Cardiorespiratory collapse at high temperature in swimming adult sockeye salmon.

Elevated summer river temperatures are associated with high in-river mortality in adult sockeye salmon (Oncorhynchus nerka) during their once-in-a-lifetime spawning migration up the Fraser River (British Columbia, Canada). However, the mechanisms underlying the decrease in whole-animal performance and cardiorespiratory collapse above optimal temperatures for aerobic scope (T opt) remain elusive for aquatic ectotherms. This is in part because all the relevant cardiorespiratory variables have rarely been measured directly and simultaneously during exercise at supra-optimal temperatures. Using the oxygen- and capacity-limited thermal tolerance hypothesis as a framework, this study simultaneously and directly measured oxygen consumption rate (MO2), cardiac output [Formula: see text], heart rate (f H), and cardiac stroke volume (V s), as well as arterial and venous blood oxygen status in adult sockeye salmon swimming at temperatures that bracketed T opt to elucidate possible limitations in oxygen uptake into the blood or internal delivery through the oxygen cascade. Above T opt, the decline in MO2max and aerobic scope was best explained by a cardiac limitation, triggered by reduced scope for f H. The highest test temperatures were characterized by a negative scope for f H, dramatic decreases in maximal [Formula: see text] and maximal V s, and cardiac dysrhythmias. In contrast, arterial blood oxygen content and partial pressure were almost insensitive to supra-optimal temperature, suggesting that oxygen delivery to and uptake by the gill were not a limiting factor. We propose that the high-temperature-induced en route mortality in migrating sockeye salmon may be at least partly attributed to physiological limitations in aerobic performance due to cardiac collapse via insufficient scope for f H. Furthermore, this improved mechanistic understanding of cardiorespiratory collapse at high temperature is likely to have broader application to other salmonids and perhaps other aquatic ectotherms.

Introduction

Temperature has been coined the ‘ecological master factor’ for ectotherms because of its influence on the biochemistry, physiology, behaviour, and ecology of animals (Fry, 1971). The optimal temperature (Topt) window for performance may be narrow in stenotherms, such as sockeye salmon (Oncorhynchus nerka), or broad in eurytherms, such as killifish (Fundulus heteroclitus; Eliason ; Healy and Schulte, 2012). Outside of the Topt window, whole-animal performance declines until death eventually occurs. The Topt for aerobic scope has been established as a useful metric for the successful return of adult sockeye salmon to their natal spawning areas in the Fraser River watershed (British Columbia, Canada; Farrell ). Indeed, Topt appears to be locally adapted among sockeye salmon populations to the temperatures that they are likely to encounter once they enter the river (Eliason ). Peak summer river temperatures have increased by around 2°C since the 1950s (Patterson ), forcing salmon to migrate at temperatures warmer than their Topt for aerobic scope. Biotelemetry studies have shown that high river temperature is correlated with excessive (>70%) mortality among migrating salmon (Crossin ; Farrell ; Mathes ; Martins ). Clearly, a conservation concern exists for future salmon migrations in this and other watersheds. As a result, there is great interest in the mechanism(s) underlying the decline of whole-animal performance and cardiorespiratory collapse above Topt.

Physiological processes are critical in defining thermal limits and temperature-induced mortality (Wang and Overgaard, 2007). Aerobic scope, defined as the difference between maximal oxygen consumption (MO2max) and resting oxygen consumption (MO2rest; Fry, 1947), represents the amount of oxygen available for activity. By definition, aerobic scope remains largely unchanged from its maximum within a defined Topt window. However, at the critical temperature (Tcrit), when MO2rest and MO2max intersect, aerobic scope is zero and survival becomes passive, time limited, and supported by anaerobic metabolism (Pörtner, 2001; Pörtner and Farrell, 2008), a situation clearly incompatible with a highly aerobic upriver migration. Here we were specifically interested in which process triggers the decline in aerobic scope at a supra-optimal temperature. In the context of the present study, the term Topt refers to the optimal temperature for aerobic scope.

While it is unclear what determines the limits of upper thermal tolerance in ectotherms, one leading possibility is the oxygen- and capacity-limited thermal tolerance (OCLTT) hypothesis, which attributes the decline in aerobic scope above Topt to capacity limitations of the organ systems delivering oxygen to the tissues (Pörtner, 2001; Pörtner and Knust, 2007). Several studies with aquatic ectotherms have found support for the OCLTT hypothesis (e.g. fish and invertebrates; Frederich and Pörtner, 2000; Mark ; Lannig ; Pörtner and Knust, 2007; Pörtner and Farrell, 2008; Eliason ; Verberk and Calosi, 2012), while recent studies with pink salmon (Oncorhynchus gorbuscha), an intertidal snail (Echinolittorina malaccana), and an air-breathing toad (Rhinella marina) found little support for the OCLTT hypothesis (Clark ; Marshall ; Overgaard ). According to the OCLTT hypothesis, any one of the five major steps in the oxygen cascade from the environment to the mitochondria might trigger the collapse above Topt (i.e. oxygen delivery by gill ventilation, oxygen diffusion across the gills, oxygen transport via the circulatory system, oxygen diffusion across tissue capillaries, and oxygen use by mitochondria; Weibel, 1984). However, simultaneous and preferably direct measurements of MO2, cardiac output , heart rate (fH), and cardiac stroke volume (Vs), as well as arterial and venous blood oxygen status [partial pressure (PO2) and content (CO2)] are required to discover the initial limitation in the oxygen cascade that triggers the decline in aerobic scope above Topt. For example, a limitation in either oxygen delivery to or oxygen diffusion across the gill would be manifest by a decrease in arterial PO2 (PaO2) and arterial CO2 (CaO2). Alternatively, a cardiac limitation would be indicated by a failure of to increase to match the increased tissue oxygen demand above Topt. Finally, a limitation in oxygen diffusion to mitochondria in the working muscles would be indicated by venous PO2 (PvO2) remaining constant with increased tissue oxygen demand above Topt. However, support for a single limiting site in the oxygen cascade of fish (gills, heart, or locomotory muscle) remains incomplete despite considerable analysis (Brett, 1971; Heath and Hughes, 1973; Taylor , 1997; Farrell, 2007a; Steinhausen ; Farrell ). This data gap exists because most studies examining temperature effects on aerobic scope in fish have not directly measured all the required variables (e.g. Fry, 1947; Brett, 1971; Taylor ), and the more comprehensive studies only used resting fish, thus failing to examine aerobic scope (e.g. Heath and Hughes, 1973; Cech ; Sartoris ; Gollock ; Clark ; Keen and Gamperl, 2012). In fact, only Steinhausen have directly measured all the required variables as a function of temperature in exercising fish.

The objective of this study was to examine the mechanism(s) that triggers the decline in MO2max and aerobic scope above Topt in sockeye salmon. Previous work suggested that sockeye salmon thermal limits are set by physiological limitations in aerobic performance, which may possibly be due to cardiac collapse at high temperatures (Steinhausen ; Eliason ). However, Eliason did not systematically examine all the possible mechanisms leading to cardiorespiratory collapse (only aerobic scope, cardiac scope, scope for heart rate, MO2rest, and MO2max were reported as a function of temperature), and Steinhausen only swam sockeye salmon up to ∼75% of their critical swim speed (Ucrit). Thus, the present study reports the first simultaneous and direct measurements of all the pertinent oxygen transport variables in adult sockeye salmon swimming maximally below, at, and above Topt. Novel data on MO2, , fH, and Vs, as well as arterial and venous PO2, CO2, and blood chemistry are presented. We hypothesize that a limitation on fH triggers cardiorespiratory collapse when temperature exceeds Topt for aerobic scope.

