Dr Wood's Commentary (Wood, 2018) provides six reasons to question the usefulness of Pcrit and proposes alternative ṀO2 versus PO2 analyses as its replacement. While we agree with some of Dr Wood's arguments, we feel that none of them warrant abandoning Pcrit, especially in favour of his proposed alternatives, which provide different information than Pcrit. A more useful way forward would involve (1) clearly defining Pcrit, to avoid misinterpretation, and (2) standardizing (or at least clearly describing) the methods used to determine and report Pcrit, to optimize its comparative value. This topic demands further discussion because Dr Wood's conclusion could have unwarranted influence on how future hypoxia research is conducted and past hypoxia research is interpreted.
Dr Wood's arguments are either theoretical (reasons 3–6) or methodological (reasons 1, 2). The theoretical arguments, if true, may warrant the abandonment of Pcrit. However, contrary to Dr Wood's claims, across species, Pcrit is strongly correlated with the environmental O2 level to which species are exposed and is therefore ecologically relevant (Childress and Seibel, 1998; Mandic et al., 2009; Wishner et al., 2018). Pcrit is also correlated with multiple steps of the O2 transport cascade, from gill surface area through haemoglobin P50 to mitochondrial P50 (Childress and Seibel, 1998; Lau et al., 2017; Mandic et al., 2009), and is a sensitive measure of an animal's overall ability to extract O2 because altering physiological traits along the cascade can change Pcrit. For example, anatomical restructuring of the gill to favour O2 diffusion [e.g. reducing gill epithelial thickness through seawater acclimation in sculpins (Henriksson et al., 2008); increasing lamellar surface area through hypoxia acclimation in crucian carp (Sollid et al., 2003)] can lower Pcrit. These relationships between Pcrit and plastic traits along the O2 transport cascade clearly reflect the physiological relevance of Pcrit and indicate that Pcrit – especially when it shifts with acclimation – does indeed represent the PO2 at which O2 uptake becomes constrained. Therefore, contrary to reason 6, Pcrit per se does carry biologically relevant information. Hence, Pcrit remains a useful tool for understanding hypoxic performance because it allows for predictive statements.
Much of Dr Wood's reasoning centres on the over-interpretation of Pcrit. Pcrit does not necessarily quantify an animal's overall hypoxia tolerance (the product of some combination of aerobic metabolism, anaerobic metabolism and metabolic depression; reason 5), reveal what biological processes the O2 consumed at Pcrit is supporting (reason 4), or indicate the onset PO2 of enhanced glycolytic reliance (reason 4) or metabolic depression (reason 5). These ideas have long been excluded from the definition of Pcrit. Simply, Pcrit defines the lowest water PO2 at which the animal can maintain some benchmark ṀO2 state (e.g. ṀO2,std, the ṀO2 of an inactive and post-absorptive ectotherm, is a useful benchmark for evaluating O2 supply capacity at low PO2), and this has physiological and ecological relevance.
Dr Wood's methodological arguments mention that Pcrit calculation requires the portion of the ṀO2 curve at PO2 above Pcrit to have a slope of zero (Box 1A in Wood, 2018), a response that not all animals display. However, this is not required with the above definition, and indeed widely used calculation methods (e.g. Yeager and Ultsch, 1989) can effectively assign Pcrit when the slope of this portion of the curve is positive or negative (Box 1B,D in Wood, 2018). In the case of true oxy-conforming species (Box 1C in Wood, 2018), Pcrit may be impossible to calculate (though some species once thought to be oxy-conformers are actually oxy-regulators when measured with modern respirometric techniques). However, this does not negate the usefulness of Pcrit for oxy-regulating species, which comprise the vast majority of animals measured to date (Steffensen, 2006).
As Dr Wood states, cross-study comparisons can become muddled in the absence of standardized methods for Pcrit calculation and experimentation [though the two-segmented lines method (Yeager and Ultsch, 1989) has been used >250 times]. However, this criticism is hardly unique to Pcrit and does not necessarily justify abandoning Pcrit or any other non-standardized measurement. Methods can be standardized, or at least carefully described and presented with all relevant data (e.g. ṀO2 versus PO2 traces; reason 6) to optimize comparative value. But even if they cannot, Pcrit would remain a useful comparator within studies exploring differences in environmental condition, treatment, genotype, strain, population and/or species.
Two important points must be made regarding Dr Wood's proposed alternatives. First, we welcome such measurements and others like them (e.g. Claireaux and Chabot, 2016; Cobbs and Alexander, 2018), as they add valuable physiological information on animal performance in hypoxia. However, caution is needed, as some measures may be less relevant to hypoxia tolerance per se than Pcrit (e.g. regulation index, which simply quantifies an animal's oxy-regulatory ability) and others unsuitable for a complex system like the O2 cascade (e.g. Michaelis–Menten approach, which is based on simple enzyme kinetics). Second, the methods for experimentation and calculation of the proposed alternatives have not been rigorously standardized, and so Dr Wood's critique of Pcrit applies to his alternatives as well. For example, to determine regulation index, how many and what types of curves are needed to best fit the data (reason 2)? And, if there is variation in ṀO2,std, which point is used to anchor the diagonal line of oxy-conformation (reason 1)?
To summarize, Pcrit should not be abandoned, but it should be carefully defined to avoid misinterpreting what it represents. The theoretical argument against Pcrit is incorrect, and the methodological argument against Pcrit can be rectified through standardization and/or clearly defining and reporting the conditions and methods of measurement. What will aid in this are published guidelines that the community accepts and uses for applicable species. Importantly, Pcrit defines the lower bound of the PO2 spectrum over which an animal supports its metabolic rate predominantly using aerobic metabolism, albeit with a diminishing aerobic scope for activity as PO2 approaches Pcrit. On its own, this information has important ecological relevance and serves as an effective comparator within and among species. When paired with other hypoxia-related physiological measurements such as lactate accumulation and calorimetry-based measurements of metabolic depression (e.g. Regan et al., 2017), Pcrit contributes to a more complete picture of an animal's total hypoxic response by capturing the suite of aerobic contributions to hypoxic survival in a single value. In other words, when properly measured and interpreted, Pcrit is useful.
