SUMMARY

The critical O2 tension of whole-animal O2 consumption rate (), or Pcrit, is the water PO2 (PwO2) at which an animal transitions from an oxyregulator to an oxyconformer. Although Pcrit is a popular measure of hypoxia tolerance in fishes because it reflects the capacity for O2 uptake from the environment at low PwO2, little is known about the interrelationships between Pcrit and blood O2 transport characteristics and increased use of anaerobic metabolism during hypoxia exposure in fishes, especially elasmobranchs. We addressed this knowledge gap using progressive hypoxia exposures of two elasmobranch species with differing hypoxia tolerance. The Pcrit of the hypoxia-tolerant epaulette shark (Hemiscyllium ocellatum, 5.10±0.37 kPa) was significantly lower than that of the comparatively hypoxia-sensitive shovelnose ray (Aptychotrema rostrata, 7.23±0.40 kPa). Plasma [lactate] was elevated above normoxic values at around Pcrit in epaulette sharks, but increased relative to normoxic values at PwO2 below Pcrit in shovelnose rays, providing equivocal support for the hypothesis that Pcrit is associated with increased anaerobic metabolism. The , arterial PO2 and arterial blood O2 content (CaO2) were similar between the two species under normoxia and decreased in both species with progressive hypoxia, but as PwO2 declined, epaulette sharks had a consistently higher and CaO2 than shovelnose rays, probably due to their significantly greater in vivo haemoglobin (Hb)–O2 binding affinity (in vivo Hb–O2P50=4.27±0.57 kPa for epaulette sharks vs 6.35±0.34 kPa for shovelnose rays). However, at PwO2 values representing the same percentage of each species' Pcrit (up to ∼175% of Pcrit), Hb–O2 saturation and CaO2 were similar between species. These data support the hypothesis that Hb–O2P50 is an important determinant of Pcrit and suggest that Pcrit can predict Hb–O2 saturation and CaO2 during hypoxia exposure, with a lower Pcrit being associated with greater O2 supply at a given PwO2 and consequently better hypoxia tolerance. Thus, Pcrit is a valuable predictor of environmental hypoxia tolerance and hypoxia exposures standardized at a given percentage of Pcrit will yield comparable levels of arterial hypoxaemia, facilitating cross-species comparisons of responses to hypoxia.

INTRODUCTION

Environmental hypoxia is a common abiotic stressor affecting the survival and distribution of aquatic species, but its occurrence varies in magnitude and spatio-temporal scale depending on habitat (Diaz and Breitburg, 2009). Consequently, many species of fish have evolved the ability to survive periods of low O2 exposure but the severity and duration of hypoxia that can be tolerated is highly species specific. Identification of the physiological responses contributing to hypoxia tolerance among fishes is an area of major research interest. The increasing occurrence worldwide of environmental hypoxia due to anthropogenic activities, in particular, has led to the desire for simple physiologic metrics of hypoxia tolerance in fishes and other aquatic organisms. Even so, relatively few comparative studies exist on this topic in fishes.

One simple metric that holds promise in this regard is the critical O2 tension of whole-animal O2 consumption rate (), or Pcrit, which is the water PO2 (PwO2) at which the of an organism transitions from oxyregulation to oxyconformation. Pcrit is thought to reflect the ability of an organism to extract O2 from the environment to maintain routine as PwO2 decreases, with a low Pcrit being associated with greater hypoxia tolerance presumably because of improved O2 uptake and transport to tissues at low PwO2. Consequently, Pcrit has been employed routinely as an important measure of hypoxia tolerance in aquatic organisms including fishes (Chapman et al., 2002; Mandic et al., 2009; Nilsson and Östlund-Nilsson, 2008; Pörtner and Grieshaber, 1993; Routley et al., 2002). Physiological modifications at any step in the respiratory cascade may affect Pcrit, including changes in ventilation, gill surface area, blood O2 capacity (including blood haemoglobin concentration [Hb] and Hb–O2 binding affinity), circulation of O2 (e.g. cardiac output), diffusion into tissues and mitochondrial O2 turnover (Dejours, 1981; Farrell and Richards, 2009). Whole-blood Hb–O2P50 (i.e. Hb–O2 binding affinity), in particular, has received attention as a determinant of Pcrit as well as hypoxia tolerance in fishes because of its important role in controlling blood O2 content and O2 uptake (Brauner and Wang, 1997). A phylogenetically independent comparison of O2 transport in sculpins showed that Pcrit is strongly correlated with Hb–O2P50; species possessing a low Pcrit typically also have a low Hb–O2P50 (Mandic et al., 2009). Other fishes known to have a high Hb–O2 binding affinity typically also have a low Pcrit and are often hypoxia tolerant (Burggren, 1982; Jensen and Weber, 1982; Sollid et al., 2005). Overall, however, the relationship between Pcrit and Hb–O2P50 has rarely been directly investigated and it remains incompletely defined. Also, it has not been unequivocally demonstrated that a low Pcrit is associated with greater arterial blood O2 transport during hypoxia exposure in fishes. In fact, little is known in fishes about the responses of blood gas transport at and around Pcrit.

At PwO2 below Pcrit, O2-independent energy production (e.g. anaerobic glycolysis) is increasingly relied upon to meet energy demands at a time when aerobic metabolism is constrained. Measurements of Pcrit and anaerobic end-product accumulation in certain animals provide evidence for the hypothesis that the increased activation of anaerobic metabolism coincides with Pcrit (Pörtner and Grieshaber, 1993), although support from other studies is equivocal (McKenzie et al., 2000; Nonnotte et al., 1993). The link between Pcrit and increased activation of anaerobic metabolism is thus uncertain, especially in fishes. Also, there are no direct comparisons of the onset of lactate accumulation in hypoxia-tolerant and -sensitive fishes that differ in Pcrit.