Materials and methods

Fish collection

All procedures were approved by the University of British Columbia's Animal Care Committee (Animal use protocols A06-0328 and A08-0388) in accordance with guidelines recommended through the Canadian Council on Animal Care. Wild adult sockeye salmon (n = 55; body mass, 2380 ± 60 g; fork length, 59.6 ± 0.4 cm) were collected in 2007, 2008, and 2009 early in their upriver spawning migration (∼100 km upstream of the Fraser River mouth) and transported to the Fisheries and Oceans Canada Cultus Lake Research Laboratory (Cultus Lake, BC, Canada), where experiments were conducted. All fish were given a unique PIT (Passive Integrated Transponder; Biomark Inc., Boise, ID, USA) tag for individual identification, a scale was removed, and <0.1 g of the adipose fin was clipped for population identification via DNA analysis (Beacham ). Only fish from Early Stuart (n = 21), Chilko (n = 22), and Quensel (n = 12) populations were used in the present study. Fish were held at 11–12°C for 1–4 weeks in outdoor 8000–12 000 l circular aquaria under seasonal photoperiod.

Surgical procedures

Individual fish were anaesthetized with buffered tricaine methanesulfonate in freshwater (0.2 g l−1 NaHCO3 and 0.1 g l−1 MS-222; Sigma, St Louis, MO, USA), weighed, and transferred onto wet foam on a surgical table, where their gills were continuously irrigated with aerated, chilled freshwater with a lower dose of buffered anaesthetic (0.15 g l−1 NaHCO3 and 0.075 g l−1 MS-222). Surgical procedures have been detailed elsewhere (Steinhausen ; Eliason ). Fish were instrumented with a 3 mm SB flowprobe (lateral cable exit; Transonic Systems, Ithaca, NY, USA) around the ventral aorta (Steffensen and Farrell, 1998) to measure , fH, and Vs. Cannulae made of PE-50 tubing were inserted into the dorsal aorta (Soivio ) and sinus venosus (Farrell and Clutterham, 2003) to sample arterial and venous blood, respectively, for measurement of oxygen status and blood chemistry.

Swim challenge to

Three days prior to the swimming test, most fish were placed in a 1400 l circular tank, and the temperature was progressively increased from the holding temperature (11–12°C) to the test temperature (12–22°C) by no more than 5°C day−1. The fish were maintained at their test temperature for 24–48 h before surgery was conducted. Following surgery, the fish were placed individually in one of two Brett-type swim tunnels (described by Lee ; Steinhausen ) and allowed to recover overnight (>8 h) at their test temperature (12–22°C) at a low water velocity of ∼0.39 body lengths per second (bl s−1). Resting measurements of all variables were made following overnight recovery from surgery in the swim tunnel. Fish then underwent two sequential ramp-Ucrit swim protocols with a 45  min recovery in between (Jain ; Lee ; Eliason ). Only data from the first swim are used here.

Fish could not be held at the temperature extremes due to logistical constraints on temperature regulation in the holding tanks. Therefore, some fish recovered overnight (>8 h) from surgery in the swim tunnel at the holding temperature (12°C); initial resting values were measured at this temperature in the morning, and then the water temperature was increased or decreased by 4°C h−1 to the test temperature of either 8–10 or 22–26°C. One hour after reaching the test temperature, resting variables were recorded, and the fish underwent a single ramp-Ucrit swim challenge.

The MO2 was measured during the second half of every 20 min velocity interval using an Oxyguard probe (Point Four Systems, Richmond, BC, Canada) attached to a Windaq box (Dataq Instruments, Akron, ON, USA) interfaced with Labview software (version 6.0; National Instruments, Austin, TX, USA). The was measured continuously at 200 Hz throughout the swim trials by connecting the flowprobe to a flowmeter (Transonic Systems) interfaced with Biopac hardware and Acknowledge software (Biopac Systems, Santa Barbara, CA, USA). The value of was calculated as the mean of three to six segments of continuous 30 s traces. Blood was strategically sampled prior to the swim test (rest), during steady-state swimming (steady), once the fish had transitioned to burst-and-coast swimming (burst), and immediately following the swim test within 5 min of fatigue (fatigue).

Analysis of whole blood and plasma

Partial pressure of oxygen (PO2), oxygen content (CO2), haemoglobin concentration ([Hb]), and haematocrit (Hct) were measured using whole blood samples. Blood PO2 was measured using a blood gas monitor (PHM 73; Radiometer, Copenhagen, Denmark), which was calibrated and maintained at the experimental temperature using a water jacket. Blood CO2 was measured according to the method of Tucker (1967). The [Hb] was measured using either a handheld haemoglobin analyser (HemoCue 201+; Ängelholm, Sweden) calibrated for fish blood (Clark ) or the spectrophotometer method with Drabkin's solution (Drabkin and Austin, 1935). Haematocrit was measured in duplicate using microhaematocrit capillary tubes spun at 10 000g. The remaining blood was centrifuged at 7000g, and the plasma was flash frozen in liquid nitrogen and stored at −80°C for subsequent analyses. Plasma glucose and lactate (YSI 2300 Stat Plus analyser), sodium and potassium (Cole-Parmer, model 41, single channel flame photometer) and chloride (Haake Buchler digital chloridometer) were measured using techniques outlined previously (Farrell ).

Data analysis and statistics

The Ucrit was calculated using established methods (Brett, 1964), after accounting for the solid blocking effect as outlined by Bell and Terhune (1970). Stroke volume (Vs) was calculated as follows: . Cost of transport (COT) was calculated as follows: COT = MO2/(U × 60), where U is the swimming speed in metres per second. Net cost of transport (COTnet) was calculated as follows: COTnet = (MO2 – MO2rest)/(U × 60). Likewise, cost of transport for cardiac output and net cost of transport for cardiac output were calculated. Oxygen extraction (A–VO2) was calculated as follows: A–VO2 = CaO2 – CvO2. Arterial oxygen transport (TaO2) to the tissues was calculated as follows: . Venous oxygen transport (TvO2) to the spongy myocardium and gills was calculated as follows: . Mean corpuscular haemoglobin concentration (MCHC) was calculated as follows: MCHC = [Hb]/(Hct/100).

In order to maximize statistical power, data were pooled for Early Stuart, Chilko, and Quesnel populations, because they undergo similar long, difficult river migrations, have a similar Topt for aerobic scope (∼17°C) and have a similar maximal aerobic scope, cardiac scope and scope for heart rate (Eliason ). Moreover, Eliason et al. (under review) clearly show that MO2, , fH, and Vs for these three populations do not differ at rest, when swimming, or during recovery at Topt for aerobic scope.

