 2007;Lüthi et al., 2008). The oceans have absorbed more than 30% of the additional CO 2 released by human activities, thus tempering the atmospheric rise in CO 2 (Sabine et al., 2004;Sabine and Feely, 2007). However, the resulting rise in seawater CO 2 partial pressure (PCO 2 ) and the associated reduction in pH, called ocean acidification, is a significant threat to marine organisms and ecosystems (Hoegh-Guldberg et al., 2007;Fabry et al., 2008).
The reduced carbonate saturation state that accompanies lower seawater pH affects the ability of calcifying marine organisms to form carbonate shells and skeletons (Orr et al., 2005;Doney et al., 2009), but rising oceanic CO 2 may also impact the respiratory physiology of many water-breathing organisms. Acid-base disturbances related to elevated environmental CO 2 can reduce oxygen uptake and delivery, which could directly impact metabolic performance. Reductions in an organism's scope for aerobic metabolic performance can result in less energy being available for crucial life-history processes, such as growth and reproduction (Pörtner, 2008;Pörtner and Farrell, 2008). For instance, Humboldt squid (Dosidicus gigas) exhibit a 30% reduction in resting metabolic rate and a 45% decrease in activity upon exposure to projected near-future CO 2 levels, owing to an impaired oxygen transport system, which would be predicted to reduce overall performance and compress their habitable depth range (Rosa and Seibel, 2008). In contrast, teleost fishes are expected to be physiologically well equipped to compensate pH and ion disturbances caused by high CO 2 (Ishimatsu et al., 2008;Brauner and Baker, 2009). Nevertheless, interspecific variation is evident in the physiological responses of teleost fish to elevated CO 2 ; for example, some fishes exhibit no change in aerobic scope in high-CO 2 environments (Ishimatsu et al., 2008;Melzner et al., 2009a;Couturier et al., 2013), whereas others reduce aerobic scope (Munday et al., 2009) and some even increase aerobic scope (Couturier et al., 2013; 2013a) when exposed to near-future CO 2 levels. Consequently, the effects of ocean acidification on a broad range of species, including vulnerable and tolerant species, should be investigated in order to identify traits that will be important for individual performance and success in near-future oceans and predict changes in community structure (Melzner et al., 2009b).
In contrast to the growing body of knowledge about the effects of ocean acidification on teleost fishes, little is known about the impacts of rising levels of oceanic CO 2 on elasmobranchs. Elasmobranchs buffer a pH disturbance, such as that associated with exposure to high CO 2 , in a manner similar to teleosts. Bicarbonate is accumulated in the blood, but in addition, elasmobranchs may also increase branchial ammonia excretion rates to ameliorate the acidosis further (Evans, 1982;King and Goldstein, 1983;Claiborne and Evans, 1992;Brauner and Baker, 2009;Tresguerres et al., 2010). The haemoglobin of elasmobranchs also has a much higher buffering capacity compared with that of most teleosts, and thus, O 2 transport and aerobic performance may be less sensitive to pH disturbances (Berenbrink et al., 2005). Yet, it is thought that the resilience of elasmobranchs to acidbase disturbances is related largely to their sophisticated acid excretion processes at the gill (Wood et al., 1995). If elasmobranchs are notably tolerant to near-future CO 2 conditions, this could potentially increase predation pressure and alter species compositions of marine environments.
The epaulette shark (Hemiscyllium ocellatum) exhibits exceptionally high tolerance to the severe hypoxia (low oxygen) that it routinely experiences while inhabiting shallow coral reef flats (Routley et al., 2002;Nilsson and Renshaw, 2004), and thus, it may not be surprising if this species is also tolerant to near-future CO 2 . However, acute responses may differ dramatically from the responses to long-term exposure; studies on H. ocellatum in response to anoxia or hypoxia have been following only minutes to hours of exposure (Wise et al., 1998;Renshaw et al., 2002;Routley et al., 2002;Chapman and Renshaw, 2009;Dowd et al., 2010;Speers-Roesch et al., 2012a). No study, to date, has examined how H. ocellatum responds to prolonged exposure to elevated CO 2 . Given that increased uptake of CO 2 by the ocean will affect both the average CO 2 levels and the magnitude of extreme CO 2 levels (Shaw et al., 2013), it is important to consider longer-term responses to elevated CO 2 beyond those that would be experienced on a diurnal basis (e.g. hours; Ohde and van Woesik, 1999;Compagno, 2002;Last and Stevens, 2009;Shaw et al., 2013). Thus, both the physiological sensitivity of the organism and the variations it may already be experiencing in its habitat are important when considering which species will exhibit positive or negative responses to rising ocean CO 2 levels. However, it is also important to consider the relationship between environmental cues and other traits, such as behaviour-which is especially relevant to species like H. ocellatum-when considering the importance of phenotypic plasticity, because this could influence selection over the longer term (Marais and Chown, 2008).