In the present study, we investigated the relationship between Pcrit and arterial O2 transport properties, including in vivo Hb–O2P50 and arterial total O2 content (CaO2), during progressive hypoxia exposure in two tropical elasmobranch species with similar lifestyles and activity levels, the epaulette shark, Hemiscyllium ocellatum (Bonnaterre), and the eastern shovelnose ray, Aptychotrema rostrata (Shaw). Epaulette sharks inhabit shallow coral reef environments where nocturnal hypoxia occurs commonly and they can tolerate hours of severe hypoxia (<1.0 kPa) exposure and up to 45 min of anoxia exposure (Renshaw et al., 2002; Routley et al., 2002). In contrast, eastern shovelnose rays are found in generally well-oxygenated coastal sandy and muddy benthic habitats in eastern Australia, including our sampling site of Moreton Bay, where hypoxic events are rare and dissolved O2 is usually close to air saturation at all depths (Dennison et al., 2004; Gabric et al., 1998; Kyne and Bennett, 2002). Indeed, preliminary observations from the present study showed that shovelnose rays are relatively hypoxia sensitive, succumbing rapidly if held at or below a PwO2 of 2.0 kPa for more than approximately 30 min. We predicted that the more hypoxia-tolerant epaulette shark would have a lower Pcrit than the shovelnose ray and that the lower Pcrit in the epaulette shark would be associated with a lower Hb–O2P50 and a correspondingly greater CaO2 at similar hypoxic PwO2. Furthermore, we predicted that at each species' Pcrit or at the same percentage of Pcrit, Hb–O2 saturation and CaO2 would be similar between the species despite being exposed to different PwO2 values. Finally, we measured arterial blood metabolic status including pH, [lactate] and CO2 status in order to further characterize the physiological correlates of Pcrit in fishes and test the hypothesis that Pcrit is associated with increased activation of anaerobic metabolism. Overall, the present study provides a comprehensive picture of the physiological responses associated with Pcrit in two fishes, elucidating for the first time the relationship between Pcrit and arterial blood O2 transport characteristics during hypoxia exposure, and providing comparative insight into the respiratory and metabolic attributes associated with hypoxia tolerance in fishes.

MATERIALS AND METHODS

Animals

Epaulette sharks and shovelnose rays of mixed sexes were supplied by Cairns Marine (Cairns, QLD, Australia) or Seafish Aquarium Life (Dunwich, QLD, Australia), respectively. The animals were collected under A1 level commercial harvest licences granted by the Department of Primary Industries, Australia. Epaulette sharks were caught on the Great Barrier Reef and transported in flow-through seawater tanks to holding tanks at Cairns Marine, where they were kept for 2 days without feeding before being transported by air and automobile to Moreton Bay Research Station, North Stradbroke Island, QLD, Australia. Shovelnose rays were caught in Moreton Bay, transferred to the mainland in flow-through seawater tanks, and transported to Moreton Bay Research Station by automobile. All fish were held in a recirculating seawater system (28°C) for at least 5 days before experimentation. Animals were fed every other day and fasted for at least 24 h before experimentation. All experiments were conducted according to guidelines set out by the Canadian Council for Animal Care and protocols approved by the University of British Columbia Animal Care Committee and the Griffith University Animal Ethics Committee.

Experimental and analytical protocols

Surgical protocol

Epaulette sharks (1.29±0.04 kg, N=7) and shovelnose rays (1.54±0.06 kg, N=8) were netted from the holding tanks and anaesthetized in water containing a final concentration of 0.1 g l–1 benzocaine (initially dissolved in 95% ethanol; 0.001% ethanol in anaesthetic bath). Fish were then moved to a surgery table where the gills were continuously irrigated with aerated seawater (28°C) containing 0.075 g l–1 benzocaine.

To permit periodic sampling of blood, a PE50 (Clay-Adams, Parsippany, NJ, USA) cannula was fitted in the caudal artery via a lateral incision in the caudal peduncle, as described previously (De Boeck et al., 2001). The cannula was filled with heparinized (50 U ml–1) elasmobranch saline (in mmol l–1: 257 NaCl, 7 Na2SO4, 6 NaHCO3, 0.1 Na2HPO4, 4 KCl, 3 MgSO4·H2O, 2 CaCl2·2H2O, 300 urea and 100 trimethylamine oxide). The cannula was exteriorized through a PE160 grommet and sutured to the skin. The incision was closed with silk sutures. The fish were also fitted with ventral aorta flow probes as described in the accompanying paper (Speers-Roesch et al., 2012).

Experimental protocol

Following surgery, the instrumented fish was immediately moved to a cylindrical acrylic respirometer (18.2 l for epaulette sharks and 28.4 l for shovelnose rays) that was submersed inside an opaque aquarium that received seawater from the same recirculating system used for the fish holding tanks (28°C). A submersible pump inside the aquarium provided a continuous flow of water to the respirometer that ensured complete water mixing inside the respirometer and maintained PwO2. The respirometer was covered with black plastic to prevent visual disturbance of the fish. The cannula and the flow probe lead from the fish were exteriorized through a hole in the respirometer fitted with a soft rubber stopper modified with a slit. The fish was allowed to recover from surgery and habituate to the respirometer for at least 12 h before any experimental procedures were performed.

Stable baseline conditions were confirmed by monitoring routine cardiovascular variables (see Speers-Roesch et al., 2012) for 1–2 h at a normoxic PwO2 of between 15 and 16 kPa (74–78% air saturation; 100% air saturation=20.4 kPa=153 Torr). An aortic blood sample (1 ml) was taken at the end of this period for immediate measurement of normoxic resting levels of arterial whole-blood pH, PO2, [Hb], haematocrit (Hct) and total O2 content as described below. Plasma was separated by centrifugation (5000 g, 5 min), frozen in liquid nitrogen within 5 min of sampling, transported to Canada in a dry shipper and kept frozen at –80°C for several weeks until analyses of metabolites and [total CO2] (see below). The red blood cell pellet was re-suspended in elasmobranch saline to a final volume of 1 ml and this was injected via the cannula to replace the blood removed. The respirometer was then closed by connecting the inflow and outflow tubes of the respirometer via a submersible pump that re-circulated the water inside the respirometer. The fish was allowed to consume O2 in the respirometer and the rate of depletion of PwO2 was used to calculate as described below. The changes in water parameters (e.g. pH, PCO2) potentially associated with the use of closed respirometry have been shown previously to have no effect on Pcrit in fish (Henriksson et al., 2008). Indeed, only modest increases in arterial PCO2 (PaCO2) were observed in the present study (see Results and Discussion). Arterial blood samples were taken and treated as described above at regular intervals including PwO2 at approximately 11.8, 7.7, 5.8, 3.8 and 2.0 kPa in both species, as well as approximately 1.0 and 0.1 kPa in epaulette sharks (see Table 1). Fish were held in the closed respirometer until PwO2 reached ∼0.1 kPa for epaulette sharks and ∼1.6 kPa for the shovelnose rays, which took 135±8 and 71±6 min, respectively, from the point at which the normoxic blood sample was taken and the respirometer was closed. These nadirs of PwO2 were chosen based on previous studies on epaulette sharks (e.g. Renshaw et al., 2002) and preliminary observations of hypoxia-exposed animals, which clearly revealed that epaulette sharks tolerated prolonged bouts of severe hypoxia whereas shovelnose rays showed distress or loss of equilibrium if held at or below 2.0 kPa for more than 30 min. The rate of O2 depletion was similar between species (see Table 1). Once the nadir in PwO2 (∼0.1 or ∼1.6 kPa) was reached, normoxic water was reintroduced to the respirometer. A final blood sample was taken at 60 min of recovery in normoxic water and analysed as previously described. In some cases, cannulae were damaged by the movements of the fish during the overnight acclimation period or during the experimental exposure and therefore sample sizes of measured parameters vary slightly (see figure captions for final N values). At the end of the trials the fishes were terminally anaesthetized in seawater containing benzocaine.