In order to permit statistical comparisons, individual fish were sorted into four temperature groupings based on performance relative to the maximal aerobic scope for a population (Fig. 1). The Topt grouping (15–20°C; n = 28) combined individuals that attained 90–100% of the population-specific maximal aerobic scope. The assumption here is that the Topt window is >90% of maximal aerobic scope. Bracketing the Topt window were groupings of individuals that attained only 50–90% of the population-specific maximal aerobic scope. Thus, the grouping below Topt was designated Tmin50–90 (n = 8; 12°C for Early Stuart, and 9–10°C for Chilko; no Quesnel fish were swum in this grouping), while the grouping above Topt was designated Tmax50–90 (n = 11; 22–23°C for Early Stuart and Quesnel, and 24–25°C for Chilko). The Tmax0–50 grouping (n = 8) combined fish that attained 0–50% of the population-specific maximal aerobic scope at a supra-optimal temperature (23–26°C for Early Stuart and Quesnel, and 25–26°C for Chilko). In addition, a limited analysis was performed with the Chilko population alone to examine whether or not pooling data in the manner described above obscured or changed major cardiorespiratory trends.

Figure 1:

a schematic representation of the four temperature groupings used to analyse cardiorespiratory performance with temperature. The 50 and 90% cut-offs were assigned to individual fish based on their performance relative to Topt for the population (see main text for details).

Grouped data are presented as means ± SEM, unless otherwise indicated. Values of P < 0.05 were considered statistically significant. Independent data were compared using one-way ANOVA. Dependent data were compared using Student's paired t-test, a one-way repeated measures ANOVA or a two-way repeated measures ANOVA, as appropriate. When the requirement for normal distribution and equal variance could not be met after transformation, data were compared using appropriate non-parametric tests (e.g. Mann–Whitney U-test, Kolmogorov–Smirnov test, or Kruskal–Wallis test). The appropriate post hoc test (Holm–Sidak or Dunn's) was used to test for differences among groups. A polynomial quadratic equation was fitted to maximal and scope data for MO2, , Vs, and fH, and an exponential equation was fitted to the resting data for MO2, , and fH for individual Chilko sockeye salmon. A quadratic equation was fitted through the individual data for resting and fatigue venous PO2 and CO2.

Results

Cardiorespiratory and swimming performance

As intended from the grouping procedure, aerobic scope was highest for the Topt grouping, significantly lower and similar for the Tmin50–90 and Tmax50–90 groupings, and lowest for Tmax0–50 grouping (Fig. 2E). Thus, the four performance groupings created the equivalent of a Fry curve for aerobic scope (Fry, 1947; Farrell, 2009). The Fry aerobic scope curve was a result of MO2rest increasing progressively from the lowest to the highest temperature grouping (Fig. 2A), while MO2max increased significantly between Tmin50–90 and Topt, was unchanged between Topt and Tmax50–90, and then decreased significantly at Tmax0–50 (Fig. 2A). Indeed, swimming to fatigue significantly increasing MO2max for the Tmin50–90, Topt, and Tmax50–90 groupings, while maximal swim speed and its corresponding MO2max were significantly lower for the Tmax0–50 grouping compared with the other groupings (Fig. 3A).

Figure 2:

resting and maximal oxygen consumption rate (MO2; A), cardiac output (; B), stroke volume (Vs; C), and heart rate (fH; D) at the four temperature categories. Scope for MO2 (E), (F), Vs (G), and fH (H) are shown. All values are presented as means ± SEM. Significant differences among temperature groupings are indicated by differing letters (P < 0.05).

Figure 3:

changes in oxygen consumption rate (A), cardiac output (B), cardiac stroke volume (C), and heart rate (D) as a function of swimming speed among the four temperature groupings. Fatigue values were obtained within 5 min after the fish stopped swimming. Means ± SEM are shown.

Cardiac output varied among temperature groupings and with swimming, following similar patterns to MO2 (Figs 2 and 3). The resting cardiac output increased progressively with increasing temperature groupings, plateauing between Tmax50–90 and Tmax0–50, while increased significantly between Tmin50–90 and Topt, was unchanged between Topt and Tmax50–90, and then decreased significantly at Tmax0–50. As a result, cardiac scope was lowest for the Tmax0–50 grouping (Fig. 2F).

Stroke volume at rest was independent of temperature except for a quantitatively small, but statistically significant decrease at Tmax0–50 when compared with Topt (Fig. 2C). Scope for Vs was maintained between the Tmin50–90 and Tmax50–90 groupings, but decreased significantly for the Tmax0–50 grouping when Vsmax also decreased significantly (Fig. 2C and G).

While fHrest increased progressively with each temperature grouping (Fig. 2D), fHmax increased with temperature only up to the Tmax50–90 grouping, plateauing between Tmax50–90 and Tmax0–50 (Fig. 2D). Therefore, scope for fH was highest at Topt (Fig. 2H). Notably, scope for fH decreased precipitously at supra-optimal temperatures, approaching zero for the Tmax50–90 grouping, and becoming negative for the Tmax0–50 grouping (Fig. 2H).

In addition to the bradycardia during swimming and thus negative scope for fH, every fish that swam at Tmax0–50 exhibited an irregular heart beat immediately following the swim test, with 57% exhibiting cardiac dysrhythmias during the swim and shortly before fatigue (Fig. 4). Despite decreasing the temperature immediately following fatigue, 29% of the Tmax0–50 fish died, even though minutes before many were swimming at 1.1–1.5 bl s−1. Post-fatigue cardiac dysrhythmia was also present in the Tmax50–90 grouping, but at a lower percentage (27% of the fish), and none died post-exhaustion. Thus, the difference between post-fatigue life and death at supra-optimal temperature was a matter of 2–3°C. No cardiac dysrhythmias or deaths accompanied or followed swimming to exhaustion at either Tmin50–90 or Topt.

Figure 4:

individual blood flow traces for two Chilko sockeye salmon at 17°C (Topt) and 26°C (Tmax0–50) at rest (A and C) and during swimming (B, D, and E). Swimming traces were recorded during the final swimming speed before each fish fatigued (measured at 2.3 and 1.5 bl s−1 for the 17 and 26°C fish, respectively). Trace D was recorded 5 min before trace E, at the same swimming speed.

Maximal scope occurred at Topt for MO2, , and fH, but at Tmin50–90 for Vs (Fig. 5). In comparison to these maximal values, scope for MO2, , and Vs decreased by 13–25%, whereas scope for fH plummeted by 80% for the Tmax50–90 grouping. Clearly, the precipitous collapse of scope for fH was a potential trigger for the general cardiorespiratory collapse, which became clear and profound for the Tmax0–50 grouping, with scope for MO2, , and Vs all declining to 16–20% of their maximal values, while scope for fH collapsed to almost −40% of its maximal value (Fig. 5).

Figure 5:

percentage of maximal aerobic scope, cardiac scope, scope for heart rate (fH), and scope for stroke volume (Vs) for each temperature grouping.

Cardiorespiratory and swimming performance of Chilko sockeye salmon

The pooling of populations accurately reflected the major cardiovascular trends seen for a single population, Chilko sockeye salmon swum at temperatures ranging from 8 to 26°C (Figs 2 and 6). Values of MO2, , Vs, and fH all changed as a function of temperature in a similar manner to those of the pooled populations that were analysed statistically (Figs 2 and 6). Resting MO2, , and fH all increased exponentially with increasing temperature (Fig. 6), whereas Vsrest was insensitive to temperature. Both MO2max and increased with increasing temperature until Topt was reached, and both declined at the warmest temperatures. Maximal heart rate increased with increasing temperatures and plateaued above Topt, resulting in a negative scope for fH at the warmest temperatures. Both Vsmax and scope for Vs steadily declined with increasing temperatures (Fig. 6).

