Metabolic rate depression (MRD) has long been proposed as the key metabolic strategy of hypoxic survival, but surprisingly, the effects of changes in hypoxic O2 tensions (PwO2) on MRD are largely unexplored. We simultaneously measured the O2 consumption rate (O2) and metabolic heat of goldfish using calorespirometry to test the hypothesis that MRD is employed at hypoxic PwO2 values and initiated just below Pcrit, the PwO2 below which O2 is forced to progressively decline as the fish oxyconforms to decreasing PwO2. Specifically, we used closed-chamber and flow-through calorespirometry together with terminal sampling experiments to examine the effects of PwO2 and time on O2, metabolic heat and anaerobic metabolism (lactate and ethanol production). The closed-chamber and flow-through experiments yielded slightly different results. Under closed-chamber conditions with a continually decreasing PwO2, goldfish showed a Pcrit of 3.0±0.3 kPa and metabolic heat production was only depressed at PwO2 between 0 and 0.67 kPa. Under flow-through conditions with PwO2 held at a variety of oxygen tensions for 1 and 4 h, goldfish also initiated MRD between 0 and 0.67 kPa but maintained O2 to 0.67 kPa, indicating that Pcrit is at or below this PwO2. Anaerobic metabolism was strongly activated at PwO2 ≤1.3 kPa, but only used within the first hour at 1.3 and 0.67 kPa, as anaerobic end-products did not accumulate between 1 and 4 h exposure. Taken together, it appears that goldfish reserve MRD for near-anoxia, supporting routine metabolic rate at sub-PcritPwO2 values with the help of anaerobic glycolysis in the closed-chamber experiments, and aerobically after an initial (<1 h) activation of anaerobic metabolism in the flow-through experiments, even at 0.67 kPa PwO2.

Aerobic pathways of ATP production yield ∼15 times more ATP than anaerobic pathways (Hochachka and Somero, 2002). Consequently, environmental hypoxia and the corresponding shift to anaerobic metabolism seriously threaten energy balance by reducing an animal's ability to generate sufficient ATP to meet metabolic demands. Despite the critical importance of aerobic respiration to the maintenance of metabolic function, many organisms inhabit and thrive in various hypoxic and even anoxic environments (Bickler and Buck, 2007; Ramirez et al., 2007). Fishes are particularly adept at surviving low-oxygen environments, having independently evolved hypoxia tolerance numerous times (Hochachka and Lutz, 2001) owing to the relatively high prevalence of hypoxia among aquatic habitats (Boesch, 2002; Diaz and Breitburg, 2009; Diaz and Rosenberg, 1995; Smith et al., 2006).

Metabolic rate depression (MRD) has been proposed as the hallmark response enabling hypoxic survival in hypoxia-tolerant animals (e.g. Boutilier and St-Pierre, 2000; Hochachka et al., 1996). MRD is achieved through reductions in whole-animal (e.g. locomotion, reproduction, feeding) and cellular (e.g. growth, repair, protein synthesis) processes (Guppy and Withers, 1999; Richards, 2010), reducing ATP demand and rates of anaerobic fuel depletion (glycogen) and waste accumulation (lactate and protons). Although MRD is a well-described response to anoxia exposure in a range of animals including fruit flies (Callier et al., 2015), goldfish (Addink et al., 1991; van Waversveld et al., 1989) and turtles (Jackson, 1968), it has been suggested that MRD would also enhance hypoxic survival (Boutilier and St-Pierre, 2000; Hochachka et al., 1996). Indirect (i.e. non-calorimetric) measurements in common frogs (Donohoe and Boutilier, 1998) and direct (i.e. calorimetric) measurements in goldfish (van Ginneken et al., 1994, 2004) and tilapia (van Ginneken et al., 1997) suggest that MRD may be employed at hypoxic O2 tensions (PwO2 for water). Indeed, goldfish reduced metabolic heat by ∼31% at ∼2.1 kPa PwO2 compared with normoxia (∼21 kPa) (van Ginneken et al., 2004), and tilapia reduced metabolic heat by ∼40% at ∼1.1 kPa PwO2 (van Ginneken et al., 1997). However, it is still unknown how these changes in metabolic heat correspond with changes in aerobic and anaerobic metabolism, and how this is affected by PwO2.

MRD would be particularly important at PwO2 values below an animal's critical PwO2 for O2 consumption rate (O2), referred to as Pcrit, which is the PwO2 at which O2 becomes dependent on environmental PO2. Pcrit is largely determined by the O2 binding affinity of hemoglobin (Hb) (Mandic et al., 2009), and at PwO2 values below Pcrit the ability to extract environmental O2 to saturate Hb is constrained and thus unable to support routine metabolic rate (MR) aerobically. The animal can attempt to sustain ATP production at routine levels through an upregulation of anaerobic glycolysis, but this comes with the depletion of carbohydrate reserves and the accumulation of deleterious anaerobic end-products (Richards, 2009), ultimately limiting hypoxic survival time (Lague et al., 2012; Speers-Roesch et al., 2013). However, if the animal is capable of reducing its energy-consuming processes through a controlled, hypoxia-induced MRD, then it could simultaneously mitigate the negative consequences of reduced ATP production and increased rates of fuel depletion and waste accumulation. We therefore hypothesized that MRD is employed at hypoxic PwO2 values and is initiated just below Pcrit, where the negative impacts of reduced aerobic capacity and increased anaerobic reliance begin to accrue.

List of symbols and abbreviations
     
  • CO2

    oxygen content

  •  
  • F

    flow rate

  •  
  • Hb

    hemoglobin

  •  
  • M

    mass

  •  
  • O2

    oxygen consumption rate

  •  
  • MR

    metabolic rate

  •  
  • MRD

    metabolic rate depression

  •  
  • P50

    PO2 at which Hb is 50% saturated with oxygen

  •  
  • Pcrit

    critical partial pressure of oxygen for O2

  •  
  • PO2

    partial pressure of oxygen

  •  
  • PwO2

    partial pressure of oxygen in water

  •  
  • T

    time

  •  
  • V

    volume

We tested this hypothesis using closed-chamber and flow-through calorespirometry to simultaneously measure O2 consumption rates, MRD (via metabolic heat) and anaerobic glycolysis (via excretion rates of the anaerobic end-product ethanol) in goldfish held at PwO2 values ranging from normoxia to anoxia. We also performed terminal sampling experiments on goldfish exposed to the same PwO2 values as used in the calorespirometry experiments to fully quantify whole-body anaerobic metabolism. Goldfish were chosen owing to their exceptional hypoxia tolerance and well-documented ability to induce MRD (e.g. Addink et al., 1991; van Waversveld et al., 1989; van Ginneken et al., 2004), something not all fish species are capable of (Stangl and Wegener, 1996). We used calorespirometry because it is the ‘gold standard’ of MR measurements and the only way to accurately measure MR on hypoxemic animals in real time (see Kaiyala and Ramsay, 2011; Nelson, 2016). Despite the superiority of calorespirometry, only a few studies have measured the metabolic heat of fishes (Addink et al., 1991; Regan et al., 2013; Stangl and Wegener, 1996; van Ginneken et al., 1994, 1997, 2004; van Waversveld et al., 1989), and only three of these (van Ginneken et al., 1994, 1997, 2004) have measured metabolic heat at PwO2 other than normoxia and anoxia. While the data from these studies suggest that MRD is employed at hypoxic PwO2 values, they exposed their organisms to progressive hypoxia over sometimes prolonged periods of time and they did not relate their measurements to Pcrit nor directly assess the contributions of anaerobic metabolism at various hypoxic PwO2 values. Furthermore, other studies that have attempted to examine the role of MRD and other metabolic and respiratory responses to hypoxia have not attempted, to our knowledge, to directly assess the relative contributions of MRD, aerobic respiration and anaerobic metabolism at different hypoxic PwO2 values over time. Consequently, we still do not have a comprehensive picture of the hypoxic survival strategies of fishes.