Table 1.

Arterial Hct, [Hb], MCHC, plasma [glucose], plasma [β-HB], [HCO 3], PCO2 and [total CO2] of epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2, and after a subsequent 60 min of recovery in normoxic water

Arterial Hct, [Hb], MCHC, plasma [glucose], plasma [β-HB], [HCO –3], PCO2 and [total CO2] of epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2, and after a subsequent 60 min of recovery in normoxic water
Arterial Hct, [Hb], MCHC, plasma [glucose], plasma [β-HB], [HCO –3], PCO2 and [total CO2] of epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2, and after a subsequent 60 min of recovery in normoxic water

Data acquisition and calculation of oxygen consumption rate and Pcrit

PwO2 in the respirometer was measured using an Oxyguard probe (Mark IV, Point Four Systems, Richmond, BC, Canada), modified to give a ±1 V output signal, that was placed in a custom-made Plexiglas® chamber connected in line with the circulation pump. The probe output was fed to a Power Lab unit (ADInstruments, Castle Hill, NSW, Australia) and subsequently analysed using LabChart Pro software (v. 6.0; ADInstruments).

Whole-animal during the respirometry trials was calculated from the rate of decline in PwO2 over 10 min periods bracketing PwO2 at regular intervals from approximately 14.8 to 0.67 kPa in epaulette sharks and approximately 13.7 to 1.9 kPa in shovelnose rays, following the methods of Henriksson et al. (Henriksson et al., 2008). Blanks without fish were run for both chambers and background was subtracted from fish . The corrected was plotted against PwO2 and the inflection point at which transitions from being independent to being dependent on PwO2 (i.e. Pcrit) was calculated using the BASIC program designed by Yeager and Ultsch (Yeager and Ultsch, 1989). At PwO2 above Pcrit, values were constant and not significantly different from one another within a species, therefore confirming that is independent of PwO2 above Pcrit. The was not measured at the initial normoxic PwO2 (∼15.3–16.0 kPa) or the normoxic recovery period because of the flow-through conditions.

Analytical protocols

Blood [Hb] was measured spectrophotometrically (Blaxhall and Daisley, 1973). Hct was determined following centrifugation at 5000 g in a sealed capillary tube. Mean cellular Hb content (MCHC) was calculated as ([Hb]/Hct)100. Arterial blood PO2 (PaO2) was measured with a Radiometer PO2 (E-5046) electrode, thermostatically controlled in a D616 cell at 28°C, in conjunction with a PHM 71 acid–base analyser (Radiometer, Copenhagen, Denmark). Whole-blood CaO2 was measured following the method of Tucker (Tucker, 1967). In order to calculate the percentage O2 saturation of Hb in arterial blood, the quantity of O2 bound to Hb was calculated by subtracting physically dissolved O2 in the blood [calculated from measured PaO2 and published blood O2 solubility coefficients (Christiforides and Hedley-Whyte, 1969)] from the CaO2 measured in whole blood. The quantity of O2 bound to Hb was then expressed as a percentage of the theoretical maximum quantity of O2 bound to Hb, which was calculated using measured [Hb] values and assuming four O2 bound per Hb tetramer at 100% saturation. In vivo blood P50 and Hill coefficient values were then calculated from Hill plots based on data for arterial PO2 and Hb–O2 saturation.

Blood pH was measured using a Radiometer pH micro-electrode thermostatically held at 28°C and displayed on a Radiometer PHM 71 acid–base analyser. True plasma [total CO2] was measured according to Cameron (Cameron, 1971), immediately following thawing of the plasma on ice. To confirm that freezing of plasma and storage at –80°C has no effect on [total CO2], we compared [total CO2] measured in freshly sampled plasma from normocarbic, normoxic tilapia (Oreochromis hybrid sp.) with an aliquot of the same plasma that was frozen in liquid nitrogen and stored at –80°C for 4 weeks. [Total CO2] was 7.1±0.7 mmol l–1 in the fresh samples vs 7.0±0.8 mmol l–1 in the frozen samples (N=6; paired t-test, P>0.05; B.S.-R., unpublished); thus, freeze–thaw of plasma does not appear to affect [total CO2]. We have seen similar results in hagfish (Eptatretus stoutii) (D. W. Baker and C.J.B., unpublished). PaCO2 and [HCO3] were calculated from measured values of pH and [total CO2] through manipulation of the Henderson–Hasselbalch equation (Brauner et al., 2000) with appropriate constants for elasmobranchs (Boutilier et al., 1984). Plasma [lactate] and [glucose] were measured on deproteinized and untreated plasma, respectively, according to the protocols outlined by Bergmeyer (Bergmeyer, 1983). Plasma [β-hydroxybutyrate] ([β-HB]) was measured on deproteinized plasma following the protocol of McMurray et al. (McMurray et al., 1984).

