# In a marine teleost, the significance of oxygen supply for acute thermal tolerance depends upon the context and the endpoint used

Julie Nati, Felipe Blasco, Charles Rodde, Alain Vergnet, François Allal, Marc Vandeputte, David Mckenzie

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## ► To cite this version:

Julie Nati, Felipe Blasco, Charles Rodde, Alain Vergnet, François Allal, et al.. In a marine teleost, the significance of oxygen supply for acute thermal tolerance depends upon the context and the endpoint used. Journal of Experimental Biology, 2023, 226 (11), pp.jeb245210. 10.1242/jeb.245210. hal-04100080

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In a marine teleost, the significance of oxygen supply for acute thermal
tolerance depends upon the context and the endpoint used

Julie J.H. Nati¹*, Felipe R. Blasco², Charles Rodde¹,³, Alain Vergnet¹, François Allal¹, Marc Vandeputte¹, David J. McKenzie¹

¹ MARBEC, Univ Montpellier, CNRS, Ifremer, INRAE, IRD, 34250 Palavas-les-Flots, France.

²Department of Physiological Sciences, Federal University of São Carlos, São Carlos, Brazil.

³UMR ISEM, Univ Montpellier, CIRAD, CNRS, Ifremer, IRD, 34398 Montpellier, France.

*Corresponding author: julienati3@gmail.com
16 Abstract

Eight juvenile European seabass were exposed to two thermal ramping protocols with different levels of aerobic activity and tolerance endpoint: the critical thermal maximum for swimming (CTSmax) while exercising aerobically until fatigue, and the critical thermal maximum (CTmax) under static conditions until loss of equilibrium (LOE). In the CTSmax, warming caused a profound increase in oxygen uptake rate (MO2) culminating in a gait transition, from steady aerobic towards unsteady anaerobic swimming, then fatigue at 30.3 ± 0.4°C (mean ± SE). Gait transition and fatigue presumably indicate an oxygen limitation, an inability to meet the combined demands of swimming plus warming. The CTmax also elicited an increase in MO2, culminating in LOE at 34.0 ± 0.4°C, significantly warmer than fatigue at CTSmax. The maximum MO2 achieved in the CTmax was, however, less than 30% of that achieved in the CTSmax. Therefore, the static CTmax did not exploit full cardiorespiratory capacity for oxygen supply, indicating that LOE was not caused by systemic oxygen limitation. Consequently, systemic oxygen supply can be significant for tolerance of acute warming in seabass but this depends upon the physiological context and the endpoint used.

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31 **Keywords:** critical thermal maximum, fish, oxygen uptake, swimming

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**Introduction**

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Global warming will cause a rise in sea surface temperatures of up to 4°C by 2100, accompanied by increased frequency and severity of acute heat waves (Frölicher et al., 2018). Temperature has direct thermodynamic effects on the physiology of ectotherms, so there has been extensive research to investigate thermal tolerance limits in fishes, and the underlying mechanisms. The Fry paradigm and Oxygen and Capacity Limited Thermal Tolerance hypothesis (Fry-OCLTT) have been proposed as a universal principle underlying thermal tolerance in ectotherms (Fry, 1971; Pörtner, 2010).

According to the Fry-OCLTT, oxygen demands of metabolism are accelerated inexorably by warming until they exceed cardiorespiratory capacity for oxygen supply, causing functional collapse (Eliason et al., 2011; Fry, 1971; Pörtner, 2010; Pörtner and Farrell, 2008; Schulte, 2015). Empirical evidence for the Fry-OCLTT paradigm is, however, mixed (Farrell, 2016; Jutfelt et al., 2018; Lefevre, 2016; Lefevre et al., 2021; Pörtner, 2021).

