Response to ‘How and how not to investigate the oxygen and capacity limitation of thermal tolerance (OCLTT) and aerobic scope – remarks on the article by Gräns et al.’

We appreciate the on-going discussion and healthy evaluation of the hypothesis of oxygen and capacity limitation of thermal tolerance (OCLTT). However, we think it is unfortunate that Pörtner (Pörtner, 2104) sees little value in our study (Gräns et al., 2014), which currently represents the largest long-term experimental test of OCLTT.

The OCLTT hypothesis emphasises the importance of oxygen delivery to aerobic processes as the major evolutionary constraint shaping organisms, their physiology and ecosystems. However, such a broad view does not easily produce testable predictions, and we argue that the value of a scientific idea lies in its ability to predict future observations. Therefore, we focused on a testable prediction that OCLTT is founded upon; that reduced aerobic scope is the physiological limitation that impairs other organismal performances, such as growth at high temperatures and high P_CO2 (Gräns et al., 2014; Pörtner and Farrell, 2008; Pörtner and Knust, 2007). We found that aerobic scope increased continuously with acclimation temperature, and even more so in CO₂-acidified seawater, whereas growth plateaued at the three intermediate temperatures and declined at the highest acclimation temperatures [fig. 1A and fig. 3 in original article (Gräns et al., 2014)]. This clear mismatch in thermal profiles for aerobic scope and growth indicates that oxygen delivery capacity does not decrease at high temperatures and cannot have limited growth, as OCLTT would have predicted.

Pörtner (Pörtner, 2014) suggests that we should change the definition of aerobic scope to include the scope for all oxygen-requiring performances: ‘use the term aerobic scope for all routine performances that draw on aerobic energy such as growth, reproduction or steady-state swimming’. We, however, use the widely adopted definition of aerobic scope: the difference between standard metabolic rate (SMR) in resting unfed animals, and maximum metabolic rate (MMR) (Fry and Hart, 1948; Pörtner and Farrell, 2008). Growth rate, reproductive output and aerobic scope are all commonly used terms with clear definitions, and we fail to see how redefining and mixing of terminology can improve our understanding of thermal biology. If anything, such a move would risk confusing the debate further with semantic misunderstandings.

Pörtner (Pörtner, 2014) proposes that the thermal mismatch between aerobic scope and growth is due to growth occurring in unstressed fish in ‘steady state’, whereas we measured MMR (and thus aerobic scope) during non-steady state recovery from exhaustive exercise. We suspect the misunderstanding may lie in our differing definitions of aerobic scope. MMR can, in most animals, only be quantified during or immediately after exercise when the fish are using, or recovering from, partly anaerobic white muscle activity. Moreover, Pörtner suggests that oxygen limitation at high temperature can occur for growth at rest, but that oxygen transport capacity can increase greatly during exercise because of catecholamine release and shifts in blood chemistry, which seems unlikely. The stimulatory effects of catecholamines on cardiac performance and oxygen transport also typically decrease with increasing temperature, as a result of blunted β-adrenergic stimulation of the myocardium (Keen et al., 1993). Therefore, our experimental protocol would be expected to have fewer stimulatory effects on aerobic performance at the higher temperatures and yet we still observed the highest aerobic scope at these temperatures.

The positive effect of CO₂ on aerobic scope that we reported (Gräns et al., 2014) is also questioned. Pörtner claims that aerobic scope could be protected by bicarbonate accumulation in fish exposed to high P_CO2, which may be possible. However, this is not a relevant argument against the positive effects of CO₂ on aerobic scope, because this would also occur in nature. Although the effect size on aerobic scope by CO₂ was arguably small, it was nonetheless confirmed by statistical tests across temperatures in the opposite direction to what OCLTT predicts and the increase in aerobic scope from CO₂ was not matched by increased growth.

We feel that Pörtner's critique of our statistical analysis might be due to misunderstandings. As stated in the original article, the trend lines added to the figures are for visual aid, and not based on the statistical models we used. The experimental design and statistical models were developed together with a mathematical statistician, and we are confident that our statistical analyses are of the highest standard.