Statistics

The effects of species and PwO2 on blood gas, acid–base and metabolite parameters were tested for samples 1–6 (at overlapping PwO2 between species; see Table 1) using a two-way ANOVA followed by Holm–Sidak post hoc (H–S) tests against species or the normoxic resting values measured at ≥15.3 kPa (PwO2 values were not statistically different between species at each sample point, allowing this two-way design; Student's t-test, P>0.05). Samples 7 and 8 in epaulette sharks (see Table 1) were tested separately for significance against the normoxic resting sample 1 (16.0 kPa) using a one-way ANOVA with H–S tests. Species differences in the in vivo blood P50 and Hill coefficient (nH) values were examined using a Student's t-test and the effect of O2 on Hb–O2 saturation was compared within species using a one-way ANOVA with H–S tests (comparisons between species were not made because arterial PO2 at sample points were not always comparable). The relationship between Hb–O2 saturation and Pcrit was examined via linear regression analysis of Hb–O2 saturation against PwO2 expressed as the percentage of Pcrit (using values from individual animals). This analysis was carried out up to a Hb–O2 saturation of ∼75% because at higher values Hb saturation curves are asymptotic. Recovery values were compared between species and with the normoxic resting values at ≥15.3 kPa within species using a two-way ANOVA with H–S test.

Fig. 1.

Oxygen consumption rate () and critical O2 tension (Pcrit) values of epaulette sharks and shovelnose rays exposed to progressive decreases in water PO2 (PwO2). See Table 1 and Materials and methods for further information on time course and starting and ending PwO2 of the exposures. Data are means ± s.e.m. (N=7 for epaulette sharks; N=8 for shovelnose rays).*Statistically significant difference from the first normoxic resting value within species. Statistically significant difference between species for Pcrit, and statistically significant difference between the epaulette shark value and the shovelnose ray value bracketed by a horizontal brace (where the two species values were taken at statistically similar PwO2); the absence of a dagger indicates that the two bracketed species values are not statistically different from each other. See Materials and methods for details on statistical methods.

Fig. 1.

Oxygen consumption rate () and critical O2 tension (Pcrit) values of epaulette sharks and shovelnose rays exposed to progressive decreases in water PO2 (PwO2). See Table 1 and Materials and methods for further information on time course and starting and ending PwO2 of the exposures. Data are means ± s.e.m. (N=7 for epaulette sharks; N=8 for shovelnose rays).*Statistically significant difference from the first normoxic resting value within species. Statistically significant difference between species for Pcrit, and statistically significant difference between the epaulette shark value and the shovelnose ray value bracketed by a horizontal brace (where the two species values were taken at statistically similar PwO2); the absence of a dagger indicates that the two bracketed species values are not statistically different from each other. See Materials and methods for details on statistical methods.

The critical PwO2 of were compared between species using a Student's t-test. The effects of species and PwO2 on were tested using a two-way ANOVA with H–S tests using data from eight sampling points of overlapping PwO2 at approximately 13.6, 12.3, 10.3, 6.1, 4.2, 3.4, 2.5 and 1.9 kPa and the points of statistical comparison are denoted by horizontal braces on the figures. Overlapping PwO2 values were not statistically different between species (Student's t-test, P>0.05). Data from other sampling points were omitted from these analyses. However, in order to fully assess the effect of PwO2 on in each species, one-way ANOVA were run across all sampling points within each species, with H–S comparisons against the first normoxic resting value. The effect of PwO2 on measured parameters was found to be similar for both two-way and one-way ANOVA designs.

Statistical significance was accepted when P<0.05 and analyses were carried out using SigmaStat 3.0 or GraphPad Prism 5.0. Data were log or square-root transformed prior to statistical analyses if assumptions of equal variance or normality were not met. Repeated measures ANOVA could not be carried out because experimental constraints negated the use of data from the same animal at every single sample period. In any case, the standard ANOVA procedures utilized here result in a conservative statistical assessment of our data.

Fig. 2.

Arterial PO2 (PaO2) (A) and arterial O2 content (CaO2) (B) of epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2 and after a subsequent 60 min of recovery in normoxic water. See Table 1 and Materials and methods for further information on time course and starting and ending PwO2 of the exposures. Data are means ± s.e.m. (N=4–6 for epaulette sharks except for recovery where N=3; N=5–6 for shovelnose rays). Recovery values are offset for clarity. *Statistically significant difference from the first, normoxic resting value at ≥15.3 kPa within species. †Shovelnose ray value is significantly different from that of the epaulette shark at the same sample point. See Materials and methods for details on statistical methods. Recovery values were not significantly different between species or when compared with resting values in both species.

Fig. 2.

Arterial PO2 (PaO2) (A) and arterial O2 content (CaO2) (B) of epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2 and after a subsequent 60 min of recovery in normoxic water. See Table 1 and Materials and methods for further information on time course and starting and ending PwO2 of the exposures. Data are means ± s.e.m. (N=4–6 for epaulette sharks except for recovery where N=3; N=5–6 for shovelnose rays). Recovery values are offset for clarity. *Statistically significant difference from the first, normoxic resting value at ≥15.3 kPa within species. †Shovelnose ray value is significantly different from that of the epaulette shark at the same sample point. See Materials and methods for details on statistical methods. Recovery values were not significantly different between species or when compared with resting values in both species.

RESULTS

Under normoxic conditions, individuals of both species were generally quiescent. As PwO2 decreased, there was a modest and temporary increase in activity level associated with exploratory behaviour in some individuals of each species. As PwO2 decreased further, the fishes again became quiescent. In a couple instances in each species, severe agitation occurred temporarily (<10 s) at low PwO2, causing the cannula to be dislodged and negating further blood sampling.

A typical relationship between and PwO2 was observed in both species, each with a zone of O2-independent occurring at higher PwO2 followed by a zone of O2 dependence below Pcrit, where decreased with decreasing PwO2 (Fig. 1). The Pcrit was significantly lower in epaulette sharks than in shovelnose rays (Fig. 1). When PwO2 was above ∼5 kPa, was similar between species. However, when PwO2 was below ∼5 kPa, was consistently greater for epaulette sharks than for shovelnose rays at any similar PwO2 (Fig. 1).