There is particular uncertainty about the role of oxygen limitation in tolerance of acute warming in fishes. Acute thermal tolerance is typically measured with the critical thermal maximum ($CT_{max}$) protocol, where fish are warmed in steps until loss of equilibrium (LOE) (Lutterschmidt and Hutchison, 1997). The LOE at $CT_{max}$ is an incipient lethal endpoint due to dysfunction of critical organs, brain and heart (Andreassen et al., 2022; Lefevre et al., 2021; Rezende et al., 2011). In some fish species, environmental oxygen availability and/or blood oxygen carrying capacity have little or no effect on $CT_{max}$, indicating that LOE is not oxygen dependent (Brijs et al., 2015; Ern et al., 2016; McArley et al., 2021). In other species, measurements of rates of oxygen uptake ($\dot{M}O_2$) during a $CT_{max}$ protocol found an initial exponential increase up to a maximum that was comparable to their maximum metabolic rate measured at initial acclimation temperature. This was then followed by a plateau and/or decline, which preceded LOE and which might indicate a limitation in capacity for tissue oxygen supply prior to the endpoint (McArley et al., 2017; Norin et al., 2019). The contrasting evidence indicates that LOE at $CT_{max}$ may have many underlying mechanisms, which may or may not be linked to oxygen limitation, and which differ among species, methodology and context (Andreassen et al., 2022; Ern et al., 2023; Lefevre et al., 2021; Rezende et al., 2014).

In a study to investigate effects of acute temperature increases on cardiorespiratory performance of sockeye salmon (*Oncorhynchus nerka*), Steinhausen et al. (2008) warmed fish progressively while they swam aerobically in a swim tunnel. A number of salmon showed a gait-transition to an anaerobic swimming mode and then fatigued at warm temperatures, which the authors attributed to an inability to meet the combined oxygen demands of swimming plus warming (Steinhausen et al., 2008). Blasco et al. (2020) proposed that a similar protocol could be used to evaluate acute thermal tolerance in fishes, warming in steps with fatigue as the endpoint. In two
tropical freshwater teleosts exercising aerobically in a swim tunnel, progressive warming caused profound increases in ˙MO₂, to levels very significantly higher than the individuals' maximum metabolic rate during a critical swimming speed (U crit) test at their acclimation temperature. At a certain warm temperature, however, all individuals transitioned from a steady aerobic to an unsteady anaerobic gait and then fatigued, at temperatures significantly lower than their LOE at CT max (Blasco et al., 2020). Dubbed the critical thermal maximum for swimming (CTS max) by Blasco et al. (2022), the mechanism underlying fatigue would presumably be oxygen supply limitation, an inability to meet the combined oxygen demands of swimming plus warming (Blasco et al., 2020; Blasco et al., 2022; Steinhausen et al., 2008).

Here, we investigated the general hypothesis that capacity for tissue oxygen supply can play a role in acute upper thermal tolerance in fishes, but that this depends upon the prevailing level of aerobic metabolic activity and consequent oxygen demand, and the tolerance endpoint used. We studied the European seabass *Dicentrarchus labrax*, a temperate marine teleost that has been the focus of investigations into the role of oxygen supply in defining CTmax (Anttila et al., 2023; Wang et al., 2014). We compared ˙MO₂ during a CTSmax and CTmax protocol in the same individuals, in particular the maximum ˙MO₂ achieved prior to each tolerance endpoint, fatigue or LOE. We reasoned that, if in the CTSmax protocol the seabass transitioned to unsteady anaerobic swimming at a certain warm temperature, and then fatigued, then capacity for tissue oxygen supply would underly this thermal tolerance endpoint in this species, as for the species studied by Blasco et al. (2020). We expected that LOE at CTmax would occur at a significantly higher temperature than fatigue at CTSmax, as observed previously (Blasco et al., 2020; Blasco et al., 2022). We, however, investigated the specific hypothesis that maximum ˙MO₂ elicited by the CTmax would be significantly less than those in the CTSmax, indicating that the LOE endpoint was not caused by limitations in capacity for systemic tissue oxygen supply in this species (Wang et al., 2014).