We exposed H. ocellatum to near-future CO 2 conditions for a minimum of 60 days and measured resting oxygen consumption rates and critical oxygen tensions as proxies for resting metabolic rate and sensitivity to hypoxia, respectively. In addition to whole-organism responses, we also measured or calculated haematological and tissue parameters, including plasma ionic (HCO 3 − , Cl − , Na + and K + ) and urea concentrations, haemoglobin (Hb), mean cell haemoglobin concentration (MCHC), haematocrit (Hct), spleen-somatic index (SSI) and citrate synthase activity in heart, brain and red muscle. The aim was to provide insight into the physiological parameters that may underpin changes in metabolic performance and sensitivity to hypoxia in this species.
We hypothesized that H. ocellatum can physiologically tolerate elevated CO 2 because it routinely experiences daily reductions in environmental O 2 (Routley et al., 2002;Nilsson and Renshaw, 2004) and probably elevations in CO 2 . However, if CO 2 tolerance is related to the diurnal patterns this species already experiences in their natural habitat, prolonged exposure (60 days) to elevated CO 2 may negatively affect metabolic rate and hypoxia tolerance.

Experimental animals
Epaulette sharks (Hemiscyllium ocelatum) were collected from the northern Great Barrier Reef by Northern Barrier and Cairns Marine (Cairns, Queensland, Australia) and transported to James Cook University (JCU). Five to six individuals were placed in each of six 700 l tanks in a recirculating seawater system. Individuals were measured [standard length, 33.38 ± 7.29 cm (mean ± SD)] to ensure an equal distribution of sizes among tanks. Unique fin clips along the margins of pectoral, pelvic and dorsal fins were used for individual identification. Shelter was provided in the form of PVC pipe sections placed within each tank. Food was provided once every 24 h and consisted of 4% of shark biomass per tank in raw prawn meat. There was no indication that any individuals or treatment groups were eating less than this amount throughout the duration of the study. Sharks were acclimated to laboratory conditions for at least 4 weeks prior to commencing CO 2 treatments.

Experimental CO 2 conditions
The experimental system comprised three 8000 l recirculating seawater systems, each set to simulate one of the following three CO 2 treatments: control (~390 µatm); medium (~600 µatm); and high (~880 µatm). Carbon dioxide levels were achieved and maintained by CO 2 infusion of seawater in 3000 l sumps attached to each recirculating seawater system. The pH NBS (National Bureau of Standards scale) levels were set to match target CO 2 concentrations and maintained using a CO 2 -infusing system (Aqua Medic GmbH, Bissendorf, Germany). If the pH rose above the set point, an electronic solenoid initiated the system to deliver a steady stream of CO 2 into a diffuser within the corresponding sump. Carbon dioxide-equilibrated seawater from each system was delivered to two replicate 700 l tanks (~25 l min −1 ) per treatment. Each tank contained five or six sharks, as described above. This central approach of pH manipulation allowed for stability in seawater pH and PCO 2 within the holding tanks. Tanks were covered with transparent plastic sheeting to reduce CO 2 loss to the atmosphere.
The pH NBS was measured daily (Hach, model #HQ40d) in each tank to ensure that it remained within ±0.05 of desired levels. Temperatures were also measured daily and maintained at 28.5°C by automated heater/chillers attached to each seawater system. Salinity and alkalinity were measured on a weekly basis. Total alkalinity (TA) was estimated using Gran titrations and certified reference materials (Dr A. G. Dickson, Scripps Institution of Oceanography). Average seawater PCO 2 was calculated using these parameters in CO2SYS (Pierrot et al., 2006) and using constants from Dickson and Millero (1987) (Table 1).