Figure 6:

resting (open circles) and maximal values (filled circles) for oxygen consumption rate (MO2; A), cardiac output (; B), stroke volume (Vs; C), and heart rate (fH; D) in Chilko sockeye salmon. Each point corresponds to a single fish. Scope for MO2 (E), (F), Vs (G), and fH (H) are shown. A polynomial quadratic equation was fitted to the maximum and scope data, an exponential equation was fitted to the resting data for MO2, , and fH, and no relationship was found with temperature for resting Vs. Data in panels A and E and the equations for the lines for F and H have been previously reported by Eliason .

Cost of swimming

The oxygen cost of transport (COT) decreased with increasing swimming speed and reached a minimum of 0.12–0.26 mg O2 kg−1 m−1 across temperature groupings (Fig. 7). Temperature effects on COT were primarily evident at low speeds (0.4 bl s−1 = ‘rest’), with the warmer groupings (Tmax50–90 and Tmax0–50) tending to be twice as high compared with the colder groupings (Topt and Tmin50–90). The pattern was reversed for the net cost of transport (COTnet), which tended to be higher in the Topt and Tmin50–90 groupings.

Figure 7:

Cost of transport (COT; A), net cost of transport (COTnet; B), cardiovascular cost of transport (; C), and net cardiovascular cost of transport (; D) with swimming speed across the four temperature groupings. Means ± SEM are shown.

The circulatory cost of transport and net circulatory cost of transport for cardiac output followed similar patterns with swimming and temperature to COT and COTnet, respectively (Fig. 7). The circulatory cost of transport decreased with increasing swimming speeds until ∼0.87 bl s−1, after which it was maintained at around 0.7–1.9 ml kg−1 m−1 for the Tmin50–90, Topt, and Tmax50–90 groupings. In contrast, declined steadily in the Tmax0–50 grouping until fish stopped swimming. Again, tended to be higher for warm temperature groupings; it was significantly lower at Tmin50–90 compared with the Tmax50–90 and Tmax0–50 groupings at the slowest speeds (Fig. 7); however, tended to be higher in the Topt and Tmin50–90 groupings.

Oxygen uptake, transport, and removal

The oxygen status of arterial blood was assessed from PaO2 and CaO2, which were found to be independent of temperature at all swimming speeds (Table 1, Fig. 8). In addition, resting PaO2 and CaO2 in the Tmax50–90 or Tmax0–50 groupings did not differ between the starting temperature (12°C) and the test temperature (22–26°C; Table 2). This result suggests that an acute temperature change while the fish were in the swim tunnel had no significant effect on arterial blood oxygen status. However, PaO2 and CaO2 tended to decrease relative to the resting values with swimming, as has been previously observed (Steinhausen ). Thus, oxygen transfer at the gills was unimpaired by supra-optimal temperature, even with the small effect of swimming on arterial oxygen status.

Table 1:

oxygen status variables across the four temperature groupings and with swimming

	Rest	Steady	Burst	Fatigue
PaO2 (torr)
Tmin50–90	–	60.7 ± 9.3	62.9 ± 3.5	49.8 ± 2.4
Topt	98.4 ± 7.4	96.5 ± 5.8	64.7 ± 9.6*	69.7 ± 4.9*
Tmax50–90	84.6 ± 7.7	71.9 ± 3.8	58.1 ± 4.3	60.7 ± 5.7
Tmax0–50	72.8 ± 4.8	70.8 ± 3.7	71.7 ± 7.4	75.4 ± 7.9
PvO2 (torr)
Tmin50–90	28.0 ± 1.3ab	24.0 ± 1.1	–	17.5 ± 2.5
Topt	41.6 ± 2.4a	32.6 ± 1.9*	17.6 ± 2.8*	23.4 ± 2.0*
Tmax50–90	28.5 ± 3.9b	21.1 ± 3.3	11.6 ± 3.7*	13.2 ± 2.18*
Tmax0–50	10.3 ± 4.6c	–	–	–
CaO2 (ml dl−1)
Tmin50–90	10.3 ± 1.7	11.6 ± 1.1	11.9 ± 1.2	11.6 ± 1.8
Topt	12.0 ± 0.6	10.9 ± 0.7	9.2 ± 0.8	9.3 ± 0.6
Tmax50–90	12.8 ± 0.6	11.2 ± 0.6	10.2 ± 0.5	9.0 ± 0.7*
Tmax0–50	10.8 ± 0.9	9.9 ± 1.6	9.2 ± 1.5	8.5 ± 0.5
CvO2 (ml dl−1)
Tmin50–90	7.7 ± 1.1a	4.9 ± 1.2	–	3.5 ± 0.4a*
Topt	8.2 ± 0.4a	5.7 ± 0.4*	2.5 ± 0.3 *	3.0 ± 0.4a*
Tmax50–90	6.9 ± 0.5a	4.3 ± 0.8	1.0 ± 0.4*	1.4 ± 0.4b*
Tmax0–50	1.4 ± 0.6b	–	–	–
A–VO2 (ml dl−1)
Tmin50–90	1.6 ± 1.6	–	–	6.8 ± 1.7*
Topt	4.2 ± 0.8	4.8 ± 0.5	6.3 ± 1.0	6.0 ± 0.7
Tmax50–90	6.1 ± 1.2	7.0 ± 0.9	9.2 ± 0.7	8.0 ± 1.4
Tmax0–50	–	–	–	–
TaO2 (ml O2 min−1 kg−1)
Tmin50–90	3.4 ± 0.5	8.6 ± 1.0	13.4 ± 0.1ab*	11.1 ± 1.1a*
Topt	5.8 ± 0.4	9.4 ± 0.4*	14.7 ± 1.2a*	10.7 ± 0.8a*
Tmax50–90	7.6 ± 0.3	9.8 ± 0.4	13.7 ± 2.0a*	8.9 ± 1.7ab
Tmax0–50	7.2 ± 0.5	8.7 ± 1.2	7.4 ± 0.7b	4.1 ± 1.4b
TvO2 (ml O2 min−1 kg−1)
Tmin50–90	2.8 ± 0.4b	3.8 ± 0.7		3.6 ± 0.4
Topt	4.1 ± 0.3a	5.1 ± 0.4	3.7 ± 0.4	3.0 ± 0.4
Tmax50–90	4.2 ± 0.4ab	3.8 ± 0.7	1.4 ± 0.5*	1.6 ± 0.5*
Tmax0–50	0.8 ± 0.6b	–	–	–

Arterial and venous partial pressures of oxygen (PaO2 and PvO2), oxygen content (CaO2 and CvO2), oxygen extraction (A–VO2), arterial oxygen transport (TaO2), and venous oxygen transport (TvO2) are indicated. Values are means ± SEM. Temperature groupings with different letters within a swimming speed are statistically different, and an asterisk indicates a statistically significant difference from rest within a temperature grouping (P < 0.05).

Figure 8:

arterial and venous oxygen content (CO2; A and B, respectively) and partial pressure of oxygen (PO2; (C and D, respectively) at rest (open symbols) and fatigue (filled symbols) in four temperature groupings (triangles = Tmin50–90; circles = Topt; squares = Tmax50–90; and diamonds = Tmax0–50). Each data point corresponds to an individual fish. A quadratic equation was fitted through the venous data. For resting CvO2, R2 = 0.37, P = 0.0007; for fatigue CvO2, R2 = 0.39, P = 0.002; for resting PvO2, R2 = 0.51, P < 0.0001; and for fatigue PvO2, R2 = 0.41 P = 0.001.