## Material and Methods

Juvenile seabass were obtained from a population at the Station Ifremer de Palavas-les-Flots (France), reared in seawater at 18 – 19 °C and tagged (PIT, under benzocaine anaesthesia) for individual identification. Fish were held in a 1 m³ tank supplied with biofiltered seawater at 18 °C for at least two weeks prior to experimentation, fed a maintenance ration with commercial pellets but fasted for 24 h before experiments. Prior to the main trials, a control was performed at 18 °C on six seabass of mean (± SE) mass 111.4 ± 9.1 g and forklength 202 ± 6 mm, which were swum at the speed of the CTSmax (2.5 BL s⁻¹) for 8 h. For the CTSmax and CTmax, eight seabass were studied, of mean mass 74.2 ± 8.0 and forklength 181 ± 6 mm at the CTSmax, then mean mass 75.3 ± 8 g at the CTmax.
Swimming trials were performed in a plexiglass Steffensen-type swim tunnel (volume 30 l) provided with biofiltered aerated seawater at 18 °C. Fish were measured for mass, width, height, and fork length, to calculate relative swimming speed in body length per second (BL s⁻¹) corrected for solid blocking effect (Bell and Terhune, 1970), then acclimated overnight at a low swimming speed of 1 BL s⁻¹. The next day, current speed was increased by 0.25 BL s⁻¹ every 2 min until 2.5 BL s⁻¹. In the control trial, the fish then swam for 8h. In the CTSmax, after 30 min at 18 °C the temperature was increased by 1°C each 30 min until fish fatigued, resting their tails for at least 10 s against the rear grid. Fish were immediately removed from the tunnel into a tub of aerated seawater at 18°C for 30 min, then returned to their holding tank. CTSmax was calculated as the last temperature step fully completed plus the proportion of the ultimate step prior to fatigue (Blasco et al., 2020). The experiments were performed over three weeks, with the last individual studied 24h before measurement of its CTmax (below).

The CTmax was performed in eight individual respirometry chambers (volume 3 l) that were supplied with aerated seawater at 19 °C, due to an overnight increase in the surface temperature of the local seawater supplying the facilities. Fish were acclimated to the chambers overnight then, the next morning, temperature was increased by 1°C every 30 min until LOE (McArley et al., 2017; Penney et al., 2014). Chambers were screened from view with an opaque plastic sheet containing a small observation hole, to monitor fish throughout. As soon as fish lost dorso-ventral equilibrium, they were removed and placed in aerated water at 19 °C for 30 min, then returned to their holding tank. CTmax was calculated as for CTSmax but using LOE as endpoint (Blasco et al., 2020).

The ṀO₂ was measured in mgO₂ kg⁻¹ h⁻¹ over the last 10 min of each temperature increment, in both protocols, by intermittent-flow respirometry (Blasco et al., 2020; Steffensen, 1989). ṀO₂ was corrected for body mass with a scaling coefficient of b = 0.8 for metabolic rate in European seabass (Rodde et al., 2021). The maximum ṀO₂ achieved in each protocol was identified for each individual. Absolute thermal scope for CTmax and CTSmax was calculated as maximum ṀO₂ minus the relevant ṀO₂ at 19°C. The proportional change in ṀO₂ was calculated as a Q₁₀ value over 19 – 29°C.

A two-way ANOVA with repeated measures was used to evaluate effects of temperature on ṀO₂, with one factor protocol (CTSmax versus CTmax), repeated factor the temperature increments, each fish as an individual, and an interaction term between protocol and temperature steps. Data were considered for a range of 19 to 30°C, which was measured on all individuals in both protocols. An error function was included to correct for repeated tests on the same individual. Tukey post hoc tests, with Holm correction for multiple comparisons, were used to identify the temperature at which ṀO₂ first increased significantly from the value at 19 °C, and to compare ṀO₂ between protocols at each temperature. Single variables, such as CTSmax and CTmax, maximum ṀO₂, thermal
scope, and Q₁₀ were compared by pairwise Welch t-tests. All statistical analyses were performed in R
4.0.2 within the lsmeans package (version 2.27-61), with significance accepted at p < 0.05.