Sharks were introduced to the CO 2 treatments following 30 days acclimation to laboratory holding conditions and were then maintained in their respective CO 2 treatment conditions for a minimum of 60 days prior to physiological experimentation.

Resting oxygen consumption rates
Resting O 2 consumption rates (Ṁ O 2Rest ) were determined for sharks following 60-68 days of exposure to control (n = 10), medium (n = 12) and high (n = 11) CO 2 conditions and a 48 h fasting period using an intermittent-flow respirometry system with purpose-built respirometry chambers. Animals were transferred individually into the cylindrical 11 or 15 l respirometry chambers (depending on animal body size) submerged in a temperature-controlled aquarium (28.5°C) within each animal's respective experimental CO 2 conditions and habituated to the chamber for 12 h before oxygen consumption measurements commenced. Submersible pumps were fitted to each chamber to supply a continuous water flow (1300 l h −1 ; WEIPRO WH-2000; Yongcheng Aquarium Co., Ltd, Guangdong, China) from the surrounding water bath through the chambers. During respirometry trials, a digital relay timer (MFRT-1 Multi Function Recycling Timer; Xiamen SUPERPRO Technology Co., Ltd, Xiamen, Fujian, China) was used to stop water flow for 15 or 20 min and then resume flushing for 15 min over a total period of 12 h. The intervals of interrupted water flow were short enough to ensure that oxygen within the chambers did not fall below 80% saturation at any time, while flush periods were long enough to eliminate accumulation of metabolic CO 2 and allow water oxygen levels to return to 100% saturation (Steffensen et al., 1984;Steffensen, 1989). A second pump (1300 l h −1 ; WEIPRO WH-2000) was connected to each respirometry chamber to recirculate water continuously within the chamber, thus ensuring complete mixing and homogeneous water PO 2 (P W O 2 ). Contactless spots (2 mm) with oxygen-sensitive REDFLASH dye were adhered to the inside of glass tubes connected to the recirculating pumps on each respirometer. These spots were then linked to a Firesting Optical Oxygen Meter (Pyro Science e. K., Aachen, Germany) via 5 m fibre-optic cables to record continuously (0.5 Hz) the temperature-compensated O 2 concentration (in milligrams per litre) of the water within each chamber over the 12 h period of time. The 0 and 100% oxygen levels of the Firesting oxygen meter were calibrated using 0 and 100% air-saturated seawater. At the end of each trial, the wet mass was taken for each shark [232.47 ± 117.98 g (mean ± SD)] prior to release back to experimental holding conditions.

Critical oxygen tension
Upon completion of Ṁ O 2Rest measurements, sharks were permitted to recover in their respective CO 2 treatment conditions for ~3 weeks. Then, the same respirometers used to determine Ṁ O 2Rest were used to determine the critical oxygen tension (P crit ) for the same sharks exposed to control (n = 9), medium (n = 12) and high CO 2 (n = 10). By this point, sharks would have been exposed to their respective experimental conditions for 85-92 days. Prior to measurements, sharks were fasted for 48 h before being introduced to the cylindrical 11-15 l respirometry chambers. Then, the Ṁ O 2 of each animal was monitored for a minimum of 4 h using an intermittent flush cycle (15 min flush-15 min closed) so that stable Ṁ O 2Rest was achieved prior to commencing the hypoxia experiment. The respirometers were then sealed by turning off flush pumps and closing previously installed ball-valves downstream of the flush pumps. Oxygen levels in the chamber were monitored continuously (0.5 Hz) and allowed to decrease to at least 0.8 mg l −1 to ensure that the critical oxygen tension for each individual was recorded (based on estimates from Routley et al., 2002). The changes in water pH and PCO 2 that occur when using closed respirometry for a short period of time have been shown previously to have no effect on P crit in fish (Henriksson et al., 2008). After this oxygen concentration was achieved, the aforementioned flush cycle was reinstated such that O 2 levels within each respirometer could quickly return to normoxic conditions (100% airsaturated seawater).