Study organisms

We obtained adult goldfish [Carassius auratus auratus (Linnaeus 1758); 2.06±0.39 g wet mass; n=264; sex unknown] from a commercial supplier (Delta Aquatics, Burnaby, BC, Canada) and held them under a 12 h:12 h light:dark cycle in a 76 litre recirculating system of aerated, dechlorinated, 17°C water at the University of British Columbia (Vancouver, BC, Canada). Stocking density was <0.4 g l−1 and water in the recirculating system was replaced weekly. We fed the fish to satiation daily (Nutrafin Max Goldfish Flakes) except for 24 h before transfer to the experimental apparatus, when feeding ceased. The University of British Columbia's Animal Care Committee approved all procedures.

Calorespirometer

We used a differential calorespirometer to simultaneously measure metabolic heat and O2 under closed and flow-through conditions. The design and operation of the calorespirometer are described in detail in Regan et al. (2013). Briefly, the metabolic heat of a fish is detected as a voltage by a collection of Peltier units (Custom Thermoelectric Peltier module 12711-5L31- 03CQ, Bishopville, MD, USA) via the Seebeck effect and converted to wattage using an empirically determined calibration coefficient (see Regan et al., 2013). The design of the calorespirometer allows for the simultaneous measurements of metabolic heat and O2 using PO2 optodes (Ocean Optics OR125, Dunedin, FL, USA) placed on the inflowing and outflowing water lines as well as in the fish chamber. To determine O2 under closed-chamber conditions, a PO2 optode within the fish chamber measured the change in PwO2 over sequential 5 min intervals and was then corrected for chamber volume and fish mass according to:
(1)
where CO2 is the O2 content of the water converted to μmol l−1 from PwO2 using the solubility factor of 1.9312 μmol l−1 mmHg−1 (Boutilier et al., 1984), T is the time period over which the change in CO2 is calculated (5 min), V is the fish chamber volume (32 ml) minus the volume displaced by the fish itself, and M is the mass of the fish. To determine O2 under flow-through conditions, the difference in PwO2 between inflowing and outflowing water lines supplying the fish chamber was measured using the same PO2 optode and corrected for flow rate and fish mass according to:
(2)

where CiO2 and CoO2 are O2 content of inflowing and outflowing water, respectively, converted from PwO2 as described above, F is water flow rate (22 ml h−1) and M is the mass of the fish. Under flow-through conditions, the chamber PwO2 could be held constant for extended time periods, allowing us to measure O2 and metabolic heat at different time points at any desired PwO2.

Hypoxic exposures

Individual fish were transferred to a flow-through calorespirometer held at 17°C and a flow rate of 22 ml h−1, and in this apparatus we performed both closed-chamber and flow-through calorespirometry experiments following a 16 h normoxic habituation period. For the closed-chamber experiments (n=8), the trial began by stopping water flow and allowing the fish to reduce PwO2 from normoxia to anoxia over 60–90 min. The experiment was ended when the chamber PwO2 reached anoxia, at which point we introduced a lethal dose of anaesthetic (buffered MS-222, final chamber concentration of 150 mg l−1) to determine the calorespirometer's baseline heat signature. For the flow-through experiments, inflowing PwO2 was manually adjusted to yield one of four chamber PwO2 values over a ∼60 min period (20, 1.3, 0.67 or 0 kPa; n=3–6 for each) and the animals were maintained at one of these PwO2 values for up to 4 h (referred to as the experimental period). We measured metabolic heat over the full 21 h period (16 h normoxia habituation, 1 h transition to exposure PwO2, 4 h experimental period) and collected effluent water samples either before (time 0) or at 1 and 4 h during the experimental period for measurements of ethanol (a glycolytic end-product excreted across goldfish gills). Following the experiment, we introduced a lethal dose of anaesthetic (buffered MS-222, see above) to determine the calorespirometer's baseline heat signature. At the end of each experiment, we recalibrated the PO2 optodes to determine any drift that had occurred over the course of the experiment (up to ∼10%) for the purpose of later correction, and then washed the calorespirometer and its water lines with a 10% bleach solution. The flow-through and closed-chamber calorespirometry experiments were performed in fall 2014 and winter 2015, respectively.

Comparison of closed-chamber and flow-through calorespirometry

To more directly compare the results of the closed-chamber and flow-through calorespirometry experiments, we conducted a back-to-back comparison of the two techniques using the same fish. This was required because our first experiments (presented in Figs 1 and 2) using these techniques were conducted at different times of year and yielded different routine normoxic O2 values, which could affect our determination of Pcrit. We measured routine O2 using both techniques and determined Pcrit during closed-chamber respirometry (Pcrit was not determined via flow-through calorespirometry because it would require the fish to undergo multiple runs at different PwO2 values). Briefly, fish were introduced to the calorespirometer and allowed to habituate under the same conditions as the calorespirometry experiments described above. Following the habituation period, we first measured the fish's routine O2 under normoxia using flow-through respirometry (as described above), then immediately closed off the respirometer chamber and measured O2 using closed-chamber respirometry (as described above). We repeated this three times for each of six fish, and the PwO2 was not allowed to drop below 16 kPa during these closed-chamber measurements. Following the final closed-chamber measurement, we allowed the fish to deplete the chamber's O2 content so as to determine its Pcrit. Metabolic heat was not measured during these back-to-back experiments.

Fig. 1.

Closed-chamber calorespirometry measurements of oxygen consumption rate (O2) and metabolic heat in goldfish. Partial pressure of oxygen in water (PwO2) was reduced from normoxia to anoxia over 60 to 90 min because of the fish's O2 consumption. Data are means±s.e.m, n=8. Data points sharing a letter are not significantly different (one-way ANOVA, P>0.05).

Fig. 1.

Closed-chamber calorespirometry measurements of oxygen consumption rate (O2) and metabolic heat in goldfish. Partial pressure of oxygen in water (PwO2) was reduced from normoxia to anoxia over 60 to 90 min because of the fish's O2 consumption. Data are means±s.e.m, n=8. Data points sharing a letter are not significantly different (one-way ANOVA, P>0.05).

Fig. 2.

Flow-through calorespirometry measurements of O2, metabolic heat and glycolytic end-products in goldfish held at different PwO2 values for different periods of time. (A) 1 h; (B) 4 h. Data are means±s.e.m, n=3–6, and data points are offset slightly on the x-axis for clarity. Data points sharing a letter are not significantly different (one-way ANOVA, P>0.05).