## Results

At a speed of 2.5 BL s⁻¹, the seabass swam with a sustained aerobic body-caudal swimming gait. In the control trial the seabass swam for 8h with no sign whatsoever of fatigue. During CTSmax, all individuals eventually transitioned in swimming gait, from an entirely steady aerobic gait with rhythmic beating of the tail, to increasing engagement of unsteady anaerobic ‘burst-and-coast’ episodes. This led to fatigue within less than one temperature increment (30 min). No fish lost equilibrium in the swimming trials. During CTmax, the fish exhibited erratic behaviour, rolling sideways, then complete loss of dorsoventral orientation. The mean CTSmax was 30.3 ± 0.4°C, significantly and almost 4°C lower than mean CTmax at 34.0 ± 0.4°C (Figure 1A).

In the control swim, MO₂ remained stable for 8h (Figure S1, supplementary materials). caused There were marked increases in MO₂ in both thermal ramping protocols (Figure 2), that became significant from 19 °C at 25°C in both cases. There was, however, a significant interaction between protocol and temperature (Two-way ANOVA, F₁₁,₇₄ = 13.71, p < 0.001). All measures of MO₂ in CTSmax, irrespective of temperature, were higher than any measure of MO₂ in the CTmax (Two-way ANOVA, Holm p < 0.001, Table S1, supplementary materials). Consequently, the maximum MO₂ achieved and absolute thermal scope were significantly higher in the CTSmax (Figure 1). Despite these differences in MO₂ and absolute thermal scope, Q₁₀ did not in fact differ between protocols (Figure 1).

## Discussion

The results demonstrate that capacity for tissue oxygen supply can be significant for upper thermal tolerance in this teleost species, but this depends upon the physiological context and the tolerance endpoint used. When warmed while exercising aerobically, all individuals eventually transitioned to unsteady anaerobic swimming, evidence of limitations in capacity for tissue oxygen supply (Steinhausen et al., 2008), leading to fatigue at CTSmax. When warmed under static conditions, the animals eventually lost equilibrium at a CTmax that was significantly higher than their CTSmax but, in line with our specific hypothesis, maximum MO₂ in the CTmax was very significantly less than achieved in the CTSmax. Therefore, the full cardiorespiratory capacity for oxygen supply was not exploited during thermal ramping in the static CTmax, indicating that LOE is not linked to limitations in systemic oxygen delivery in this species (Wang et al., 2014).

Fatigue in the CTSmax was not due to limited aerobic endurance because, at their acclimation temperature, seabass swam at 2.5 BL s⁻¹ for longer than the duration of the warming challenge.
Similar aerobic endurance was observed in Nile tilapia *Oreochromis niloticus* and pacu *Piaractus mesopotamicus* (Blasco et al., 2020). This confirms that the fatigue endpoint in the CTSmax is due to the progressive warming. The swimming responses to the CTSmax protocol, gait transition followed by fatigue, were also very similar in tilapia and pacu (Blasco et al., 2020). The gait transition is qualitatively indistinguishable to that observed prior to fatigue in a critical swimming speed (Ucrit) protocol, where it is stimulated by increments in current velocity rather than temperature. This indicates that the transition occurs due to a similar mechanism. In the Ucrit, it is typically assumed that gait transition and then fatigue occur because a fish has reached its maximum cardiorespiratory capacity for oxygen supply (Brett, 1964; Claireaux et al., 2005; Farrell, 2007; Webb, 1998). Although it is not known what causes the gait transition in a Ucrit test, one plausible theory focuses on the heart. An aerobic organ, the teleost heart receives much of its oxygen supply from venous return in the single circulation, after the blood has unloaded oxygen to respiring tissues (Farrell and Jones, 1992; Jones and Randall, 1978). It has been suggested that during an incremental swim challenge, fishes engage the gait transition towards anaerobic swimming when tissue oxygen extraction outstrips capacity for supply, and oxygen levels in venous blood returning to the heart drop below a critical level. By engaging the white muscle, the fish reduces rates of oxygen extraction by the working red muscle and so venous oxygen supply to the heart is assured (Farrell and Clutterham, 2003; McKenzie and Claireaux, 2010). The gait transition preceding fatigue in the CTSmax may reflect a similar decline in venous oxygen levels, that risked compromising oxygen supply to the working heart (Blasco et al., 2020).