Haematological and tissue analyses
Following P crit measurements, animals were returned to their treatment tanks to recover for ~1 week. After this time, blood was sampled from sharks exposed to control (n = 8), medium (n = 10) and high (n = 8) CO 2 conditions by inserting a 23 gauge needle posterior to the cloaca into the caudal vein and collecting the blood (<1% of body volume) into heparinized syringes. Animals were then euthanized by severing the spinal cord using the method described by Speers-Roesch et al. (2012b) so that tissues could be sampled. Whole blood [Hb] was determined using the HemoCue ® (Hb 201 System, Australia Pty Ltd) with 10 µl of whole blood and was reported as grams per 100 ml using a calibration curve according to Clark et al. (2008) corrected for tropical reef species by Rummer et al. (2013b). The Hct was determined by centrifuging 60 µl of whole blood in heparinized microcapillary tubes for 3 min at 17 000g and calculated as the ratio of packed red blood cells to total blood volume (as a percentage). Both [Hb] and Hct were used to calculate the MCHC. The spleen was dissected from each shark and weighed to the nearest 0.001 g. The SSI was calculated as the ratio of the spleen to body mass (as a percentage). Plasma was flash frozen immediately in liquid nitrogen and then stored at −80°C until analysis for [HCO 3 − ] via colorometrically linked enzyme assay and for [Na + ], [K + ], [Cl − ] (1:1 dilution with deionized water) and [urea] (1:19 dilution with deionized water) via ion-specific electrodes (ISE; Beckman Coulter System AU480). Heart, brain and red muscle samples were also collected and frozen in liquid N 2 for citrate synthase enzyme analysis according to McClelland et al. (2005). Briefly, frozen tissues were homogenized in a standard buffer solution containing 5 mm EDTA, 0.1% Triton X-100, 0.2 mm dithiothreitol and 50 mm Hepes (adjusted to pH 7.4) and stored at −80°C. The citrate synthase assay buffer contained (mm): 20 Tris (pH 8.0), 0.1 5,5-dithiobis (2-nitrobenzoic acid) and 0.3 acetyl-CoA. The reaction was initiated by the addition of 0.5 mm oxaloacetate, and absorbance was measured for 5 min at 412 nm. Control samples were assayed without oxaloacetate to control for background hydrolase activity.

Calculations and statistical analyses
Raw text files created for the Firesting recordings were imported offline into LabChart version 6.1.3 (ADInstruments, Colorado Springs, CO, USA), which was used to analyse data. A modified version of equations from Bushnell et al. (1994) and Schurmann and Steffensen (1997)  proportion of background O 2 consumption, which was measured 2-3 h before and after each trial for each respirometer and assumed linear, was subtracted. This value was then multiplied by the volume of the respirometer (in litres; minus the volume of the fish), all of which was divided by the mass of the fish (in kilograms). Respirometers were cleaned daily to ensure that background (microbial) respiration did not exceed 10% of the Ṁ O 2Rest of the sharks. Means and SEM for Ṁ O 2Rest were calculated for each of the three CO 2 treatments.
A similar data extraction and calculation protocol was followed for determining P crit . Again, Ṁ O 2Rest was calculated for each shark from the shallowest 10% of slopes that were recorded prior to sealing the respirometer. Then, the mean slope for every 5 min period of time while the respirometer was sealed was extracted (usually 20-30 slopes), and Ṁ O 2 values were calculated from those slopes. To determine P crit , all Ṁ O 2 values were plotted against the oxygen concentration within the chamber for each shark. A horizontal (regression) line was fitted to the mean Ṁ O 2Rest prior to sealing the respirometer. Then, a linear regression was applied to all of the points that consecutively fell below Ṁ O 2Rest once the respirometer had been sealed. The point at which both regression lines intersected was reported as the critical oxygen tension or P crit (in milligams of O 2 per litre) for that individual ( Fig. 1; Ott et al., 1980;Collins et al., 2013). Means and SEM for P crit were calculated for each CO 2 treatment.