Fig. 2.

Flow-through calorespirometry measurements of O2, metabolic heat and glycolytic end-products in goldfish held at different PwO2 values for different periods of time. (A) 1 h; (B) 4 h. Data are means±s.e.m, n=3–6, and data points are offset slightly on the x-axis for clarity. Data points sharing a letter are not significantly different (one-way ANOVA, P>0.05).

Terminal sampling experiments

To better estimate the effects of PwO2 on anaerobic metabolism, we ran parallel hypoxia exposures where we euthanized animals to measure whole-body concentrations of lactate and ethanol. For each PwO2, we exposed 24 goldfish spread across six tanks (four fish per tank) and sampled two replicate tanks at each of 0, 1 and 4 h to match the experimental periods of the calorespirometry experiments (n=8 per time point). We sampled fish by inconspicuously introducing a lethal dose of anaesthetic (buffered MS-222, see above), weighing the individual fish, freezing them immediately in liquid N2, and then storing them at −80°C for later metabolite analyses. We ensured that the conditions between these experiments and the calorespirometry experiments were similar by including a 16 h habituation period followed by a 1 h transition period to the desired PwO2, conducting exposures in the dark, and by preventing the fish in the tank from accessing the air–water interface (which was not available to the calorespirometry fish). Thus, the main difference between this experiment and the calorespirometer experiment was vessel size (calorespirometer chamber was 32 ml and exposure tanks were 10 litres), which could affect the ability of the fish to move during our hypoxia exposures and yield different levels of lactate and ethanol accumulation. We are, however, confident that fish movement is minimal in the calorespirometer based on the relatively smooth heat traces observed over the habituation and experimental periods, and periodic visual inspection of the fish in the 10 litre tanks revealed little to no movement, especially during the hypoxia exposures. Thus, despite the differences in exposure regimes, the fish from both the calorespirometry and the terminal sampling experiments likely responded to hypoxia in a similar manner.

Lactate and ethanol analyses

To link our whole-body calorespirometry measurements of MR to the activation of anaerobic metabolism, we measured whole-body concentrations of lactate and ethanol. Entire goldfish from the 10 litre tank exposures were ground into a fine powder using a liquid N2-chilled mortar and pestle. To extract the metabolites (lactate and ethanol) from the powder, an aliquot of powder was weighed and transferred to a 2 ml centrifuge tube containing 1 ml of ice-cold 30% HClO4 and immediately homogenized at 0°C using a Polytron homogenizer set to the highest setting for 30 s. The resulting homogenate was then centrifuged at 20,000 g for 5 min at 4°C and the supernatant was transferred to a new 1.5 ml centrifuge tube and neutralized using 3 mol l−1 Tris base to avoid the volatilization of ethanol that occurs in association with vigorous CO2 production when HClO4 is neutralized with K2CO3. We confirmed that neutralization with Tris base does not affect our enzymatic analysis. We measured ethanol immediately following neutralization using a commercial kit designed for biological ethanol analysis (Diagnostic Chemical Ltd., PEI, Canada), and then froze the unused portion of the sample extract for later lactate analysis. Lactate concentration was measured using the LDH reaction according to the protocols outlined in Bergmeyer (1983).

O2 equilibrium curves

To understand how the goldfish's Hb–O2 affinity is related to Pcrit and MRD, we constructed O2 equilibrium curves for the whole blood of five normoxia-acclimated goldfish using the thin film spectrophotometric technique (Lilly et al., 2013). Blood was collected from the caudal artery of anaesthetized fish using 60 μl heparinized capillary tubes. We then centrifuged the tubes and resuspended the red blood cells in HEPES buffer (pH 7.8) to ensure a consistent blood pH across all samples. A Wöstoff gas mixing pump (H. Wösthoff Messtechnik GmbH, Bochum, Germany) mixed compressed O2 and N2 to each of seven PO2 values for the construction of the O2 equilibrium curves, and Hb P50 values (the PO2 at which Hb is 50% saturated with O2) were calculated using the equation of each sigmoidal curve as calculated by SigmaStat 11.0.

Pcrit calculation

Pcrit is defined as the PwO2 at which an organism's routine O2 transitions from being independent of to being dependent upon PwO2. We determined Pcrit for each individual in the closed-chamber calorespirometry experiments using the BASIC program (Yeager and Ultsch, 1989), which uses a two-segment linear regression model to determine Pcrit as the PwO2 at which the two linear trend lines intersect on a graph plotting O2 as a function of PwO2. Some individuals' O2 values increased above routine O2 levels at hypoxic PwO2 values close to Pcrit, and including these O2 values would overestimate Pcrit. To prevent this, we excluded from our routine O2 estimation any O2 value that exceeded 1.5 times the standard deviation of an individual's average O2 between 13 and 21 kPa PwO2.

Data analysis and statistics

O2 and ethanol production rates were calculated at each time point, while metabolic heat was represented by averaging the continual heat measurements made over the 20 min straddling the time point (e.g. 50–70 min for 1 h time point). All data are presented as means±s.e.m. The effects of PwO2 on each variable were determined using one-way ANOVA (SigmaStat 11.0).

Closed-chamber calorespirometry experiments

We used closed-chamber calorespirometry to measure Pcrit and to characterize the effects of a progressive reduction in PwO2 on O2 and metabolic heat. PwO2 in the closed-chamber experiments was decreased from normoxia to anoxia by the fish's own O2 over 60–90 min (depending on the fish's O2). Pcrit was calculated to be 3.0±0.3 kPa (Fig. 1). At PwO2 values above Pcrit, there were no significant effects of changes in PwO2 on the average routine O2, while at PwO2 values below Pcrit (at which the fish spent ∼30 min), O2 progressively fell to zero as the goldfish depleted the available oxygen. Metabolic heat was maintained at routine levels at all PwO2 values between 20 and 0.5 kPa (Fig. 1) but was depressed upon reaching anoxia, eventually stabilizing at ∼21% of routine normoxic values (an MRD of 79%; Fig. 1) after ∼20 min.

Flow-through calorespirometry experiments

We used flow-through calorespirometry to characterize the effects of PwO2 and time on O2, metabolic heat and excreted ethanol. We held individuals at one of four PwO2 values (20, 1.3, 0.67 or 0 kPa) for 1 and 4 h. For 1 h exposures, O2 and metabolic heat were maintained at routine levels to PwO2 of 0.67 kPa, while at PwO2 values below this, O2 fell to zero and metabolic heat fell to ∼32% of routine levels (an MRD of 68%; Fig. 2A). Similarly, for 4 h exposures, O2 and metabolic heat were maintained at routine levels to PwO2 of 0.67 kPa, while at PwO2 values below this, O2 fell to zero and metabolic heat fell to ∼20% of routine levels (an MRD of 80%; Fig. 2B).

Ethanol excretion rates were undetectable following 1 h exposure at all PwO2 values (Fig. 2A). These rates increased following 4 h exposure, and higher rates were generally detected at lower PwO2 values (Fig. 2B), but these increases were not statistically significant (Fig. 2B).