PaO2 decreased linearly with decreasing PwO2 and was not different between species, except at the two highest PwO2 where epaulette sharks had higher PaO2 compared with shovelnose rays (Fig. 2A). Total CaO2 decreased with decreasing PwO2 with the exception of between 16.01±0.43 and 11.80±0.06 kPa in epaulette sharks. Total CaO2 was the same in both species at the highest PwO2, but at all lower PwO2 it was greater in epaulette sharks than in shovelnose rays (Fig. 2B). Recovery in normoxic water returned PaO2 and CaO2 to values similar to those measured at the beginning of the progressive hypoxia trial (Fig. 2). Hb–O2 saturation decreased as PaO2 fell below approximately 9.0 and 6.0 kPa in shovelnose rays and epaulette sharks, respectively (Fig. 3). The in vivo Hb–O2 binding affinity was significantly higher (lower P50) in epaulette sharks than in shovelnose rays (Fig. 3). The Hill coefficient was similar between species (Fig. 3). At each species' P50, arterial pH was ∼7.82 in epaulette sharks and ∼7.81 in shovelnose rays, and PaCO2 was ∼0.23 kPa in both species (extrapolated from pH and PaCO2 data at the extrapolated PwO2 at P50).

A significant linear relationship was found for each species when Hb–O2 saturation was regressed against PwO2 expressed as a percentage of Pcrit, and because the slopes and y-intercepts of these regression lines were not significantly different between species, one line of best fit was applied to the pooled data for the two species (Fig. 4). Above ∼175% of Pcrit, the Hb–O2 saturation became asymptotic so these data were excluded from the linear regression. Thus, Hb–O2 saturation was similar between species at PwO2 values representing the same percentage of each species' Pcrit (up to ∼175% of Pcrit). Because there were no major differences in [Hb] between species (see below), the same relationship existed for CaO2 (data not shown). Overall, these data show that Pcrit is predictive of Hb–O2 saturation and CaO2 at PwO2 lower than about 175% of Pcrit.

In both species progressive hypoxia had no effect on Hct, [Hb] or MCHC (Table 1). Hct and [Hb] were significantly higher in epaulette sharks at sampling points 4 (PwO2=5.73±0.02 kPa in epaulette sharks and 5.83±0.09 kPa in shovelnose rays) and 6 (PwO2=1.85±0.03 kPa in epaulette sharks and 2.07±0.03 kPa in shovelnose rays) and [Hb] was also higher at sampling point 5 (PwO2=3.81±0.12 kPa in epaulette sharks and 3.82±0.07 kPa in shovelnose rays) (Table 1). Recovery [Hb] and MCHC were similar to normoxic resting values but Hct was higher in epaulette sharks during recovery (Fig. 2; Table 1).

Fig. 3.

Arterial haemoglobin–O2 (Hb–O2) saturation as a function of arterial PO2 (PaO2) of epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2 and after a subsequent 60 min of recovery in normoxic water. In vivo Hb–O2P50 values and Hill coefficients (nH) were calculated from Hill plots (see Materials and methods) and are presented in the figure. At each species' P50, arterial pH was ∼7.82 in epaulette sharks and ∼7.81 in shovelnose rays, and PaCO2 was ∼0.23 kPa in both species (extrapolated from pH and PaCO2 data at the extrapolated PwO2 at P50). See Materials and methods for details on calculation of Hb–O2 saturation. See Table 1 and Materials and methods for further information on time course and starting and ending PwO2 of the exposures. Data are means ± s.e.m. (N=4–6 for epaulette sharks except for recovery where N=3; N=5–6 for shovelnose rays). Recovery values are offset for clarity. *Statistically significant difference from the first, normoxic resting value at ≥15.3 kPa within species. Shovelnose ray value for Hb–O2P50 is significantly different from that of the epaulette shark. The Hill coefficients were not significantly different between species. See Materials and methods for details on statistical methods. Recovery values were not significantly different between species or when compared with resting values in both species.

Fig. 3.

Arterial haemoglobin–O2 (Hb–O2) saturation as a function of arterial PO2 (PaO2) of epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2 and after a subsequent 60 min of recovery in normoxic water. In vivo Hb–O2P50 values and Hill coefficients (nH) were calculated from Hill plots (see Materials and methods) and are presented in the figure. At each species' P50, arterial pH was ∼7.82 in epaulette sharks and ∼7.81 in shovelnose rays, and PaCO2 was ∼0.23 kPa in both species (extrapolated from pH and PaCO2 data at the extrapolated PwO2 at P50). See Materials and methods for details on calculation of Hb–O2 saturation. See Table 1 and Materials and methods for further information on time course and starting and ending PwO2 of the exposures. Data are means ± s.e.m. (N=4–6 for epaulette sharks except for recovery where N=3; N=5–6 for shovelnose rays). Recovery values are offset for clarity. *Statistically significant difference from the first, normoxic resting value at ≥15.3 kPa within species. Shovelnose ray value for Hb–O2P50 is significantly different from that of the epaulette shark. The Hill coefficients were not significantly different between species. See Materials and methods for details on statistical methods. Recovery values were not significantly different between species or when compared with resting values in both species.

Plasma [lactate] was low at PwO2 above 8 kPa, then significantly increased above normoxic resting levels by ∼3.7 kPa in both species and increased further with decreasing PwO2. Plasma [lactate] remained elevated after 60 min recovery in normoxic water (Fig. 5A). Plasma [lactate] was generally similar between species at all PwO2 during progressive hypoxia but was greater in epaulette sharks during recovery (Fig. 5A). A significant decrease in blood pH first occurred at ∼3.8 kPa in both species, decreasing further with declining PwO2 and remaining low after 60 min recovery in normoxic water (Fig. 5B). Blood pH was similar between species but was significantly lower in the shovelnose rays at the final sampling point for this species at ∼2.0 kPa (Fig. 5B). Arterial [HCO 3] and [total CO2] were unchanged during progressive hypoxia exposure in both species (Table 1). An increase in PaCO2 occurred in both species and it remained after 60 min recovery in normoxic water (Table 1). In epaulette sharks only, normoxic recovery was associated with significantly lower [HCO 3] and [total CO2] compared with normoxic resting levels as well as compared with the same parameters in shovelnose rays in recovery (Table 1). Plasma [glucose] or [β-HB] were similar between species and were unaffected by either progressive hypoxia or recovery in normoxic water (Table 1).