Table 2:

arterial partial pressure of oxygen (PaO2) and oxygen content (CaO2) in resting fish, measured at 12°C and at the test temperature

	n	PaO2 (torr)		CaO2 (ml dl−1)
		12°C	Test temp.	12°C	Test temp.
Tmax50–90	5	67.5 ± 3.3	81.0 ± 5.6	14.2 ± 0.6	13.4 ± 0.5
Tmax0–50	7	63.5 ± 6.4	72.8 ± 4.8	12.0 ± 0.4	10.8 ± 0.9

Means ± SEM are presented. There were no significant differences within a temperature group (P > 0.05).

Haemoglobin concentration, haematocrit, and mean corpuscular haemoglobin concentration were also independent of temperature and swimming speed, except that MCHC decreased at fatigue in the Tmax50–90 grouping relative to rest (Table 3). Given the stability of both CaO2 and [Hb] as a function of temperature, the changes in arterial oxygen transport (TaO2) as a function of temperature (and swimming speed) were primarily a result of changes in . Notably, TaO2 for the Tmax0–50 grouping was significantly lower for burst swimming and at fatigue when compared with Topt (Table 1). Indeed, while TaO2 increased during burst swimming by 294, 153, and 80% over the resting values for the Tmin50–90, Topt, and Tmax50–90 groupings, respectively, TaO2 was unchanged for the Tmax0–50 grouping (Table 1). Thus, the ability of the circulatory system to transport blood to active locomotory muscles was greatly diminished at supra-optimal temperatures because of a decline in , not CaO2.

Table 3:

arterial haematological variables across the four temperature groupings and with swimming

	Rest	Steady	Burst	Fatigue
[Hb] (g l−1)
Tmin50–90	92.3 ± 9.0	89.9 ± 10.6	93.5 ± 11.8	81.5 ± 12.6
Topt	92.4 ± 4.6	90.3 ± 4.2	91.8 ± 5.4	87.3 ± 3.0
Tmax50–90	100.3 ± 10.9	95.8 ± 2.7	89.3 ± 6.3	89.1 ± 6.3
Tmax0–50	89.0 ± 5.0	98.3 ± 6.0	91.6 ± 7.5	91.8 ± 2.3
Hct (%)
Tmin50–90	30.7 ± 3.4	29.2 ± 1.6	30.6 ± 3.5	29.6 ± 5.2
Topt	31.0 ± 1.7	30.4 ± 1.6	33.9 ± 2.5	31.1 ± 1.4
Tmax50–90	32.9 ± 1.8	31.4 ± 0.8	34.2 ± 2.2	36.1 ± 1.4
Tmax0–50	32.6 ± 1.5	32.1 ± 0.9	35.8 ± 3.4	37.5 ± 3.3
MCHC (g l−1)
Tmin50–90	302.2 ± 9.1	306.4 ± 19.7	305.1 ± 6.3	278.3 ± 10.9
Topt	300.5 ± 6.6	299.4 ± 6.8	273.1 ± 8.1	282.3 ± 4.9
Tmax50–90	308.4 ± 32.7	306.7 ± 13.5	267.7 ± 25.1	249.0 ± 22.0*
Tmax0–50	274.5 ± 15.8	305.7 ± 11.7	259.1 ± 19.4	250.4 ± 16.4
Glucose (mmol l−1)
Tmin50–90	–	9.6 ± 0.7	9.0 ± 0.3	9.8 ± 0.7
Topt	5.6 ± 0.5	5.1 ± 0.5	6.6 ± 0.5	6.2 ± 0.6
Tmax50–90	7.6 ± 1.9	8.3 ± 1.2	6.9 ± 1.0	7.8 ± 1.1
Tmax0–50	10.2 ± 2.0	–	5.4 ± 1.9*	7.3 ± 1.6*
Lactate (mmol l−1)
Tmin50–90	0.8 ± 0.4a	1.3 ± 0.3	2.3 ± 0.5	4.0 ± 0.9a
Topt	1.3 ± 0.2a	1.4 ± 0.2	3.5 ± 0.5	5.3 ± 0.6ab*
Tmax50–90	2.3 ± 0.5ab	2.5 ± 0.4	5.0 ± 0.6	9.5 ± 0.9c*
Tmax0–50	4.5 ± 0.7b	–	5.0 ± 1.4	8.0 ± 0.7b*
Na+ (mmol l−1)
Tmin50–90	142.3 ± 2.9a	138.1 ± 1.1	133.1 ± 6.0	–
Topt	140.1 ± 1.7a	143.8 ± 1.6	145.9 ± 2.1	152.9 ± 2.0a*
Tmax50–90	120.7 ± 6.8b	140.4 ± 5.2*	138.9 ± 3.2*	139.6 ± 4.7ab*
Tmax0–50	137.7 ± 4.1ab	–	135.9 ± 2.6	137.6 ± 2.6b
K+ (mmol l−1)
Tmin50–90	3.3 ± 0.1	3.9 ± 0.5	4.3 ± 0.8	4.0 ± 1.8
Topt	4.9 ± 0.4	4.6 ± 0.4	2.7 ± 0.7	2.6 ± 0.3*
Tmax50–90	6.2 ± 1.0	4.5 ± 0.7	2.8 ± 0.6*	1.7 ± 0.3*
Tmax0–50	3.8 ± 0.3	–	3.9 ± 0.9	1.7 ± 0.6
Cl− (mmol l−1)
Tmin50–90	121.0 ± 2.0ab	122.0 ± 5.2	117.4 ± 2.3ab	123.6 ± 3.8ab
Topt	127.5 ± 0.9a	129.9 ± 1.4	130.2 ± 1.3a	133.3 ± 1.2a
Tmax50–90	119.3 ± 6.0ab	119.8 ± 2.1	118.3 ± 4.5ab	119.9 ± 5.2b
Tmax0–50	112.0 ± 3.1b	–	113.4 ± 3.9b	111.9 ± 2.7b

Haemoglobin concentration ([Hb]), haematocrit (Hct), mean cell haemoglobin concentration (MCHC), plasma sodium (Na+), plasma potassium (K+), and plasma chloride (Cl−) are indicated. Values are means ± SEM. Temperature groupings with different letters within a swimming speed are statistically different, and an asterisk indicates a statically significant difference from rest within a temperature grouping (P < 0.05).

Unlike arterial blood, PvO2 and CvO2 both varied significantly with temperature and swimming (Table 1). The changes in CvO2 directly reflected changes in haemoglobin saturation, because [Hb] was unchanged (Table 3). The effect of temperature on resting PvO2 and CvO2 was profound. Indeed, resting PvO2 for the Tmax0–50 grouping was 10 torr, 25% of the Topt value, and resting CvO2 was only 16–20% of the CvO2 for the other three temperature groupings. When temperature was treated as a continuous independent variable, the significant polynomial relationships also revealed decreases in resting PvO2 and CvO2 at the highest temperature (Fig. 8).