Whole-body anaerobic end-product concentrations

Whole-body concentrations of lactate significantly increased over time 0 values following 1 and 4 h at 1.3 kPa, 0.67 kPa and anoxia (Table 1). Whole-body concentrations of ethanol significantly increased over time 0 values following 1 and 4 h of anoxia exposure (Table 1). The total anaerobic end-product concentrations at 1.3 and 0.67 kPa following 4 h were similar to those following 1 h, suggesting that the rate of anaerobic end-product accumulation fell to near-zero levels after 1 h (Fig. 2). A similar result was observed for the anoxia-exposed fish, though to a lesser extent, with anaerobic end-product concentrations being ∼1.8-fold higher following 4 h exposure than following 1 h exposure (Fig. 2).

Table 1.

Whole-body concentrations (μmol g−1) of lactate and ethanol in goldfish exposed to different PwO2 (partial pressure of oxygen in water) values for 1 and 4 h following a 16 h normoxic habituation period (0 h)

Whole-body concentrations (μmol g−1) of lactate and ethanol in goldfish exposed to different PwO2 (partial pressure of oxygen in water) values for 1 and 4 h following a 16 h normoxic habituation period (0 h)
Whole-body concentrations (μmol g−1) of lactate and ethanol in goldfish exposed to different PwO2 (partial pressure of oxygen in water) values for 1 and 4 h following a 16 h normoxic habituation period (0 h)

Closed-chamber versus flow-through calorespirometry

Individual O2 values determined in the same fish in a back-to-back comparison of closed-chamber and flow-through respirometry were positively correlated (n=18, r=0.925, P<0.0001; Fig. 3A) and yielded similar mean O2 values (t=0.423, P=0.678; Fig. 3B). The closed-chamber portion of these experiments yielded a Pcrit of 2.7±0.2 kPa (n=6).

Fig. 3.

A comparison of O2 measurements of goldfish made using closed-chamber and flow-through calorespirometry. Both respirometric techniques were performed in the same apparatus following a ≥16 h habituation period, at the same time of day (∼10:00 h PST), and at PwO2 ≥16 kPa. (A) O2 values resulting from closed and flow-through techniques, with measurements made back-to-back on the same fish (n=18, r=0.925, P<0.0001). (B) Average O2 measurements for each technique, with error bars representing s.e.m. (n=18, t=0.423, P=0.678).

Fig. 3.

A comparison of O2 measurements of goldfish made using closed-chamber and flow-through calorespirometry. Both respirometric techniques were performed in the same apparatus following a ≥16 h habituation period, at the same time of day (∼10:00 h PST), and at PwO2 ≥16 kPa. (A) O2 values resulting from closed and flow-through techniques, with measurements made back-to-back on the same fish (n=18, r=0.925, P<0.0001). (B) Average O2 measurements for each technique, with error bars representing s.e.m. (n=18, t=0.423, P=0.678).

Hb–O2 equilibrium curves

The Hb of goldfish displayed a very high affinity for O2, resulting in a steep O2 equilibrium curve and an average whole-blood P50 of 0.49±0.12 kPa (Fig. 4).

Fig. 4.

O2 equilibrium curve for the whole blood of five normoxia-acclimated goldfish. Red blood cells were separated from plasma and resuspended in HEPES buffer (pH 7.8). O2 levels were achieved using a Wösthoff gas mixing pump attached to cylinders of compressed O2 and N2.

Fig. 4.

O2 equilibrium curve for the whole blood of five normoxia-acclimated goldfish. Red blood cells were separated from plasma and resuspended in HEPES buffer (pH 7.8). O2 levels were achieved using a Wösthoff gas mixing pump attached to cylinders of compressed O2 and N2.

We hypothesized that goldfish employ MRD at hypoxic PwO2 values and initiate it at PwO2 values just below Pcrit. This hypothesis predicted that metabolic heat would decrease from routine levels at a PwO2 below Pcrit, when the fish's ability to take up environmental O2 to support a routine O2 was compromised. Our closed-chamber calorespirometry experiments yielded a Pcrit of 3.0±0.3 kPa (Fig. 1), consistent with the Pcrit values reported in other studies on goldfish (Fry and Hart, 1948; Fu et al., 2011). However, contrary to our hypothesis that MRD is initiated at hypoxic PwO2 values just below Pcrit, metabolic heat was maintained at routine normoxic levels to a PwO2 of 0.67 kPa, and MRD was only evident in goldfish exposed to anoxia. The magnitude of the anoxia-induced MRD [79% depression in closed-chamber experiments; 68% (1 h) and 80% (4 h) in flow-through experiments; Figs 1, 2] was very similar to what has been shown previously for anoxia-exposed goldfish using calorimetry (Addink et al., 1991; Stangl and Wegener, 1996; van Ginneken et al., 1994).

Metabolic responses to hypoxia

Goldfish maintained routine MR at severely hypoxic PwO2 values under both closed-chamber and flow-through conditions (Figs 1, 2), but appear to have used different strategies to do so. In the closed-chamber experiments, metabolic heat was maintained at routine normoxic levels to 0.67 kPa despite a decrease in O2 at 3.0 kPa (Fig. 1), suggesting that anaerobic glycolysis was upregulated to support MR (though lactate and ethanol could not be measured in closed-chamber experiments as a function of PwO2). In the flow-through experiments, metabolic heat was similarly maintained at routine normoxic levels to 0.67 kPa at both 1 and 4 h, but unlike the closed-chamber experiments, O2 was maintained at near-routine levels at all hypoxic PwO2 values tested. This suggests that MR was supported aerobically even at severely hypoxic PwO2 values and that MRD is reserved for all but severely hypoxic (<0.67 kPa) or near-anoxic environments. This is different than the results of van Ginneken and colleagues (1994, 2004), who showed moderate 27% and 33% decreases in heat production along with lower O2 in goldfish exposed to 3.5 and 2.1 kPa, respectively. These incongruent results are likely due to differences in experimental design and study goals. van Ginneken et al. (1994, 2004) exposed each fish in their studies to progressive hypoxia over prolonged periods of time (e.g. 8.4, 4.2, 2.1 and finally 0.63 kPa over a 16 h period in van Ginneken et al., 2004), which does not allow the authors to disentangle the effects of PwO2 and time on metabolic heat and O2. In contrast, our flow-through calorespirometry experiments exposed goldfish to only a single hypoxic PwO2 (after a 1 h adjustment period) for up to 4 h and we assessed the effects of varying hypoxic PwO2 values using different individuals, allowing us to independently assess the effects of PwO2 and time on metabolic responses. Using this approach, we clearly show that within 1 h of exposure, goldfish are capable of maintaining oxygen uptake under severely hypoxic conditions (0.67 kPa), obviating the need for hypoxia-induced MRD.