DISCUSSION

The present study demonstrates a link between Pcrit and arterial blood O2 transport characteristics during hypoxia exposure in two elasmobranch species, adding significantly to a growing body of evidence showing that Pcrit is an important indicator of hypoxia tolerance in fish (Chapman et al., 2002; Mandic et al., 2009; Nilsson and Östlund-Nilsson, 2008). We provide the first comparative evidence that a lower Pcrit is associated with maintenance of greater CaO2 during hypoxia exposure, which benefits hypoxia tolerance. Similarly, we show that Pcrit is predictive of Hb–O2 saturation and CaO2 (in the observed absence of changes in [Hb]) during hypoxia exposure (Fig. 4), supporting the notion that differences in Hb–O2 binding affinity determine differences in Pcrit. Indeed, the in vivo Hb–O2P50 of the epaulette shark was lower than that of the shovelnose ray and this is the likely explanation for the greater CaO2 observed in the former species at low PwO2. The impressive hypoxia tolerance of the epaulette shark is probably attributable, in part, to its enhanced O2 transport characteristics compared with those of the less tolerant shovelnose ray.

Fig. 4.

Hb–O2 saturation as a function of PwO2 represented as percentage of Pcrit in epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2 (i.e. Pcrit of each species occurs at 100%). Data are means ± s.e.m. (N=4–6 for epaulette sharks; N=5–6 for shovelnose rays). The linear regression is statistically significant (P<0.0001, linear regression ANOVA).

Fig. 4.

Hb–O2 saturation as a function of PwO2 represented as percentage of Pcrit in epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2 (i.e. Pcrit of each species occurs at 100%). Data are means ± s.e.m. (N=4–6 for epaulette sharks; N=5–6 for shovelnose rays). The linear regression is statistically significant (P<0.0001, linear regression ANOVA).

Fig. 5.

Arterial plasma lactate (A) and arterial blood pH (B) of epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2 and after a subsequent 60 min of recovery in normoxic water. See Table 1 and Materials and methods for further information on time course and starting and ending PwO2 of the exposures. Data are means ± s.e.m. (N=4–6 for epaulette sharks except for recovery where N=3; N=5–6 for shovelnose rays). Recovery values are offset for clarity. *Statistically significant difference from the first, normoxic resting value at ≥15.3 kPa within species. Shovelnose ray value is significantly different from that of the epaulette shark at the same sample point.

Fig. 5.

Arterial plasma lactate (A) and arterial blood pH (B) of epaulette sharks and shovelnose rays exposed to progressive decreases in PwO2 and after a subsequent 60 min of recovery in normoxic water. See Table 1 and Materials and methods for further information on time course and starting and ending PwO2 of the exposures. Data are means ± s.e.m. (N=4–6 for epaulette sharks except for recovery where N=3; N=5–6 for shovelnose rays). Recovery values are offset for clarity. *Statistically significant difference from the first, normoxic resting value at ≥15.3 kPa within species. Shovelnose ray value is significantly different from that of the epaulette shark at the same sample point.

Oxygen uptake and blood oxygen transport properties

Under normoxic resting conditions (i.e. PwO2>Pcrit) the of epaulette sharks matches closely with that measured for this species by Routley et al. (Routley et al., 2002). Epaulette sharks and shovelnose rays had similar resting , which fell within the range of temperature-corrected for elasmobranchs of a similar activity level (0.9–3.5 μmol g–1 h–1) (Butler and Metcalfe, 1988). In both species, exposure to progressive hypoxia caused a pronounced reduction of below Pcrit (Fig. 1), similar to many teleosts (Mandic et al., 2009; Speers-Roesch et al., 2010) as well as at least one other elasmobranch, the spotted catshark (Scyliorhinus canicula) (Butler and Taylor, 1975). The Pcrit for epaulette sharks (Fig. 1) matches the lower range of Pcrit measured for this species by Routley et al. (Routley et al., 2002). The shovelnose ray had a significantly higher Pcrit compared with the epaulette shark (Fig. 1), but it was lower than that of other previously studied elasmobranchs (see Routley et al., 2002). Although the level of O2 demand can affect Pcrit (Thuy et al., 2010), this probably does not explain the observed difference in Pcrit because under normoxic conditions was the same in each species (Fig. 1). The difference in Pcrit between the hypoxia-tolerant epaulette shark and the comparatively hypoxia-sensitive shovelnose ray is consistent with the notion that a lower Pcrit is associated with greater hypoxia tolerance in fish (Mandic et al., 2009; Nilsson and Östlund-Nilsson, 2008). The Pcrit of the epaulette shark is generally similar to those of hypoxia-tolerant teleosts and it is the lowest known Pcrit among elasmobranchs (Routley et al., 2002; Speers-Roesch et al., 2010). The lower Pcrit of epaulette sharks was associated not only with maintenance of routine to a lower PwO2 compared with shovelnose rays but also with maintenance of greater at all comparable hypoxic PwO2 (i.e. below Pcrit), where depression of occurred in both species (Fig. 1). Thus, a low Pcrit allows a fish to maintain as high as possible during hypoxia exposure, minimizing reliance on inefficient anaerobic metabolism.

Haematological (i.e. Hct, [Hb], MCHC) and O2 transport properties (i.e. PaO2, CaO2 and Hb–O2 saturation) in arterial blood of epaulette sharks and shovelnose rays under normoxic resting conditions (Figs 2 and 3, values at highest PwO2 or PaO2; Table 1, sample 1) were generally similar between species and the values were typical for elasmobranchs (e.g. Hct, 10–20%; [Hb], 0.46–0.62 mmol l–1; MCHC, ∼3.3; PaO2, 8–15 kPa; CaO2, 3–5 vol. %; Hb–O2 saturation, 75–100%) (Butler and Metcalfe, 1988; Butler and Taylor, 1975; De Boeck et al., 2001; Lai et al., 1990; Perry and Gilmour, 1996; Routley et al., 2002). The lack of 100% Hb–O2 saturation in normoxia is consistent with other studies on elasmobranchs (e.g. Lai et al., 1990) and the presence of up to 27% methaemoglobin in fishes has been put forward as an explanation as to why some fish haemoglobins are not saturated to their theoretical maximum in vivo (Graham and Fletcher, 1986). The in vivo P50 values for epaulette sharks and shovelnose rays are higher than previously measured in other elasmobranchs (1.9–2.7 kPa) (Butler and Taylor, 1975; Cooper and Morris, 2004; Lai et al., 1990), but in the previous studies these were measured at lower environmental temperatures compared with the present study and environmental temperature appears to be positively correlated with P50 in marine fishes (Wells, 2005). The Hill coefficients were similar between species and relatively low, which is typical of elasmobranchs (Butler and Metcalfe, 1988).