Steinhausen et al., (2008) found that fatigue from exercise during acute warming in the sockeye salmon coincided with cardiac pumping capacity reaching its maximum. Cardiac performance plays a major role in defining acute thermal tolerance in many fish species (Ekström et al., 2014; Ekström et al., 2016; Farrell, 2007; Gilbert et al., 2019), so represents a focus for future studies regarding the mechanisms for the CTSmax endpoint. Other mechanisms may also contribute, progressive warming can impair locomotor coordination in fishes, possibly as a consequence of limitation in oxygen supply to the brain (Andreassen et al., 2022). Further research is therefore required to understand what causes gait transition and fatigue in a CTSmax but it is interesting that the test solicits similar physiological responses in multiple species (Blasco et al., 2020).

Although the CTSmax results provide evidence that tissue oxygen supply can play a role in thermal tolerance in swimming seabass when fatigue is the endpoint, the CTmax data support the hypothesis that LOE is not due to limitations in systemic oxygen supply in this species (Wang et al., 2014). The mean CTmax of 34.0 °C in our juvenile seabass reared at 18° C, was comparable to previous reports of 34.7 and 35.6 °C for juveniles acclimated for 30 days to 15 or 20 °C, respectively (Kir and
Demirci, 2018). The slightly higher $CT_{max}$ reported by Kir and Demirci (2018) may also be linked to a more rapid thermal ramping of 0.3 °C min⁻¹ compared to 0.033 °C min⁻¹ in this study, as more rapid ramping is known to lead to higher $CT_{max}$ values in fishes (Desforges et al., 2023; Ern et al., 2023). We ramped at 1°C every 30 min to ensure that fish core temperature was equilibrated with the water at each step, and that we could measure $M_{O_2}$ over the last 10 min. While $M_{O_2}$ increased markedly with warming in the static $CT_{max}$, as observed in other studies (McArley et al., 2017; Norin et al., 2019), and the $Q_{10}$ was similar in both protocols, the actual rates were always significantly lower than at any temperature in the $CTS_{max}$. That is, LOE occurred at $M_{O_2}$ levels lower than those of the same individuals exercising at 2.5 BL s⁻¹ at 19 °C. Static respirometry does not constrain gas exchange in seabass, juveniles achieved similar high maximum rates of oxygen uptake when either swum in a $U_{crit}$ test or chased to exhaustion and then placed in a static chamber (Killen et al., 2017). Therefore, warming did not challenge capacity for oxygen supply in stationary fish, it caused LOE by an oxygen-independent mechanism. There are a variety of possible contenders, particularly direct temperature-induced effects on critical organs such as heart and brain, which cause their dysfunction. There is evidence of this in fishes, including that such effects can interact with oxygen limitation within these organs (Andreassen et al., 2022; Friedlander et al., 1976; Vornanen, 2020). Overall, the consensus is that LOE can have multiple causes and that systemic oxygen limitation is not a universal mechanism across fish species (Andreassen et al., 2022; Ern et al., 2023; McArley et al., 2021).

The Fry-OCLTT argues that capacity for oxygen supply is a mechanism underlying thermal tolerance in fishes (Fry, 1971; Pörtner, 2010), the $CTS_{max}$ test indicates that it can be in multiple species, if fatigue from aerobic activity is the tolerance endpoint. Here we show that this is true for a species where LOE in a $CT_{max}$ protocol appears to occur by an oxygen-independent mechanism. The $CT_{max}$ is easy and rapid to perform but the endpoint has a complex multitude of potential mechanisms. The $CTS_{max}$ is more technically demanding but the mechanism underlying tolerance may be similar across fish species.