Elevated levels of lactate and ethanol at PwO2 ≤1.3 kPa at 1 and 4 h indicate that anaerobic glycolysis also contributed to maintaining MR, though in slightly different ways in anoxia and hypoxia. In anoxia, lactate and ethanol levels continued to increase throughout the 4 h exposure, but their rate of accumulation decreased from 5.81 µmol h−1 g−1 during the first hour to 1.73 µmol h−1 g−1 during the subsequent 3 h. These results are consistent with those observed in tissues from anoxia-exposed turtles (Trachemys scripta elegans), where lactate production rates were elevated during the first hour of anoxia exposure and subsequently decreased between 1 and 5 h anoxia in brain, liver and white muscle (Kelly and Storey, 1988). Combined, these results suggest that there is an initial reliance on anaerobic metabolism upon anoxia exposure that may compensate for the anoxia-induced limitations on aerobic ATP production while MRD is initiated. In hypoxia, the early reliance on anaerobic metabolism was temporally even more profound than in anoxia. Lactate and ethanol accumulation was confined entirely to the first hour of hypoxia exposure at 1.3 and 0.67 kPa, while O2 was concurrently maintained at routine normoxic levels throughout the hypoxic exposures. Taken together, these data suggest that total ATP turnover is higher over the first hour of hypoxia exposure than in normoxia. Indeed, whole-body estimates of total ATP turnover during this period indicate that it increases from ∼10 µmol h−1 g−1 in normoxia to ∼17 and 15 µmol h−1 g−1 at 1.3 and 0.67 kPa, respectively (assuming P:O2 of 6 and ATP:lactate/ethanol of 1), while heat production does not change. These inconsistencies are likely a consequence of not being able to temporally match our measurements of anaerobic metabolism (taken as the delta accumulation of lactate and ethanol over the entire hour plus the PwO2 adjustment period) with those of O2 and heat, which were taken at the end of the 1 h (between 50 and 70 min exposure). As such, it is possible there are temporal shifts in fuel selection within the first hour of hypoxia exposure, with lactate and/or ethanol accumulating during the initial descent towards the target PwO2 as O2-sustaining mechanisms are upregulated. Finer-scale studies are needed to confirm this idea.

The Pcrit values derived from the closed-chamber and flow-through calorespirometry experiments differed substantially, with Pcrit shifting from 3.0 kPa in the closed-chamber experiments to somewhere between 0 and 0.67 kPa in the flow-through experiments (the exact value cannot be determined). These technique-specific differences in Pcrit are consistent with a recent study comparing closed and intermittent-flow respirometry (Snyder et al., 2016), which attributed the higher Pcrit in closed respirometry to metabolic waste accumulation and a faster decline in PwO2. Similar factors may be at play in our closed-chamber calorespirometry experiments, resulting in an overestimation Pcrit. Another possible explanation might be that the routine normoxic O2 (and heat) in the closed-chamber experiments was approximately twofold higher than in the flow-through experiments (cf. Figs 1 and 2). All else being equal, this would necessitate the fish from the closed-chamber experiments adopting an oxyconforming strategy at a higher PwO2, yielding a higher Pcrit. However, our back-to-back comparison of calorespirometry techniques suggests that technique per se does not explain the twofold change in O2 and heat production, which might instead be explained by the time of year. In any case, the differences in O2 do not appear to affect Pcrit and therefore do not explain why Pcrit is higher in the closed-chamber experiments than in the flow-through experiments.

Another factor possibly contributing to the lower Pcrit values obtained from the flow-through experiments versus those from the closed-chamber experiments is time. Fishes possess many mechanisms that enhance O2 uptake with decreasing PwO2, including increases to gill surface area (Sollid et al., 2003), Hb synthesis (Gracey et al., 2001) and concentration in the blood (Affonso et al., 2002), hematocrit (Lai et al., 2006; Turko et al., 2014), Hb–O2 affinity (Turko et al., 2014), ventilation frequency and amplitude (Holeton and Randall, 1967; Itazawa and Takeda, 1978; Tzaneva et al., 2011; Vulesevic and Perry, 2006), as well as redistributed blood supply to critical tissues (Sundin et al., 1995). While these mechanisms effectively enhance the uptake of environmental O2 and its distribution throughout the body, their induction takes time, varying from minutes to days depending on the physiological response examined. Because the fish in the flow-through experiments had spent 1 or 4 h at each PwO2 when their O2 was measured (in addition to the ∼1 h required to reduce the PwO2 from normoxia to the target PwO2), they may have had additional time to initiate some of these mechanisms of enhanced O2 uptake compared with the closed-chamber fish that saw only ∼30 min of continually decreasing sub-Pcrit hypoxic conditions. If Pcrit is in fact influenced by the rate and duration of hypoxia induction over relatively short time scales, then it becomes important to apply similar methodology both within and between studies (something that is not currently done; see Rogers et al., 2016) to ensure Pcrit values are comparable. This is especially true when Pcrit is used as a reference point for models that, for example, predict how climate change will reshape the distribution of fishes around the world (Deutsch et al., 2015).

Hb–O2 affinity and initiation of MRD

Our results show that MRD is initiated in goldfish at a PwO2 somewhere between 0 and 0.67 kPa. Interestingly, our analysis of whole-blood Hb–O2 affinity reveals a Hb P50 value of 0.49±0.12 kPa (Fig. 4; consistent with Burggren, 1982), within the PwO2 range that goldfish appear to reduce O2 in the flow-through experiments and initiate MRD. It is therefore tempting to think of a causal link between the supply of O2 to the tissues and the initiation of MRD. Considerable debate exists regarding the signal for MRD, with some data supporting signals residing on the energy production side of the cellular energy flux pathways (de Zwaan and Wijsman, 1976; Hochachka, 1982, 1985; Plaxton and Storey, 1984; Rees and Hand, 1991; Bishop and Brand, 2000; Bishop et al., 2002) and some data supporting signals on the energy consumption side (Caligiuri et al., 1981; Flanigan and Withers, 1991; Robin et al., 1979; Sick et al., 1982; see reviews by Guppy, 2004; Guppy and Withers, 1999; Storey and Storey, 1990). If Hb–O2 affinity were in fact a signal for MRD, this would place the signal on the energy production side, consistent with some of the more recent views in the field (see Guppy, 2004). Similarly, Coulson (1977) postulated that MR was directly proportional to the circulatory system's ability to supply the tissues with O2, and this idea gained empirical support when van Ginneken et al. (2004) showed a correlation between hypoxia-induced decreases in MR and heart rate. All told, it is not unreasonable to speculate that a signal for hypoxia-induced MRD involves the supply of O2 to the tissue. The association between Hb P50 and the PwO2 of MRD initiation is therefore enticing and worth further investigation.

Ecological implications of MRD

The fact that goldfish appear to initiate MRD only near anoxia and maintain O2 without a long-term activation of anaerobic metabolism is well suited to the goldfish's (and the closely related crucian carp's, Carassius carassius) natural lake habitat. While these lakes become ice-covered in winter and eventually anoxic, they are severely hypoxic (Vornanen, 2004) for most of the winter at PwO2 values at which our study reveals goldfish remain aerobic. Goldfish can therefore maintain routine MR for most of the winter without relying on anaerobic glycolysis and/or MRD until it is entirely necessary. This strategy conserves the goldfish's finite anaerobic fuel stores (glycogen), reduces the accumulation of deleterious anaerobic end-products (lactate, protons and ethanol), and allows the goldfish to retain routine function and behaviour under most natural conditions.