Nested ANOVA, with holding tanks nested within CO 2 treatments, was first used to test whether there was a significant effect of holding tank on mean Ṁ O 2Rest (in milligams per kilogram per hour) or mean P crit (in milligams of O 2 per litre). As there was no significant effect of tanks on either parameter, data from the two tanks within treatments were pooled for further analyses. ANCOVA was used to compare Ṁ O 2Rest among the three CO 2 treatments, with standard length as a covariate. To compare P crit among treatment groups, a robust regression analysis was performed with standard length as a covariate. Robust regression analysis was chosen over ANCOVA for P crit analysis due to potential outliers that could otherwise be solely responsible for significant outcomes. The removal of such outliers was rejected owing to the relatively small sample size. Instead, robust regression weighs values differently based on their chance of being an outlier. Hence, the further away a single data point was from the mean, the less influential it became for the statistical outcome of the analysis. There was no interaction between the main effect (CO 2 ) and the covariate in either analysis; therefore, to increase statistical power, the analyses were run again without this term included. Standard length was not included in haematological and tissue data analyses because it had no significant effect on the outcomes. ANOVAs were then used together with Holm-Sidak post hoc tests to compare haematological and tissue parameters between animals acclimated to control, medium and high CO 2 conditions. Statistical significance was accepted when P < 0.05. All analyses were carried out using S-Plus (TIBCO Software Inc., Palo Alto, CA, USA). Figure 1: Representative trace illustrating the changes in oxygen consumption rate of an individual epaulette shark (Hemiscyllium ocellatum) as the oxygen concentration of the water decreased and the time over which this occurred. The parallel line represents the resting oxygen consumption rate. After the respirometry chamber was sealed, the oxygen consumption rate began to decrease below resting levels. The diagonal line is a trend line, with the intersection of both lines demarcating the critical oxygen tension (P crit ).

Resting oxygen consumption rates
There were no significant differences in Ṁ O 2Rest values between CO 2 treatment groups (F 2,28 = 0.578; P = 0.568). However, Ṁ O 2Rest depended on the standard length of the individuals, with larger animals having a higher Ṁ O 2Rest than smaller animals (F 2,28 = 6.70; P = 0.0151; Fig. 2A). Values for Ṁ O 2Rest ranged from 46.8 to 95.4 mg O 2 kg −1 h −1 with a mean of 65.2 ± 2.13 mg O 2 kg −1 h −1 across all treatments.

Critical oxygen tension
The P crit values did not differ significantly between CO 2 treatment groups (t 4,26 = −0.170; P = 0.866). However, standard length had a significant effect on the P crit of individuals (t 4,26 = 2.26; P = 0.0323; Fig. 2B), with larger animals reaching P crit at a higher seawater O 2 concentration than smaller animals. The P crit values ranged from 1.32 to 5.07 mg O 2 l −1 with a mean of 2.51 ± 0.122 mg O 2 l −1 across all treatment groups.

Discussion
Long-term exposure to near-future CO 2 conditions did not significantly affect metabolic performance or hypoxia sensitivity of epaulette sharks. In contrast, changes in [Hb] and MCHC were evident after ~90 days of exposure to 600 µatm CO 2 levels, and plasma [HCO 3 − ] was elevated in both the moderate and high CO 2 treatment groups, suggesting that physiological adjustments were being made to cope with elevated CO 2 at the level of oxygen transport and ion regulation. However, there was no increase in metabolic capacity at the level of the mitochondria, as indicated by the lack of change in citrate synthase activity. Our findings suggest that, for this reef-inhabiting benthic elasmobranch, neither the energetic costs of basic maintenance nor sensitivity to hypoxia may be compromised in the elevated CO 2 conditions projected for the end of this century.