Swimming increased muscle oxygen extraction from the blood, as reflected by decreases of PvO2 and CvO2 (Table 1). At fatigue, CvO2 and PvO2 were 114 and 77%, respectively, lower in the Tmax50–90 grouping compared with Topt. Likewise, the significant polynomial relationship with temperature for both PvO2 and CvO2 at fatigue further illustrates the point that tissue oxygen extraction must have increased above Topt (Fig. 8). Tissue oxygen extraction (A–VO2) was calculated only for individuals with paired, simultaneous arterial and venous samples. The tendency for A–VO2 to increase with swimming and at warmer temperatures did not reach statistical significance, probably due to low statistical power (Table 1).

Even though the tissues extracted more oxygen from the blood with swimming, the amount of oxygen leaving the tissues and returning to the heart (venous oxygen transport; TvO2) was independent of swimming effort at Topt (Table 1). Thus, the increase in matched the decrease in CvO2. However, this situation changed at supra-optimal temperatures, because TvO2 significantly decreased at burst and fatigue for the Tmax50–90 grouping (Table 1). Even at rest, TvO2 was reduced by over 4-fold in the Tmax0–50 grouping compared with Topt (Table 1).

Other blood variables

Plasma lactate varied significantly with both temperature and swimming (Table 3). The effect of supra-optimal temperatures on plasma lactate was profound, because resting plasma lactate was >3-fold higher in the Tmax0–50 grouping compared with Topt (Table 3). This result suggests that oxygen supply was insufficient to meet tissue oxygen demand even at rest for the highest test temperature. As expected, burst swimming to fatigue increased plasma lactate by 2- to 4-fold relative to resting levels for all temperature groupings. Moreover, plasma lactate was highest in fish at Tmax50–90 at fatigue compared with the other groups (Table 3). Plasma glucose was independent of temperature and swimming, except for a significant decrease in plasma glucose with swimming for the Tmax0–50 grouping (Table 3), perhaps reflecting a greater drain on this fuel for anaerobic metabolism.

Plasma sodium varied significantly with both temperature and swimming speed (Table 3), tending to be highest for the Topt grouping and increasing with swimming speed for the Topt and Tmax50–90 groupings. Plasma chloride also varied significantly with temperature, but not with swimming speed (Table 3), and was significantly higher at Topt relative to the Tmax0–50 grouping. Plasma potassium did not vary significantly with temperature (Table 3), but tended to decrease with swimming speed, and was lowest at fatigue.

Discussion

In order to apply physiological knowledge to conservation problems, scientists require a mechanistic understanding and the support of guiding principles. The present study sought to make such a contribution by examining the mechanism of the decline in MO2max and aerobic scope above Topt in sockeye salmon using the OCLTT hypothesis as a framework. Earlier temperature studies with fishes either did not measure all the required variables (i.e. MO2, , CaO2, and CvO2) or the fish were not swum maximally, if at all. For example, many studies have examined the collapse of cardiorespiratory performance during warming of resting fish (Heath and Hughes, 1973; Cech ; Sartoris ; Gollock ; Clark ; Keen and Gamperl, 2012), but their results relate to what is triggering events close to Tcrit as opposed to the collapse of aerobic scope above Topt.

Brett (1971) first described a parallel association of swimming performance, aerobic scope, and cardiac scope as a function of temperature between 5 and 22°C. He noted a decrease in aerobic scope, cardiac scope, and scope for heart rate above a Topt of 15°C. However, was calculated using the Fick principle, and Brett concluded that the cardiorespiratory collapse above Topt was likely to be due to limited oxygen availability, even though blood gases were not reported (Brett, 1964, 1971). Taylor examined aerobic swimming of rainbow trout at three acclimation temperatures (4, 11, and 18°C), and indirectly estimated using microspheres. By coupling these cardiac measurements with an earlier study of blood gases (Taylor ), they concluded that aerobic swimming performance may have been limited at high temperature because of numerous factors, including reduced oxygen solubility in water and plasma, decreased arterial blood oxygen content and haematocrit, elevated energy expenditure for reproduction, reduced aerobic muscle mass or decreased cardiac scope, and reduced blood flow to swimming muscles. Steinhausen was the first to measure MO2, , CaO2, and CvO2 simultaneously and directly in sockeye salmon as they were progressively warmed from 15 to 24°C while swimming steadily at ∼1.35 bl s−1 or ∼75% of maximum. They concluded that a cardiac limitation developed at warm temperatures in swimming fish, because , fH, and TaO2 reached maxima and failed to continue to increase with increasing temperatures in order to meet the increased tissue oxygen demand.

The present study is the most comprehensive assessment to date of aerobic scope and the associated cardiovascular and blood gas variables across a range of temperatures in an aquatic ectotherm. We conclude that a limitation in maximal cardiac performance inhibits MO2max and aerobic scope at supra-optimal temperatures in salmonids, which is apparently triggered by a precipitous decrease in scope for fH. Thus, the present study with sockeye salmon found support for the OCLTT hypothesis, though we caution that the OCLTT hypothesis may not be broadly applicable to all ectotherms [e.g. pink salmon (Clark ), intertidal snails (Marshall ), and air-breathing toads (Overgaard )] and warrants further investigation. Of particular conservation concern for sockeye salmon is the additional finding that post-fatigue mortality occurred at temperatures only 3–5°C above Topt.

Swimming performance

Salmonids are known to increase their reliance on anaerobic swimming in Ucrit tests at supra-optimal temperatures (Brett, 1964; Jain and Farrell, 2003), a finding that lends support to the OCLTT hypothesis. This was clearly evidenced here by the elevated lactate levels at fatigue in the warmest temperature grouping, supporting the idea that oxygen delivery to tissues in swimming fish becomes limited at temperatures near the critical maximum. A novel finding in the present study was the elevated plasma lactate even at rest and the depletion of plasma glucose with swimming in the highest temperature grouping, providing further indications that tissue oxygen delivery was compromised. The difficulty of swimming at supra-optimal temperature was evident from the increased COT and with temperature, indicating higher oxygen and cardiovascular costs to swimming at a given speed compared with cooler temperatures. In fact, the true COT and may be even higher, because the metabolic costs of anaerobic swimming require the characterization of excess post-exhaustion oxygen consumption (see Lee ), which was not done here.

Gill oxygen delivery and uptake

Water oxygen content decreases by around 2% °C−1 with increasing water temperature (Dejours, 1975), which limits environmental oxygen availability. Furthermore, the affinity of haemoglobin for oxygen decreases with increasing temperature (Perry and Reid, 1994; Jensen ; Clark ), which may hamper oxygen uptake at the gill and potentially prevent full haemoglobin saturation. Therefore, strong theoretical arguments exist for a limitation in oxygen uptake at the gills to explain the collapse of aerobic scope above Topt (through either insufficient water delivery to the gills or reduced diffusion of oxygen across the gills). If this were the case, PaO2 and CaO2 would decrease and potentially reduce TaO2 at temperatures above Topt.

There is considerable evidence for such changes in arterial oxygen status in resting, but not in exercising fishes. Keen and Gamperl (2012) showed a reduction in CaO2 and PaO2 by ∼25 and 35%, respectively, in resting steelhead trout warmed from 12°C to their critical thermal maximum. However, they noted that a limited capacity for ram ventilation in the test apparatus could have been a contributing factor. Clark found that resting adult Chinook salmon slightly decreased CaO2 and PaO2 when acutely warmed, but this was more pronounced in larger individuals. Heath and Hughes (1973) showed a large and parallel decrease in CaO2 and CvO2 with acute heat stress up to 25°C in resting rainbow trout, but did not measure Hct or [Hb] to check for haemodilution with repetitive blood sampling. The only report of CaO2 becoming lower during swimming was for seasonally acclimatized rainbow trout at 18°C when compared with 4 and 11°C, but haematocrit was also halved at 18°C (Taylor ).