Another benefit of a near-anoxic induced MRD is a delayed accumulation of MRD's inherent physiological and ecological costs (Humphries et al., 2003). These include oxidative stress resulting from the production of reactive O2 species (Carey et al., 2000), impaired immunocompetence resulting from reduced lymphocyte production (Burton and Reichman, 1999), impaired cognitive and memory function resulting from reductions in synaptic contacts and dendritic branching (Popov et al., 1992), and significant reductions in sensory and motor activity (Choi et al., 1998) that increase predation susceptibility. Because the costs associated with each of these likely accumulate with time, a hypoxic survival strategy that involves an extended bout of MRD is likely to cause significant damage regardless of its effectiveness to balance cellular energy supply and demand. The goldfish's predominant reliance on aerobic respiration is therefore the ideal strategy for surviving long-term hypoxic bouts because it minimizes the time the fish is forced to rely on MRD and the associated physiological costs.

Taken together, the goldfish's overall hypoxia tolerance strategy appears finely tuned to its particular hypoxic environment, which is characterized by long, protracted descents into eventual anoxia. This may be the case with other species too; because hypoxic environments vary greatly in severity, duration and rate of hypoxic induction, the hypoxia tolerance strategies employed by organisms native to these different environments are likely to be just as variable.

Conclusions

By demonstrating that goldfish prioritize O2 uptake over MRD in all but nearly anoxic environments, our results suggest two things. First, the exceptional hypoxia tolerance of goldfish owes more to its O2 extraction abilities than to MRD. Second, MRD is not necessarily a key mechanism of hypoxic survival, as has been hypothesized (Hochachka et al., 1996), but of anoxic survival. While MRD is an effective means of balancing energy supply and demand, the potential costs associated with reducing cellular and whole-body processes may threaten organismal fitness and preclude its selection in all but the most extreme environments.

We thank the reviewers for their insightful comments, as well as Milica Mandic, Bruce Gillespie and Joe Veneri. We dedicate this paper to John M. Gosline (1943–2016), a friend and mentor who always found the joy in science.

Author contributions

M.D.R. and J.G.R. designed the study. M.D.R. performed all experiments and data analyses. I.S.G. assisted with metabolite assays. M.D.R. drafted the manuscript and all authors commented.

Funding

This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to J.G.R. M.D.R. was supported by an NSERC postgraduate scholarship and a University of British Columbia Zoology Graduate Fellowship.

Data availability

Data have been deposited in the Dryad Digital Repository (Regan et al., 2017): http://dx.doi.org/10.5061/dryad.719bt