The changes in PaO2 and CaO2 during progressive hypoxia (Fig. 2) were similar to those seen in other elasmobranchs (Butler and Taylor, 1975; Perry and Gilmour, 1996; Routley et al., 2002). The roughly linear decrease in PaO2 was of similar magnitude in epaulette sharks and shovelnose rays, suggesting ventilatory responses to hypoxia are comparable between species (Fig. 2A). However, at all matched PwO2 below the normoxic resting level, CaO2 was greater in epaulette sharks than in shovelnose rays, which appears to be due to greater Hb–O2 saturation resulting from the epaulette shark's higher in vivo Hb–O2 binding affinity (i.e. lower P50) (Fig. 2B; Fig. 3). This result is consistent with findings suggesting that Hb–O2P50 is an important component of hypoxia tolerance in fishes, with the most tolerant species possessing the lowest P50, which result in greater blood O2 loading at low PwO2 (Jensen and Weber, 1982; Mandic et al., 2009; Perry and Reid, 1992).

Interestingly, the species differences in Hb–O2 saturation and CaO2 disappeared when these parameters were plotted against PwO2 expressed as a percentage of Pcrit (i.e. the relationships overlapped between species) (Fig. 4). Thus, when measured at a PwO2 of the same percentage of each species' Pcrit, values of Hb–O2 saturation or CaO2 (because changes in [Hb] were not apparent) were the same in epaulette sharks and shovelnose rays. These results suggest that Pcrit is predictive of Hb–O2 saturation and CaO2 during hypoxia exposure, with species with lower Pcrit having a greater capacity for arterial blood O2 transport at similar hypoxic PwO2. These results also suggest that the differences in arterial Hb–O2 saturation between epaulette sharks and shovelnose rays may represent the basis of species differences in Pcrit. This observation, as well as the fact that at Pcrit the PaO2 in both species was similar to their respective in vivo Hb–O2P50 (Fig. 2A; Fig. 3), agrees with Mandic and colleagues' (Mandic et al., 2009) discovery of a close relationship between Pcrit and Hb–O2P50 in sculpins and supports the idea that P50 is an important determinant of Pcrit in fishes.

The lack of a change in Hct or [Hb] during progressive hypoxia (Table 1) is consistent with previous studies on epaulette sharks and other elasmobranchs (Perry and Gilmour, 1996; Routley et al., 2002; Short et al., 1979) and thus adjustments of these parameters may have a minimal role in improving O2 transport in elasmobranchs exposed to hypoxia. Nonetheless, Hct and [Hb] were approximately 25% higher at some hypoxic PwO2 values in epaulette sharks compared with shovelnose rays (Table 1), probably contributing to the higher CaO2 seen in the former species. However, the difference of CaO2 between species was greater (40–100% higher in epaulette sharks) at these hypoxic PwO2, affirming an important role for Hb–O2P50 in enhancing O2 supply in the epaulette shark.

Metabolites and acid–base status

An increased reliance on anaerobic glycolysis and a consequent metabolic acidosis during hypoxia exposure was indicated by a large increase in plasma [lactate] and a decrease in blood pH at or below Pcrit in both species (Fig. 5), similar to results observed in other hypoxia-exposed fishes (Routley et al., 2002; Scott et al., 2008). At the lowest PwO2 exposure of shovelnose rays, blood pH was significantly lower compared with epaulette sharks (Fig. 5B), suggesting that the latter species may possess more effective acid–base regulation or less reliance on anaerobic energy production, potentially due to a superior O2 supply. Nevertheless, at lower PwO2 epaulette shark blood pH dropped precipitously and like other hypoxia-tolerant vertebrates this species must be able to tolerate severe metabolic acidosis during hypoxia exposure (Driedzic and Gesser, 1994). The PwO2 at which epaulette sharks first showed a significant increase in plasma [lactate] was approximately the same as found previously for this species (Routley et al., 2002) and coincided with its Pcrit (Fig. 5A). In shovelnose rays, however, the increase in plasma [lactate] did not coincide with its Pcrit but rather occurred at a similar PwO2 to that of the epaulette sharks and accumulated at a similar rate (Fig. 5A). Although our data do not provide solid evidence for the hypothesis that Pcrit correlates with increased activation of anaerobic metabolism in fishes (Pörtner and Grieshaber, 1993), they are consistent with this activation not occurring above Pcrit. It is also possible that a non-equilibrium state during early progressive hypoxia exposure including interspecific differences in blood volume or lactate handling may have obscured our ability to detect the onset of an increase of anaerobic metabolism at Pcrit in shovelnose rays. This may also explain why the rate of plasma lactate accumulation was similar between species despite lower blood O2 content in shovelnose rays. We consider the effect of changes in fish activity (see Results) on plasma [lactate] to be negligible because of the low measurement variability and the consistently low values at PwO2 above Pcrit (Fig. 1). Also, most fish activity was moderate and in the few instances of excessive activity, cannula dislodgement meant that plasma [lactate] was no longer measured.

The metabolic acidosis seen in epaulette sharks and shovelnose rays during progressive hypoxia resulted in a downward trend in [HCO 3] and [total CO2] (Table 1). In both species PaCO2 increased, possibly due to a build-up of CO2 in the closed respirometer. This elevation in CO2 probably had no major effect on other measured parameters, because the magnitude of the PaCO2 increase was not large until the final sample point and no major differences were observed between the final sample point and the previous sample point for any measured parameters. In any case, hypercarbia commonly accompanies environmental hypoxia so in this regard the present exposures are more ecologically relevant. The responses of blood CO2 parameters to progressive hypoxia in the present study differ from the respiratory alkaloses seen in hypoxic spiny dogfish (Squalus acanthias) (Perry and Gilmour, 1996) or spotted catshark (Butler et al., 1979), but these other studies utilized moderate rather than severe hypoxia that probably mitigated metabolic acidosis – Butler et al. (Butler et al., 1979) found no increase in plasma [lactate]. The relatively low normoxic resting levels of PaCO2, [HCO 3] and [total CO2] in epaulette sharks and shovelnose rays are typical of elasmobranchs (Butler and Metcalfe, 1988; De Boeck et al., 2001; Lai et al., 1990).