In contrast to these works, both the present study and that of Steinhausen found that CaO2, PaO2, and Hct were maintained independent of temperature in swimming adult sockeye salmon. Likewise, Sartoris found that PaO2 remained constant during acute warming in resting Atlantic cod. A telling discovery here was that CaO2 was maintained even when cardiac dyssynchrony and post-fatigue delayed mortality occurred at extremely warm temperatures. As such, the present study clearly provides no support for an oxygen limitation developing at the gills at warm temperatures.

Given the different conclusions that have been reached for the effects of temperature on oxygen delivery and uptake at the gill, future studies must pay close attention to the response of Hct and [Hb] to temperature, which directly affects CaO2. Haematocrit has been shown to increase by up to 27% due to splenic contraction in acutely warmed resting rainbow trout (Sandblom and Axelsson, 2007), to decrease by 50% in warm-acclimated rainbow trout (Taylor ), and to be minimally affected by temperature (present study and see Farrell, 1997; Clark ; Steinhausen ). Future studies must consider haemodilution, because repeated blood sampling can reduce Hct, [Hb], and CaO2, though this was not a problem in the present study. In addition, future studies should carefully consider the role of water velocity in assisting gill ventilation (ram ventilation), as was possible here in the swim tunnels, and the possibility that fish holding devices may restrict buccal and opercular movements during gill ventilation.

Circulatory transport of oxygen

All studies that have measured (directly or indirectly) and MO2 as a function of temperature in swimming salmonids have reported parallel declines in and MO2 above Topt (Brett, 1971; Taylor ; Steinhausen ; Eliason ). Here we present additional evidence in support of a cardiac limitation developing at supra-optimal temperatures, because maximal TaO2, MO2, and all failed to increase above Topt. In addition, all three variables decreased at Tmax0–50, at which point scope for fH completely collapsed without any compensation by Vs. Therefore, we conclude that a perfusion limitation develops above Topt, resulting in insufficient oxygen delivery to meet the increased locomotory tissue oxygen demand. Notably, scope for fH collapsed at a lower temperature compared with scope for , aerobic scope, and scope for Vs (Fig. 5). Therefore, we propose that reduced scope for fH is the triggering mechanism that limits above Topt, supporting an earlier proposal (Farrell, 2009). It is becoming clear that fH is a central mechanism setting upper temperature tolerance and thus may be a key limiting factor regulating fish distribution globally.

Changes in and TaO2 as a function of temperature are almost entirely mediated by changes in fH in both resting and swimming fish (present study and Cech ; Gollock ; Sandblom and Axelsson, 2007; Clark , 2011; Steinhausen ), underscoring the central importance of fH in determining cardiac performance and oxygen delivery with temperature. More broadly, the central importance of resting fH in setting temperature tolerances for intertidal invertebrates is also gaining support (Somero, 2011). Direct effects of temperature on the cardiac pacemaker rate (Randall, 1970) are likely to account for temperature-induced cardiac acceleration. Here, resting fH continued to increase until lethal temperatures were approached in the highest temperature group (range, 117–135 beats min−1; mean, 123.9 beats min−1). Moreover, fHmax plateaued above Topt, decreasing from rest at the highest test temperatures. Therefore, we suggest that factors that (i) determine fHrest and (ii) limit fHmax may be of particular importance for future studies.

The bradycardia followed by dysrhythmia in fish swimming at the highest temperature is a novel observation that adds to the observation of irregular heart beats in resting rainbow trout, Atlantic cod, and Chinook salmon acutely warmed to their Tcrit (Heath and Hughes, 1973; Gollock ; Clark ). Here, severe dysrhythmias were associated with dramatic decreases in PvO2, CvO2, and TvO2, although these sockeye salmon displayed an impressive tenacity to continue swimming, albeit at lower speeds and with a lower MO2, , and TaO2. Such tenacity, however, may have severe consequences, because there was significant post-fatigue mortality shortly after exhaustion despite cooling the salmon. At temperatures a few degrees cooler, there was no such immediate consequence. However, we cannot rule out the possibility of delayed mortality, because it can occur hours to days after exhaustive exercise, both in the laboratory (e.g. Black, 1957; Wood ) and in nature (e.g. Donaldson ), especially at elevated temperature.

The mechanisms limiting maximal fH and triggering bradycardia and cardiac dysrhythmia remain unclear. Maximal fH is clearly unable to continue increasing with a Q10 of ∼2 at temperatures above Topt. The initial bradycardia during swimming may have been vagally mediated and could act as a protective mechanism by (i) reducing and cardiac oxygen demand (Farrell and Steffensen, 1987), (ii) increasing blood residence time in the heart to favour oxygen extraction by spongy myocardium (Farrell, 2007b), and (iii) reducing coronary vascular compression to favour a more continuous coronary blood flow to compact myocardium (Axelsson and Farrell, 1993; Gamperl ). The possibility of a centrally mediated bradycardia should be examined using atropine injection or vagotomy. Alternatively, synergistically or perhaps secondarily, the deleterious venous blood environment associated with anaerobic swimming at high temperature could have triggered cardiac problems, including the observed decrease in fHmax and Vsmax. Specifically, anaerobic swimming makes venous blood acidotic (low pH), hypoxaemic (low PvO2) and hyperkalaemic (high K+; Kiceniuk and Jones, 1977; Holk and Lykkeboe, 1998), all of which become exacerbated at high temperature (Brett, 1964; Jain and Farrell, 2003; Steinhausen ). Notably, hyperkalaemia was absent in the present study. These extracellular changes can impair cardiac contractility (Driedzic and Gesser, 1994), and even more so at high temperature (Hanson and Farrell, 2007). At extremely high temperatures, the decreases in PvO2 and TvO2 at warm temperatures may not have guaranteed sufficient oxygen supply to the avascular spongy myocardium (Farrell and Clutterham, 2003; Farrell, 2007a), resulting in reduced contractility and perhaps dysrhythmias. In fact, the lowest PvO2 observed at the highest temperature (10 torr) is around the suggested lower limit for adequate oxygen delivery to the fish spongy myocardium (Davie and Farrell, 1991). In contrast, the compact myocardium (supplied with oxygen by the coronary circulation) probably would not have suffered in a similar manner, because CaO2 and PaO2 were maintained. All told, the exact mechanisms and sequence of events leading to the fH limitation are unclear and warrant further study.

Why resting fish do not increase Vs with acute warming remains unknown (present study and Cech ; Brodeur ; Gollock ; Sandblom and Axelsson, 2007; Clark ; Steinhausen ; Mendonça and Gamperl, 2010; Gamperl ). Elevated temperature, per se, does not prevent an increase in resting Vs, because zatebradine treatment, which reduces fH at the level of the pacemaker, triggered a compensatory increase in Vs in resting rainbow and steelhead trout at high temperature (Gamperl ; Keen and Gamperl, 2012). All the same, supra-optimal temperatures can reduce Vsmax in swimming salmon (present study and Clark ), something that was not revealed when sockeye salmon were swimming at ∼75% of Ucrit (Steinhausen ). The mechanism of the decline in Vsmax at extremely warm temperatures is unknown, but may be related to the deleterious venous blood environment associated with anaerobic swimming at high temperature, as outlined above.