Addink
,
A. D. F.
,
van den Thillart
,
G.
,
Smit
,
H.
and
van Waversveld
,
J.
(
1991
).
A novel 1 liter flow-through calorimeter for heat production measurements on aquatic animals without stress
.
Thermochim. Acta
193
,
41
-
48
.
Affonso
,
E. G.
,
Polez
,
V. L. P.
,
Corrêa
,
C. F.
,
Mazon
,
A. F.
,
Araújo
,
M. R. R.
,
Moraes
,
G.
and
Rantin
,
F. T.
(
2002
).
Blood parameters and metabolites in the teleost fish Colossoma macropomum exposed to sulfide or hypoxia
.
Comp. Biochem. Physiol. C
133
,
375
-
382
.
Bergmeyer
,
H. U.
,
Bergmeyer
,
J.
and
Grassl
,
M.
(
1983
).
Methods of Enzymatic Analysis
.
Weinheim, FL
:
Verlag Chemie
.
Bickler
,
P. E.
and
Buck
,
L. T.
(
2007
).
Hypoxia tolerance in reptiles, amphibians, and fishes: life with variable oxygen availability
.
Annu. Rev. Physiol.
69
,
145
-
170
.
Bishop
,
T.
and
Brand
,
M. D.
(
2000
).
Processes contributing to metabolic depression in hepatopancreas cells from the snail Helix aspersa
.
J. Exp. Biol.
203
,
3603
-
3612
.
Bishop
,
T.
,
St-Pierre
,
J.
and
Brand
,
M. D.
(
2002
).
Primary causes of decreased mitochondrial oxygen consumption during metabolic depression in snail cells
.
Am. J. Physiol. Regul. Integr. Comp. Physiol.
282
,
R372
-
R382
.
Boesch
,
D. F.
(
2002
).
Challenges and opportunities for science in reducing nutrient over-enrichment of coastal ecosystems
.
Estuaries
25
,
886
-
900
.
Boutilier
,
R. G.
and
St-Pierre
,
J.
(
2000
).
Surviving hypoxia without really dying
.
Comp. Biochem. Physiol. A Mol. Integr. Physiol.
126
,
481
-
490
.
Boutilier
,
R. G.
,
Heming
,
T. A.
and
Iwama
,
G. K.
(
1984
).
Appendix: physicochemical parameters for use in fish respiratory physiology
.
Fish Physiol.
10
,
401
-
430
.
Burggren
,
W. W.
(
1982
).
‘Air gulping’ improves blood oxygen transport during aquatic hypoxia in the goldfish Carassius auratus
.
Physiol. Zool.
55
,
327
-
334
.
Burton
,
R. S.
and
Reichman
,
O. J.
(
1999
).
Does immune challenge affect torpor duration?
Funct. Ecol.
13
,
232
-
237
.
Caligiuri
,
M.
,
Robin
,
E. D.
,
Hance
,
A. J.
and
Robin
,
D. A.
(
1981
).
Prolonged diving and recovery in the freshwater turtle, Pseudemys scripta—II. Magnitude of depression of O2 requirements and the relation to body O2 stores
.
Comp. Biochem. Physiol. A
70
,
365
-
369
.
Callier
,
V.
,
Hand
,
S. C.
,
Campbell
,
J. B.
,
Biddulph
,
T.
and
Harrison
,
J. F.
(
2015
).
Developmental changes in hypoxic exposure and responses to anoxia in Drosophila melanogaster
.
J. Exp. Biol.
218
,
2927
-
2934
.
Carey
,
H. V.
,
Frank
,
C. L.
and
Seifert
,
J. P.
(
2000
).
Hibernation induces oxidative stress and activation of NF-κB in ground squirrel intestine
.
J. Comp. Physiol. B
170
,
551
-
559
.
Choi
,
I. H.
,
Cho
,
Y.
,
Oh
,
Y. K.
,
Jung
,
N. P.
and
Shin
,
H. C.
(
1998
).
Behavior and muscle performance in heterothermic bats
.
Physiol. Zool.
71
,
257
-
266
.
Coulson
,
R. A.
,
Hernandez
,
T.
and
Herbert
,
J. D.
(
1977
).
Metabolic rate, enzyme kinetics in vivo
.
Comp. Biochem. Physiol.
56
,
251
-
262
.
Deutsch
,
C.
,
Ferrel
,
A.
,
Seibel
,
B.
,
Pörtner
,
H.-O.
and
Huey
,
R. B.
(
2015
).
Climate change tightens a metabolic constraint on marine habitats
.
Science
348
,
1132
-
1135
.
de Zwaan
,
A.
and
Wijsman
,
T. C.
(
1976
).
Anaerobic metabolism in Bivalvia (Mollusca). Characteristics of anaerobic metabolism
.
Comp. Biochem. Physiol. B
54
,
313
-
324
.
Diaz
,
R. J.
and
Breitburg
,
D. L.
(
2009
).
The hypoxic environment
.
Fish Physiol..
27
,
1
-
23
.
Diaz
,
R. J.
and
Rosenberg
,
R.
(
1995
).
Marine benthic hypoxia: a review of its ecological effects and the behavioural responses of benthic macrofauna
.
Oceanog. Mar. Biol.
33
,
245
-
303
.
Donohoe
,
P. H.
and
Boutilier
,
R. G.
(
1998
).
The protective effects of metabolic rate depression in hypoxic cold submerged frogs
.
Resp. Physiol.
111
,
325
-
336
.
Flanigan
,
J. E.
and
Withers
,
P. C.
(
1991
).
In vitro metabolic depression of tissues from the aestivating frog Neobatrachus pelobatoides
.
J. Exp. Biol.
161
,
273
-
283
.
Fry
,
F. E. J.
and
Hart
,
J. S.
(
1948
).
The relation of temperature to oxygen consumption in the goldfish
.
Biol. Bull.
91
,
66
-
77
.
Fu
,
S.-J.
,
Brauner
,
C. J.
,
Cao
,
Z.-D.
,
Richards
,
J. G.
,
Peng
,
J.-L.
,
Dhillon
,
R.
and
Wang
,
Y.-X.
(
2011
).
The effect of acclimation to hypoxia and sustained exercise on subsequent hypoxia tolerance and swimming performance in goldfish (Carassius auratus)
.
J. Exp. Biol.
214
,
2080
-
2088
.
Gracey
,
A. Y.
,
Troll
,
J. V.
and
Somero
,
G. N.
(
2001
).
Hypoxia-induced gene expression profiling in the euryoxic fish Gillichthys mirabilis
.
Proc. Natl. Acad. Sci. USA
98
,
1993
-
1998
.
Guppy
,
M.
(
2004
).
The biochemistry of metabolic depression: a history of perceptions
.
Comp. Biochem. Physiol. B Biochem. Mol. Biol.
139
,
435
-
442
.
Guppy
,
M.
and
Withers
,
P.
(
1999
).
Metabolic depression in animals: physiological perspectives and biochemical generalizations
.
Biol. Rev.
74
,
1
-
40
.
Hochachka
,
P. W.
(
1982
).
Metabolic arrest as a mechanism of protection against hypoxia
. In
Protection of Tissues Against Hypoxia
(ed. A. Wauquier), pp.
1
-
12
. Oxford: Elsevier.
Hochachka
,
P. W.
(
1985
).
Assessing metabolic strategies for surviving o-2 lack-role of metabolic arrest coupled with channel arrest
.
Mol. Physiol.
8
,
331
-
350
.
Hochachka
,
P. W.
and
Lutz
,
P. L.
(
2001
).
Mechanism, origin, and evolution of anoxia tolerance in animals
.
Comp. Biochem. Physiol. B Biochem. Mol. Biol.
130
,
435
-
459
.
Hochachka
,
P. W.
and
Somero
,
G. N.
(
2002
).
Biochemical Adaptation: Mechanism and Process in Physiological Evolution
.
Oxford
:
Oxford University Press
.
Hochachka
,
P. W.
,
Buck
,
L. T.
,
Doll
,
C. J.
and
Land
,
S. C.
(
1996
).
Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack
.
Proc. Natl. Acad. Sci. USA
93
,
9493
-
9498
.
Holeton
,
G. F.
and
Randall
,
D. J.
(
1967
).
The effect of hypoxia upon the partial pressure of gases in the blood and water afferent and efferent to the gills of rainbow trout
.
J. Exp. Biol.
46
,
317
-
327
.
Humphries
,
M. M.
,
Thomas
,
D. W.
and
Kramer
,
D. L.
(
2003
).
The role of energy availability in mammalian hibernation: a cost-benefit approach
.
Physiol. Biochem. Zool.
76
,
165
-
179
.
Itazawa
,
Y.
and
Takeda
,
T.
(
1978
).
Gas exchange in the carp gills in normoxic and hypoxic conditions
.
Resp. Physiol.
35
,
263
-
269
.
Jackson
,
D. C.
(
1968
).
Metabolic depression and oxygen depletion in the diving turtle
.
J. Appl. Physiol.
24
,
503
-
509
.
Kaiyala
,
K. J.
and
Ramsay
,
D. S.
(
2011
).
Direct animal calorimetry, the underused gold standard for quantifying the fire of life
.
Comp. Biochem. Physiol. A Mol. Integr. Physiol.
158
,
252
-
264
.
Kelly
,
D. A.
and
Storey
,
K. B.
(
1988
).
Organ-specific control of glycolysis in anoxic turtles
.
Amer. J. Physiol.
255
,
R774
-