Pörtner (Pörtner, 2014) highlights two examples ‘of how to investigate OCLTT, performance and aerobic scope more successfully’ (Eliason et al., 2011; Pörtner and Knust, 2007). They are, however, like any study (including ours) not without limitations. In Eliason et al. (Eliason et al., 2011), aerobic and cardiac scope was measured in instrumented sockeye salmon using swim tunnels. Although this is an impressive experimental endeavour, we do not understand why these animals should be considered to be in ‘steady state’. First, SMR was obtained from highly instrumented salmon after an overnight recovery from surgery and during intermittent blood samplings. Second, the thermal challenges were acute for fish at the upper and lower thermal extremes (4°C h^–1 and left for 1 h), whereas the intermediate temperatures were tested after short-term thermal acclimation (5°C day^–1 and left for 1 day), presumably leaving the fish in different stages of the thermal acclimation process. For MMR measurements, a U_crit swim protocol was used with an electric motivator grid. Thus, whereas the MMR measured in Eliason et al. (Eliason et al., 2011), and our study probably consisted of a combination of aerobic and anaerobic metabolism because white muscle is increasingly recruited with increasing swimming speed (Clark et al., 2013a; Jayne and Lauder, 1994), the metabolic measurements in our study were not affected by surgery and variable thermal test protocols.

The other suggested example, Pörtner and Knust (Pörtner and Knust, 2007), combines results from many earlier publications, none of which measured aerobic scope according to conventional definitions. The study reports, in our view, rather weak thermal associations and claims a causal link to oxygen limitation. For example, the authors conclude that oxygen transport limitation was the reason for the drop in field abundance at 19°C, yet in the same paper, growth rate remained high at 20°C (∼80% of max) and markers of anaerobic metabolism only became elevated after 72 h at 24°C (in liver, but not heart muscle). In addition, arterial blood flow was reported from only one individual during thermal ramping in a NMR setup with the fish confined in a space half the length of the fish. It is not clear to us how this can represent ‘a similar physiological status as organisms experiencing thermal limitation in their natural environment’.

Curiously, both the suggested examples of how to correctly investigate OCLTT include fish with zero oxygen transport in their datasets, representing either dead fish or measurement errors [no arterial blood flow in Pörtner and Knust, fig. 1C (Pörtner and Knust, 2007); zero MMR in Eliason et al., fig. S2A (Eliason et al., 2011)].

For these reasons, we fail to see how these two studies can be considered to represent aerobic scope measurements under ‘routine steady-state as in the field’, whereas our study is not, as proposed by Pörtner. If these publications represent the best empirical evidence for OCLTT, then the hypothesis is standing on loose ground, and it may not be surprising that a growing number of studies are questioning the generality of OCLTT (Clark et al., 2013a; Clark et al., 2013b; Ern et al., 2014; Norin et al., 2014; Overgaard et al., 2012).

In our paper (Gräns et al. 2014) we aimed to test the core proposition of the OCLTT hypothesis; that tissue oxygen limitation is the mechanism behind reductions in other performances at high temperatures. We demonstrated that the thermal windows for aerobic scope and growth differ, and that reduced aerobic scope was not associated with the decline in growth at high temperatures. This forced us to question the tissue oxygen limitation mechanism that is central to the OCLTT hypothesis. We agree with Pörtner that the ecophysiological community needs to find out at which conditions a species becomes thermally limited. However, we argue that it is unlikely that we will ever identify a single physiological mechanism explaining the complex subject of thermal tolerance and climate change vulnerability in ectothermic animals. Instead, we suggest that the physiological cause of limitation can vary and will depend on a number of biological and environmental factors including the rate of temperature change, species, lifestyle and physiological state of the organism. We therefore encourage other researchers to look for thermal limitation mechanisms beyond oxygen supply and not to feel obligated to fit their experimental findings into the framework of OCLTT.