Tissue oxygen extraction

The elevation in plasma lactate in both resting and swimming fish is a clear indication that the increased tissue oxygen demand was not being met by oxygen delivery at supra-optimal temperature. This mismatch between oxygen supply and demand could have resulted from a perfusion and/or a diffusion limitation to the locomotory muscles. The present study clearly showed that arterial oxygen transport was compromised above Topt due to reduced , leading to a muscle capillary perfusion limitation. However, insufficient tissue oxygen extraction could also lead to increased anaerobic metabolism if a diffusion limitation for oxygen existed at the locomotory muscles. Factors leading to a diffusion limitation are inadequate capillary density, ineffective muscle cellular morphology (e.g. poor mitochondria density or location), and an insufficient driving force for oxygen diffusion (low PaO2; Egginton and Cordiner, 1997; Taylor ; Egginton, 2002). Thus, a diffusion limitation for oxygen is more likely for white rather than red muscle given the attendant ∼10 times lower capillary density of white compared with red muscle in salmonids (Mosse, 1978; Egginton and Sidell, 1989). However, none of these morphometric features was studied here, and so assessing the role of a diffusion limitation relative to a perfusion limitation is difficult with in vivo studies.

Nevertheless, Steinhausen suggested that a constant PvO2 during acute warming of resting and swimming sockeye salmon was evidence for a diffusion limitation. However, PvO2 and CvO2 both decreased above Topt in resting and maximally swimming sockeye salmon in the present study. Likewise, PvO2 and/or CvO2 decreased with warming to high temperature in resting rainbow trout, Atlantic cod, and Chinook salmon (Heath and Hughes, 1973; Sartoris ; Clark ). Thus, a tissue diffusion limitation may not have occurred immediately, because the tissues were able to extract more oxygen from the blood at temperatures above Topt. We caution, however, that the decrease in CvO2 may not have been proportional to the increased oxygen demand with warming (Wagner, 1996). Assessments of a diffusion limitation are complicated by the decrease in , which potentially slows capillary transit time for blood, favouring oxygen diffusion. Furthermore, the decrease in blood pH accompanying anaerobic metabolism, which may facilitate oxygen unloading into the tissues via Bohr and Root effects, would provide further temporary relief from a diffusion limitation (Rummer and Brauner, 2011). All told, further research examining the possibility of a tissue diffusion limitation at warm temperature is warranted. Specifically, studies on the role of muscle morphology in limiting oxygen diffusion at high temperature in exercising fish remain a ripe area for future research.

Limitations of the study

This study overcame the difficult challenge of swimming a sufficient number of large fish equipped with cannulae and probes to measure critical cardiorespiratory variables directly and simultaneously in order to resolve the subtle changes in the oxygen cascade when fish swim above supra-optimal temperature. Adult sockeye salmon have proved to be an effective model, because they are large enough to carry a flow probe and support repeated but strategic arterial and venous blood sampling without haemodilution. However, migrating adult sockeye salmon naturally senesce and die 4–6 weeks after they enter freshwater, which places a logistic constraint on the duration of recovery periods. Previous work has shown that MO2rest, MO2max, and aerobic scope did not differ significantly between sockeye salmon that had undergone surgery and those that had not (Eliason ), suggesting that sockeye salmon can recover quickly from surgery.

Another challenge with wild Fraser River sockeye salmon is that different populations co-migrate upstream, and individual fish are impossible to differentiate until scale and DNA analysis have been conducted several days later. Therefore, in order to ensure good statistical power, we pooled three sockeye salmon populations that all face long and difficult river migrations, have a similar Topt for aerobic scope (∼17°C), and have a similar maximal aerobic scope, cardiac scope, and scope for heart rate (Eliason ). These three populations do not differ in MO2, , fH, or Vs at rest, when swimming, or during recovery at Topt. Also, great care was taken to create temperature groupings relative to the population-specific Topt such that the temperature differences were only 1–3°C across the populations within a temperature grouping above Topt. As a result, the variances for MO2rest, MO2max, and aerobic scope were small for each temperature grouping (Fig. 2), which would not be the case if large population differences existed. Moreover, the Chilko population had temperature responses that paralleled those for the four temperature groupings (Figs 2 and 6).

Given the above constraints, sex-specific differences were not considered, but each temperature grouping contained approximately equal numbers of male and female sockeye salmon. Previous work (Eliason ) found no sex-specific differences in cardiorespiratory physiology and blood oxygen status at Topt for sockeye salmon, while a study on pink salmon showed that males maintain a higher MO2rest and achieve greater MO2max, , and aerobic scope (Clark ). Notably, female sockeye salmon suffer higher mortality compared with males in stressful migratory conditions, such as high temperature, elevated flow, and challenging migratory obstacles (Nadeau ; Roscoe ; Jeffries ; Martins ). Given that spawning success for a population is governed by females, this raises potential conservation concerns for any sex differences in the physiological response to temperature, which should be considered in future studies.

Summary and conclusions

The results from the present study greatly expand upon the idea of a ‘death spiral’ for salmon swimming at supra-optimal temperatures (Farrell ), which proposes a mechanistic explanation of the failure of aerobic scope above Topt. Here we provide definitive evidence that the collapse of aerobic scope above Topt is associated with a cardiac limitation, triggered by a collapse in scope for fH. Accordingly, a perfusion limitation for oxygen delivery to swimming muscles developed at temperatures above Topt, resulting in a mismatch between oxygen supply and demand, as evidenced by elevated lactate levels. The severity of the cardiac constraint increased with temperature to the point that elevated resting lactate, cardiac dysrhythmias, a negative scope for fH, and even post-exhaustion mortality were observed at the highest temperatures. Both PaO2 and CaO2 were unchanged with warming above Topt, suggesting no limitation in oxygen uptake at the gill with the present experimental design. With PvO2 and CvO2 (and probably blood pH, given the elevation in plasma lactate) decreasing at the highest test temperature, the possibility of a diffusion limitation also developing at the muscle remains unclear and warrants further study. We propose that a noxious venous blood environment (low pH and low PvO2) further impairs cardiac function (reducing cardiac oxygen delivery, contractility, and perhaps fH), which could initiate a positive feedback loop to exacerbate the perfusion limitation to locomotory muscle and perhaps lead to delayed mortality at extremely warm temperatures.

In terms of salmon conservation, climate change-induced increases in Fraser River temperatures have been associated with massive mortality in sockeye salmon during their spawning migration. Nevertheless, a detailed mechanistic understanding of the causes of migration failure has remained elusive. Previous work suggests that the high en route mortality may be partly attributed to a decrease in aerobic scope (Farrell ; Eliason ). The present study expands on this idea and suggests that salmon are unable to swim at warm temperature due to a decrease in aerobic scope caused by a cardiac limitation via insufficient scope for heart rate. While some populations of sockeye salmon have an exceptionally wide Topt window (e.g. from 13 to 21°C for Chilko sockeye salmon; Eliason ), all populations studied to date are currently experiencing temperatures that exceed their population-specific Topt window, and this is expected only to worsen in the future (Eliason ). The central importance of fH in determining cardiac performance and oxygen delivery at warm temperature and the possibility of fHmax setting upper temperature limits may provide a useful, but simple measurement to move physiology into the field (Casselman ) and improve our knowledge of temperature tolerance of fishes as we try to conserve the natural resources that are left.