R779
.
Lague
,
S. L.
,
Speers-Roesch
,
B.
,
Richards
,
J. G.
and
Farrell
,
A. P.
(
2012
).
Exceptional cardiac anoxia tolerance in tilapia (Oreochromis hybrid)
.
J. Exp. Biol.
215
,
1354
-
1365
.
Lai
,
J. C. C.
,
Kakuta
,
I.
,
Mok
,
H. O. L.
,
Rummer
,
J. L.
and
Randall
,
D.
(
2006
).
Effects of moderate and substantial hypoxia on erythropoietin levels in rainbow trout kidney and spleen
.
J. Exp. Biol.
209
,
2734
-
2738
.
Lilly
,
L. E.
,
Blinebry
,
S. K.
,
Viscardi
,
C. M.
,
Perez
,
L.
,
Bonaventura
,
J.
and
McMahon
,
T. J.
(
2013
).
Parallel assay of oxygen equilibria of hemoglobin
.
Anal. Biochem.
441
,
63
-
68
.
Mandic
,
M.
,
Todgham
,
A. E.
and
Richards
,
J. G.
(
2009
).
Mechanisms and evolution of hypoxia tolerance in fish
.
Proc. R. Soc. B Biol. Sci.
276
,
735
-
744
.
Nelson
,
J. A.
(
2016
).
Oxygen consumption rate v. rate of energy utilization of fishes: a comparison and brief history of the two measurements
.
J. Fish. Biol.
88
,
10
-
25
.
Plaxton
,
W. C.
and
Storey
,
K. B.
(
1984
).
Phosphorylation in vivo of red-muscle pyruvate kinase from the channelled whelk, Busycotypus canaliculatum, in response to anoxic stress
.
Eur. J. Biochem.
143
,
267
-
272
.
Popov
,
V. I.
,
Bocharova
,
L. S.
and
Bragin
,
A. G.
(
1992
).
Repeated changes of dendritic morphology in the hippocampus of ground squirrels in the course of hibernation
.
Neuroscience
48
,
45
-
51
.
Ramirez
,
J.-M.
,
Folkow
,
L. P.
and
Blix
,
A. S.
(
2007
).
Hypoxia tolerance in mammals and birds: from the wilderness to the clinic
.
Annu. Rev. Physiol.
69
,
113
-
143
.
Rees
,
B. B.
and
Hand
,
S. C.
(
1991
).
Regulation of glycolysis in the land snail Oreohelix during estivation and artificial hypercapnia
.
J. Comp. Physiol. B
161
,
237
-
246
.
Regan
,
M. D.
,
Gosline
,
J. M.
and
Richards
,
J. G.
(
2013
).
A simple and affordable calorespirometer for assessing the metabolic rates of fishes
.
J. Exp. Biol.
216
,
4507
-
4513
.
Regan
,
M.
,
Gill
,
I. S.
and
Richards
,
J. G.
(
2017
).
Data from: Calorespirometry reveals that goldfish prioritize aerobic metabolism over metabolic rate depression in all but near-anoxic environments
.
Dryad Digital Repository
.
Richards
,
J. G.
(
2009
).
Metabolic and molecular responses of fish to hypoxia
. In Hypoxia (ed.
J. G.
Richards
,
A. P.
Farrell
and
C. J.
Brauner
), pp.
443
-
485
.
San Diego, CA
:
Academic Press
.
Richards
,
J. G.
(
2010
).
Metabolic rate suppression as a mechanism for surviving environmental challenge in fish
.
Prog. Mol. Subcell. Biol.
49
,
113
-
139
.
Robin
,
E. D.
,
Lewiston
,
N.
,
Newman
,
A.
,
Simon
,
L. M.
and
Theodore
,
J.
(
1979
).
Bioenergetic pattern of turtle brain and resistance to profound loss of mitochondrial ATP generation
.
Proc. Natl. Acad. Sci USA
76
,
3922
-
3926
.
Rogers
,
N. J.
,
Urbina
,
M. A.
and
Reardon
,
E. E.
(
2016
).
A new analysis of hypoxia tolerance in fishes using a database of critical oxygen level (Pcrit)
.
Conserv. Physiol.
4
,
1
-
19
.
Sick
,
T. J.
,
Rosenthal
,
M.
,
LaManna
,
J. C.
and
Lutz
,
P. L.
(
1982
).
Brain potassium ion homeostasis, anoxia, and metabolic inhibition in turtles and rats
.
Am. J. Physiol.
243
,
281
-
288
.
Smith
,
V. H.
,
Joye
,
S. B.
and
Howarth
,
R. W.
(
2006
).
Eutrophication of freshwater and marine ecosystems
.
Limnol. Oceanogr.
51
,
351
-
355
.
Snyder
,
S.
,
Nadler
,
L. E.
,
Bayley
,
J. S.
,
Svendsen
,
M. B. S.
,
Johansen
,
J. L.
,
Domenici
,
P.
and
Steffensen
,
J. F.
(
2016
).
Effect of closed v. intermittent-flow respirometry on hypoxia tolerance in the shiner perch Cymatogaster aggregata
.
J. Fish Biol.
88
,
252
-
264
.
Sollid
,
J.
,
De Angelis
,
P.
,
Gundersen
,
K.
and
Nilsson
,
G. E.
(
2003
).
Hypoxia induces adaptive and reversible gross morphological changes in crucian carp gills
.
J. Exp. Biol.
206
,
3667
-
3673
.
Speers-Roesch
,
B.
,
Mandic
,
M.
and
Groom
,
D. J. E.
(
2013
).
Critical oxygen tensions as predictors of hypoxia tolerance and tissue metabolic responses during hypoxia exposure in fishes
.
J. Exp. Mar. Biol. Ecol.
449
,
239
-
249
.
Stangl
,
P.
and
Wegener
,
G.
(
1996
).
Calorimetric and biochemical studies on the effects of environmental hypoxia and chemicals on freshwater fish
.
Thermochim. Acta
271
,
101
-
113
.
Storey
,
K. B.
and
Storey
,
J. M.
(
1990
).
Metabolic rate depression and biochemical adaptation in anaerobiosis, hibernation and estivation
.
Q. Rev. Biol.
65
,
145
-
174
.
Sundin
,
L.
,
Nilsson
,
G. E.
,
Block
,
M.
and
Lofman
,
C. O.
(
1995
).
Control of gill filament blood-flow by serotonin in the rainbow-trout, Oncorhynchus mykiss
.
Am. J. Physiol. Regul. Integr. Comp. Physiol.
268
,
1224
-
1229
.
Turko
,
A. J.
,
Robertson
,
C. E.
,
Bianchini
,
K.
,
Freeman
,
M.
and
Wright
,
P. A.
(
2014
).
The amphibious fish Kryptolebias marmoratus uses different strategies to maintain oxygen delivery during aquatic hypoxia and air exposure
.
J. Exp. Biol.
217
,
3988
-
3995
.
Tzaneva
,
V.
,
Gilmour
,
K. M.
and
Perry
,
S. F.
(
2011
).
Respiratory responses to hypoxia or hypercapnia in goldfish (Carassius auratus) experiencing gill remodelling
.
Resp. Physiol. Neurobiol.
175
,
112
-
120
.
van Ginneken
,
V. J. T.
,
Gluvers
,
A.
,
van der Linden
,
R. W.
,
Addink
,
A. D. F.
and
van den Thillart
,
G. E. E. J. M.
(
1994
).
Direct calorimetry of aquatic animals: automated and computerized data-acquisition system for simultaneous direct and indirect calorimetry
.
Thermochim. Acta
247
,
209
-
224
.
van Ginneken
,
V. J. T.
,
Addink
,
A. D. F.
and
van den Thillart
,
G. E. E. J. M.
(
1997
).
Metabolic rate and level of activity determined in tilapia (Oreochromis mossambicus Peters) by direct and indirect calorimetry and videomonitoring
.
Thermochim. Acta
291
,
1
-
13
.
van Ginneken
,
V. J. T.
,
Snelderwaard
,
P.
,
van der Linden
,
R.
,
van der Reijden
,
N.
,
van den Thillart
,
G. E. E. J. M.
and
Kramer
,
K.
(
2004
).
Coupling of heart rate with metabolic depression in fish: a radiotelemetric and calorimetric study
.
Thermochim. Acta
414
,
1
-
10
.
van Waversveld
,
J.
,
Addink
,
A. D. F.
and
van den Thillart
,
G.
(
1989
).
Simultaneous direct and indirect calorimetry on normoxic and anoxic goldfish
.
J. Exp. Biol.
142
,
325
-
335
.
Vornanen
,
M.
(
2004
).
Seasonality of dihydropyridine receptor binding in the heart of an anoxia-tolerant vertebrate, the crucian carp (Carassius carassius L.)
.
Am. J. Physiol. Regul. Integr. Comp. Physiol.
287
,
R1263
-
R1269
.
Vulesevic
,
B.
and
Perry
,
S. F.
(
2006
).
Developmental plasticity of ventilatory control in zebrafish, Danio rerio
.
Respir. Physiol. Neurobiol.
154
,
396
-
405
.
Yeager
,
D. P.
and
Ultsch
,
G. R.
(
1989
).
Physiological regulation and conformation: a BASIC program for the determination of critical points
.
Physiol. Zool.
62
,
888
-
907
.

Competing interests

The authors declare no competing or financial interests.