The compensatory mechanisms used by H. ocellatum to maintain resting metabolic rates in normoxic and hypoxic conditions after prolonged exposure to elevated CO 2 may be linked to maintaining oxygen uptake and delivery and ion regulation. Following ~90 days of CO 2 exposure, epaulette sharks exhibited a significant increase in [Hb] and MCHC. Short-term changes in haematological parameters have been documented in teleosts and elasmobranchs following capture, cannulation and exercise (Soivio et al., 1973;Wood et al., 1977;Turner et al., 1983;Wells et al., 1986), upon acclimation to elevated temperature (adult horn sharks, Heterodontus francisci; Neale et al., 1977) and in response to anoxia (grey carpet shark, Chiloscyllium punctatum, and epaulette shark; Chapman and Renshaw, 2009). In teleosts, acute changes can be associated with adrenergic red blood cell (RBC) swelling (Caldwell et al., 2006), a mechanism in place to protect RBC pH and oxygen transport during stress, 6 Figure 2: The relationship between resting oxygen consumption rate (A) and critical oxygen tension (P crit ; B) with the standard length of individual sharks from control, medium and high CO 2 treatment groups. Please refer to Materials and methods for further details regarding calculations.
but not known to occur in elasmobranchs (Berenbrink et al., 2005). Both teleosts and elasmobranchs do, however, use their spleen to produce and store RBCs (Turner et al., 1983;Fänge and Nilsson, 1985;Lai et al., 2006) and can contract it to increase the proportion of RBCs in the circulation ( Ken-Ichi, 1988;Lai et al., 2006), presumably to aid in oxygen transport (Jensen et al., 1992). We observed a decrease, although non-significant, in the SSI in sharks exposed to both medium and high CO 2 , suggesting that splenic contraction(s) may have occurred at some point during the CO 2 exposure period. Periodic splenic contractions could also increase the proportion of immature RBCs in circulation, which could explain the slight increase in MCHC without significant changes in Hct. The temporal scale of splenic RBC release and subsequent increases in erythropoietin, the glycoprotein responsible for regulating RBC numbers, is well understood for teleosts (Lai et al., 2006) and could be similar in elasmobranchs exposed to elevated CO 2 over extended periods of time, which is worth further investigation.

Plasma [HCO 3
− ] was elevated in sharks upon 90 days of exposure to elevated CO 2 , which indicates some level of long-term acid-base compensation. This finding is supported by studies by Deigweiher et al. (2008), in which acclimation to elevated CO 2 over 6 weeks in a marine teleost resulted in upregulation of Na + /HCO 3 − cotransporters (NBC1) and Na + -K + -ATPase at higher densities. Given the relationship between bicarbonate availability and synthesis of urea (the predominant osmolyte used by most elasmobranchs), acidbase compensatory mechanisms could have affected [urea] and therefore the efficiency of osmoregulatory pathways (Wood et al., 1995). As [urea] did not change with CO 2 exposure, this may not be problematic at the CO 2 levels used here and/or over the 90 day duration. The activity of citrate synthase, the first enzyme of the Krebs cycle located within the mitochondria, can be a good indicator of aerobic capacity. Unchanged citrate synthase activity after prolonged CO 2 exposure further suggests that there is no limitation at the level of aerobic energy production in any of the tested tissues (McClelland et al., 2005). Although there may have been no changes to aerobic capacity, changes may have been occurring in anaerobic pathways (e.g. activity of lactate dehydrogenase, the last enzyme of anaerobic glycolysis) to maintain energy production. This would be worthy of further investigation. , and spleen-somatic index (D) after sharks were exposed to control, medium or high CO 2 for ~90 days. Different letters within a panel demarcate significant differences between treatment groups, and statistical significance is noted in the top right corner of each panel. Abbreviation: NS, not significant.
As Esbaugh et al. (2012) suggest, species that are already adapted to low levels of CO 2 may no longer rely on traditional short-term acid-base compensation strategies but instead use morphological changes (e.g. gill permeability, diffusion distances) or alter chemical equilibrium constraints in the blood over longer periods to maintain oxygen transport.