Plasma [glucose] was unaffected by progressive hypoxia (Table 1), which is unlike most teleosts but mirrors previous findings in hypoxia-exposed epaulette sharks and spiny dogfish (Routley et al., 2002; Speers-Roesch and Treberg, 2010). In some hypoxia-tolerant teleosts, large decreases in plasma [non-esterified fatty acid], an important aerobic fuel, occur during hypoxia exposure (Speers-Roesch et al., 2010). There was no similar response in epaulette sharks or shovelnose rays for plasma concentration of β-HB, which in elasmobranchs serves an important role as an alternative lipid-derived aerobic fuel (Speers-Roesch and Treberg, 2010).

Recovery from progressive hypoxia exposure

Epaulette sharks and shovelnose rays showed comparable recovery of respiratory parameters after progressive hypoxia and followed a trajectory similar to that of other fishes (Hughes and Johnston, 1978; Van Raaij et al., 1996). Following 60 min of recovery in normoxic water, normoxic arterial O2 parameters were restored in both species (Fig. 2B; Fig. 3); however, recovery from metabolic acidosis and high [lactate] was incomplete, particularly in epaulette sharks, which had experienced more severe hypoxia and hypoxia-induced acidosis and lactate accumulation (Fig. 5B). Arterial [HCO 3] in recovery remained lower than normoxic levels in epaulette sharks, probably as a consequence of the persistence of acidosis, whereas shovelnose rays had recovered (Table 1).

CONCLUSIONS

The present study on the hypoxia-tolerant epaulette shark and the hypoxia-sensitive shovelnose ray provides the first evidence that Pcrit is predictive of arterial Hb–O2 saturation and CaO2 during hypoxia exposure in fishes. At the same level of hypoxia, fishes with a low Pcrit can maintain higher CaO2 than fishes with a higher Pcrit and this appears to be due to the presence of a lower in vivo Hb–O2P50 that allows greater Hb–O2 saturation (Fig. 2B; Fig. 3). Additionally, at PwO2 of the same percentage of Pcrit, Hb–O2 saturation (and CaO2) is the same between species (Fig. 4). These results suggest that the interspecific differences in Hb–O2 saturation may represent the basis of species differences in Pcrit, providing strong support for the notion that Hb–O2P50 is a major determinant of Pcrit as well as hypoxia tolerance in fishes (Mandic et al., 2009).

Our finding of a link between Pcrit, Hb–O2 saturation and CaO2 provides a mechanistic explanation for the argument that Pcrit is a good indicator of hypoxia tolerance in fishes. A low Pcrit is associated with greater CaO2 and therefore improved O2 delivery to tissues during hypoxia exposure, which presumably enhances energy supply and reduces the accumulation of deleterious anaerobic end products, thus improving hypoxia tolerance. Importantly, our findings also show that hypoxia exposures that are standardized to Pcrit will yield comparable levels of arterial hypoxaemia, facilitating cross-species comparative analyses. However, the results of the accompanying study (Speers-Roesch et al., 2012) warn that even when hypoxia exposures are scaled to Pcrit, tissue-specific differences may occur in the metabolic response to the same amount of circulating O2.

Although our measurements of plasma [lactate] showed no difference in the onset PwO2 or the rate of accumulation of lactate between species despite differing Pcrit, our ability to detect such a difference may have been obscured by non-steady-state lactate dynamics during initial hypoxia exposure. Accumulation of lactate in each species did occur at or below Pcrit, providing equivocal support for the hypothesis that Pcrit is associated with increased activation of anaerobic metabolism (Pörtner and Grieshaber, 1993). Investigations of lactate turnover in hypoxia-exposed fish, including consideration of the potential effect of metabolic depression (which could blunt lactate accumulation), are needed to further test this hypothesis.

The superior O2 uptake and blood O2 capacity during hypoxia exposure in epaulette sharks probably explains in part the renowned hypoxia tolerance of this elasmobranch (Nilsson and Renshaw, 2004). Epaulette sharks also show enhanced hypoxic cardiovascular function compared with shovelnose rays (Speers-Roesch et al., 2012), which further improves O2 delivery to tissues. Maintenance rather than depression of O2 supply and aerobic metabolism at low levels of PwO2 may be an important component of hypoxia tolerance in fishes.

LIST OF SYMBOLS AND ABBREVIATIONS

     
  • β-HB

    β-hydroxybutyrate

  •  
  • CaO2

    arterial blood O2 content

  •  
  • Hb

    haemoglobin

  •  
  • Hb–O2P50

    haemoglobin–O2 binding affinity

  •  
  • Hct

    haematocrit

  •  
  • MCHC

    mean cellular haemoglobin content

  •  
  • whole-animal O2 consumption rate

  •  
  • nH

    Hill coefficient

  •  
  • PaCO2

    arterial blood PCO2

  •  
  • PaO2

    arterial blood PO2

  •  
  • PCO2

    partial pressure of CO2

  •  
  • Pcrit

    critical O2 tension of whole-animal O2 consumption rate

  •  
  • PO2

    partial pressure of O2

  •  
  • PwO2

    water PO2

FOOTNOTES

FUNDING

Funding was provided by the Discovery Grant Program from the Natural Sciences and Engineering Research Council of Canada (NSERC) [to J.G.R., C.J.B., A.P.F., and Y.X.W.]. Funding was also provided from Sea World Research and Rescue Foundation [to G.M.C.R.]. A.J.R.H. was supported by the University of Auckland Early Career Research Excellence award. C.J.B. was supported by a Killam Faculty Research Fellowship. B.S.-R. was supported by a Pacific Century Graduate Scholarship from the University of British Columbia and the Province of British Columbia, a War Memorial Scholarship from IODE Canada, a Journal of Experimental Biology Travelling Fellowship from the Company of Biologists, a Comparative Physiology and Biochemistry Student Research Grant from the Canadian Society of Zoologists, and a Graduate Travel Award from the Department of Zoology, University of British Columbia.

Acknowledgements

Kevin and Kathy Townsend and their staff at the Moreton Bay Research Station provided exemplary logistical and technical assistance. Craig Franklin kindly lent us equipment needed for a portion of this study. Miles Gray assisted with hypoxia exposures and sampling.

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