## Acknowledgements

We are grateful to two anonymous referees for comments that significantly improved the presentation of this manuscript.

## Ethics.

Experimental procedures were approved by the ethics committee for animal experimentation n° 036 of the French Ministère de l'Enseignement Superieur, de la Recherche et de l'Innovation, with reference number APAFIS#19567-2018100910598940 v2.

**Data accessibility.** Data are available in the electronic supplementary material
238 **Author contribution.** All authors contributed to study conception, design, securing funding, animal
239 husbandry. J.J.H.N., F.R.B. and C.R. carried out the experiments and completed the data analyses.
240 J.J.H.N and D.J.M. drafted the initial manuscript; all authors then reviewed and revised the
241 manuscript.

242 **Competing interests.** We declare no competing interests.

243 **Funding.** J.J.H.N. was funded by an individual Marie Curie Fellowship (MSCA-IF-EF-ST 839038,
244 INDITOL), F.R.B. was supported by the AquaExcel Transnational²⁰²⁰ Access project AE120016
245 PhenoBass and by a Company of Biologists Travel Grant. The research was also supported by the
246 FEAMP Mesure n° 47 Aquaculture Innovation project SELFIE.

247

248 **References**

249 **Andreassen, A. H., Hall, P., Khatibzadeh, P., Jutfelt, F. and Kermen, F.** (2022). Brain dysfunction during warming is linked to oxygen limitation in larval zebrafish. *Proc. Natl. Acad. Sci.* **119**, e2207052119.

252 **Bell, W. H. and Terhune, L. D. B.** (1970). Water tunnel design for fisheries research. *Fish. Resour. Board Can. Tech. Rep.* **195**, 1-69.

254 **Blasco, F. R., Esbaugh, A. J., Killen, S. S., Rantin, F. T., Taylor, E. W. and McKenzie, D. J.** (2020). Using aerobic exercise to evaluate sub-lethal tolerance of acute warming in fishes. *J. Exp. Biol.* **223**,.

257 **Blasco, F. R., Taylor, E. W., Leite, C. A. C., Monteiro, D. A., Rantin, F. T. and McKenzie, D. J.** (2022). Tolerance of an acute warming challenge declines with body mass in Nile tilapia: evidence of a link to capacity for oxygen uptake. *J. Exp. Biol.* **225**, jeb244287.

260 **Brett, J. R.** (1964). The respiratory metabolism and swimming performance of young sockeye salmon. *J. Fish. Res. Board Can.* **21**, 1183-1226.

262 **Brijs, J., Jutfelt, F., Clark, T. D., Grans, A., Ekstrom, A. and Sandblom, E.** (2015). Experimental manipulations of tissue oxygen supply do not affect warming tolerance of European perch. *J. Exp. Biol.* **218**, 2448-2454.

265 **Claireaux, G., McKenzie, D. J., Genge, a G., Chatelier, A., Aubin, J. and Farrell, A. P.** (2005). Linking swimming performance, cardiac pumping ability and cardiac anatomy in rainbow trout. *J. Exp. Biol.* **208**, 1775-84.

268 **Desforges, J. E., Birnie-Gauvin, K., Jutfelt, F. M., Gilmour, K. M., Eliason, E. J., Dressler, T. L., McKenzie, D. J., Bates, A. E., Lawrence, M. J., Fangue, N. A., et al.** (2023). The ecological relevance of critical thermal maxima methodology (CTM) for fishes. *J. Fish Biol.* **00**,.

248 **References**

249 **Andreassen, A. H., Hall, P., Khatibzadeh, P., Jutfelt, F. and Kermen, F.** (2022). Brain dysfunction during warming is linked to oxygen limitation in larval zebrafish. *Proc. Natl. Acad. Sci.* **119**, e2207052119.

252 **Bell, W. H. and Terhune, L. D. B.** (1970). Water tunnel design for fisheries research. *Fish. Resour. Board Can. Tech. Rep.* **195**, 1-69.