While there were no changes in metabolic performance in the sharks upon long-term CO 2 exposure, there was an unexpected pattern of mass-specific metabolic rates, with larger sharks exhibiting higher mass-specific metabolic rates than smaller sharks. This contradicts the usual pattern exhibited by ectotherms, but may be related to their feeding patterns. For example, we examined sharks ranging in size from ~20 to 50 cm. However, we used a set 48 h fasting period prior to determining oxygen consumption rates and prior to blood and tissue sampling because of their small size and benthic lifestyle and previous feeding patterns while in 8 Figure 4: Changes in plasma parameters after sharks were exposed to control, medium or high CO 2 for ~90 days. Different letters within a panel demarcate significant differences between treatment groups, and statistical significance is noted in the top right corner of each panel. Abbreviation: NS, not significant.
captivity. It could have been that 48 h was sufficient fasting time for the smaller animals but not for the larger animals of that size range (Wood et al., 2007). Therefore, the larger animals could have been exhibiting slightly increased metabolic rates due to specific dynamic action, which could also mask any acid-base processes occurring due to CO 2 exposure. The relationship between acid-base disturbances originating from feeding and those due to elevated water CO 2 has ecological relevance and should be investigated in future studies.
Environment and lifestyle play an important role in physiological tolerance to changing environmental conditions (Pörtner and Farrell, 2008), and this study confirms that H. ocellatum is no exception. It is already known that H. ocellatum exhibits the lowest value of P crit shown for any elasmobranch tested to date, suggesting an exceptional tolerance to short-term hypoxia, which is unique among chondrichthyans (Wise et al., 1998;Routley et al., 2002). Hemiscyllium ocellatum occupies shallow reef platforms that are subject to dramatic diurnal fluctuations in environmental O 2 and CO 2 conditions (Routley et al., 2002;Diaz and Breitburg, 2009;Last and Stevens, 2009). During calm nights, the low O 2 tension encountered on coral reefs can drop below 10% air saturation (Routley et al., 2002), usually as a result of respiration by reef organisms and especially during nocturnal low tides. This can also result in elevations in PCO 2 , which have been reported to exceed 1000 µatm on shallow reef flats at night (Ohde and van Woesik, 1999;Shaw et al., 2013). The CO 2 levels may even be higher in caves, reef crevices and restricted-flow habitats, which are used by H. ocellatum for shelter (Compagno, 2002;Last and Stevens, 2009). Indeed, diurnal or acute fluctuations in O 2 and CO 2 may play a role in signalling metabolism in species using such habitats. However, acute responses often differ dramatically from responses to prolonged exposure, and it is important to make this distinction. The increased uptake of CO 2 by the ocean will affect both the average CO 2 level and the magnitude of extreme CO 2 fluctuations (Ohde and van Woesik, 1999;Shaw et al., 2013). This makes our finding that H. ocellatum exhibited no change in metabolic performance, including sensitivity to hypoxia, after prolonged exposure to projected future CO 2 levels even more important.
Adaptation to life on shallow reef platforms and lagoons may be the key to species like H. ocellatum for maintaining performance in projected future CO 2 concentrations (Melzner et al., 2009b). While noteworthy, what was previously known about the physiological tolerance of the epaulette shark to challenging environmental conditions was related to acute exposure of minutes to hours. This is extremely relevant to a shelter-seeking, benthic, reef-dwelling species like the epaulette shark that would experience such conditions burrowing into coral caves to avoid predation or to exploit food sources, activities vital to biological fitness. Pelagic shark species, many of which function as apex predators in their respective environments (Last and Stevens, 2009), however, do not typically exhibit shelter-seeking behaviours in areas that would experience the routine fluctuations in water chemistry experienced by H. ocellatum and therefore may not tolerate prolonged periods of elevated CO 2 . Given that increased uptake of CO 2 by the ocean may mean that the high CO 2 levels that the epaulette shark may already routinely experience could be the new average ocean CO 2 levels, some species may be able to tolerate future conditions better. Future studies should investigate the importance of fluctuating 9 Figure 5: Changes in red muscle, heart and brain citrate synthase enzyme activity after sharks were exposed to control, medium or high CO 2 for ~90 days. Statistical significance is noted in the top right corner of each panel. Abbreviation: NS, not significant. environmental conditions in shaping an organism's tolerance. Differential effects on functional groups could impact predator-prey dynamics, affect the population structure of elasmobranchs and other aquatic organisms inhabiting coral reefs and, ultimately, impact ecosystem health. Investigating both sensitive and tolerant species from an array of habitat types would help to tease apart the role of the environment from other factors, including evolutionary history and behaviour, all of which is important when considering conservation measures under future climate change scenarios.