254 **Blasco, F. R., Esbaugh, A. J., Killen, S. S., Rantin, F. T., Taylor, E. W. and McKenzie, D. J.** (2020). Using aerobic exercise to evaluate sub-lethal tolerance of acute warming in fishes. *J. Exp. Biol.* **223**,.

257 **Blasco, F. R., Taylor, E. W., Leite, C. A. C., Monteiro, D. A., Rantin, F. T. and McKenzie, D. J.** (2022). Tolerance of an acute warming challenge declines with body mass in Nile tilapia: evidence of a link to capacity for oxygen uptake. *J. Exp. Biol.* **225**, jeb244287.

260 **Brett, J. R.** (1964). The respiratory metabolism and swimming performance of young sockeye salmon. *J. Fish. Res. Board Can.* **21**, 1183-1226.

262 **Brijs, J., Jutfelt, F., Clark, T. D., Grans, A., Ekstrom, A. and Sandblom, E.** (2015). Experimental manipulations of tissue oxygen supply do not affect warming tolerance of European perch. *J. Exp. Biol.* **218**, 2448-2454.

265 **Claireaux, G., McKenzie, D. J., Genge, a G., Chatelier, A., Aubin, J. and Farrell, A. P.** (2005). Linking swimming performance, cardiac pumping ability and cardiac anatomy in rainbow trout. *J. Exp. Biol.* **208**, 1775-84.

268 **Desforges, J. E., Birnie-Gauvin, K., Jutfelt, F. M., Gilmour, K. M., Eliason, E. J., Dressler, T. L., McKenzie, D. J., Bates, A. E., Lawrence, M. J., Fangue, N. A., et al.** (2023). The ecological relevance of critical thermal maxima methodology (CTM) for fishes. *J. Fish Biol.* **00**.

248 **References**

249 **Andreassen, A. H., Hall, P., Khatibzadeh, P., Jutfelt, F. and Kermen, F.** (2022). Brain dysfunction during warming is linked to oxygen limitation in larval zebrafish. *Proc. Natl. Acad. Sci.* **119**, e2207052119.

252 **Bell, W. H. and Terhune, L. D. B.** (1970). Water tunnel design for fisheries research. *Fish. Resour. Board Can. Tech. Rep.* **195**, 1-69.

254 **Blasco, F. R., Esbaugh, A. J., Killen, S. S., Rantin, F. T., Taylor, E. W. and McKenzie, D. J.** (2020). Using aerobic exercise to evaluate sub-lethal tolerance of acute warming in fishes. *J. Exp. Biol.* **223**,.

257 **Blasco, F. R., Taylor, E. W., Leite, C. A. C., Monteiro, D. A., Rantin, F. T. and McKenzie, D. J.** (2022). Tolerance of an acute warming challenge declines with body mass in Nile tilapia: evidence of a link to capacity for oxygen uptake. *J. Exp. Biol.* **225**, jeb244287.

260 **Brett, J. R.** (1964). The respiratory metabolism and swimming performance of young sockeye salmon. *J. Fish. Res. Board Can.* **21**, 1183-1226.

262 **Brijs, J., Jutfelt, F., Clark, T. D., Grans, A., Ekstrom, A. and Sandblom, E.** (2015). Experimental manipulations of tissue oxygen supply do not affect warming tolerance of European perch. *J. Exp. Biol.* **218**, 2448-2454.

265 **Claireaux, G., McKenzie, D. J., Genge, a G., Chatelier, A., Aubin, J. and Farrell, A. P.** (2005). Linking swimming performance, cardiac pumping ability and cardiac anatomy in rainbow trout. *J. Exp. Biol.* **208**, 1775-84.

268 **Desforges, J. E., Birnie-Gauvin, K., Jutfelt, F. M., Gilmour, K. M., Eliason, E. J., Dressler, T. L., McKenzie, D. J., Bates, A. E., Lawrence, M. J., Fangue, N. A., et al.** (2023). The ecological relevance of critical thermal maxima methodology (CTM) for fishes. *J. Fish Biol.* **00**.

248 **References**

249 **Andreassen, A. H., Hall, P., Khatibzadeh, P., Jutfelt, F. and Kermen, F.** (2022). Brain dysfunction during warming is linked to oxygen limitation in larval zebrafish. *Proc. Natl. Acad. Sci.* **119**, e2207052119.

252 **Bell, W. H. and Terhune, L. D. B.** (1970). Water tunnel design for fisheries research. *Fish. Resour. Board Can. Tech. Rep.* **195**, 1-69.

254 **Blasco, F. R., Esbaugh, A. J., Killen, S. S., Rantin, F. T., Taylor, E. W. and McKenzie, D. J.** (2020). Using aerobic exercise to evaluate sub-lethal tolerance of acute warming in fishes. *J. Exp. Biol.* **223**,.

257 **Blasco, F. R., Taylor, E. W., Leite, C. A. C., Monteiro, D. A., Rantin, F. T. and McKenzie, D. J.** (2022). Tolerance of an acute warming challenge declines with body mass in Nile tilapia: evidence of a link to capacity for oxygen uptake. *J. Exp. Biol.* **225**, jeb244287.

260 **Brett, J. R.** (1964). The respiratory metabolism and swimming performance of young sockeye salmon. *J. Fish. Res. Board Can.* **21**, 1183-1226.

262 **Brijs, J., Jutfelt, F., Clark, T. D., Grans, A., Ekstrom, A. and Sandblom, E.** (2015). Experimental manipulations of tissue oxygen supply do not affect warming tolerance of European perch. *J. Exp. Biol.* **218**, 2448-2454.

265 **Claireaux, G., McKenzie, D. J., Genge, a G., Chatelier, A., Aubin, J. and Farrell, A. P.** (2005). Linking swimming performance, cardiac pumping ability and cardiac anatomy in rainbow trout. *J. Exp. Biol.* **208**, 1775-84.

268 **Desforges, J. E., Birnie-Gauvin, K., Jutfelt, F. M., Gilmour, K. M., Eliason, E. J., Dressler, T. L., McKenzie, D. J., Bates, A. E., Lawrence, M. J., Fangue, N. A., et al.** (2023). The ecological relevance of critical thermal maxima methodology (CTM) for fishes. *J. Fish Biol.* **00**.

248 **References**

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373 **Figure legends**

374 Figure 1. Boxplots of data for critical temperature of endpoint (A); maximum rate of oxygen 375 uptake achieved (B); thermal scope, calculated as maximum rate of oxygen uptake minus 376 rate at 19 °C (C), and Q10 for effects of temperature on oxygen uptake between 19 °C and 377 29 °C (D). Two thermal ramping protocols are compared in n = 8 European seabass, either 378 critical thermal maximum for swimming (CTSmax) exercising aerobic with fatigue as endpoint, 379 or critical thermal maximum (CTmax) under static conditions with loss of equilibrium as 380 endpoint. Each point is an individual, the lines connecting the two protocols show how that 381 individual responded to each. Boxplot lower and upper hinges represent the 25th and 75th 382 percentiles. The horizontal line within the box is the median. The length of whiskers 383 represents the range of data points between each hinge and 1.5× the difference between 384 the 25th and 75th percentiles. Data beyond these limits are outliers.

385 Figure 2. Rate of oxygen uptake in n = 8 European seabass during exposure to two thermal 386 ramping protocols, either critical thermal maximum for swimming (CTSmax) exercising 387 aerobic with fatigue as endpoint (19 °C to 31 °C, orange symbols and curve), or critical 388 thermal maximum (CTmax) under static conditions with loss of equilibrium as endpoint (19 to 389 35°C, red symbols and curve). Each point represents an individual, the temperature ranges 390 are those for which oxygen uptake data was collected on all individuals, the shaded area 391 shows the 95% confidence interval.

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