The use of DLW for measuring FMR (Lifson, Gordon & McClintock 1955; Lifson & McClintock 1966) has been widely recognised as a revolution in ecophysiology (Speakman 1997; Bradshaw & Bradshaw 2007). The technique permits informative measures of the energetics of free-ranging animals. Subsequent refinements and broad application of the technique to a range of taxa, however, have revealed some limitations to the technique (Bevan, Speakman & Butler 1995b; Bevan et al. 1995a; Speakman 1997; Bradshaw & Bradshaw 2007). The aim of the present study was to test the reliability of an alternative method for estimating FMR by making the first quantitative study of the correlation between 86Rb kb and metabolic rates estimated by DLW and respirometry simultaneously. The work can be considered as an extension of previous validation work that compared 86Rb kb with DLW (Peters et al. 1995; Bradshaw & Bradshaw 2007) or respirometry (Peters 1996).
When we compared our measures of metabolism () made by DLW and FTR , we found that DLW overestimated FTR by about 12% for S. macroura and 24% for S. ooldea. The larger error for S. ooldea likely reflects the higher variability of metabolic rates reported for this species compared with S. macroura due to more labile thermoregulation (Tomlinson, Withers & Maloney 2012a,b), and the large discrepancies may not reflect uncontrolled methodological errors, but represent genuine variability in the metabolic rates of S. ooldea. Some previous studies have also reported that DLW overestimates , generally by about 8% (Speakman & Racey 1988; Nagy et al. 1990; Tiebout & Nagy 1991; Speakman 1998), but other studies report underestimation of metabolic rate (e.g. Williams & Nagy (1984), Williams (1985), Nagy et al. (1990), and Speakman (1998) and references therein). Most of these studies based on their comparisons on the species mean of measured by DLW compared to the mean of another method, while our correlation of individual responses using several techniques to estimate revealed high levels of inter- and intra-individual variation, congruent with Speakman's (1998) suggestion that there is low repeatability of DLW for reasons yet to be understood.
Generality of 86Rb as a measure of FMR
The rationale for the present study was to investigate whether 86Rb kb was correlated with for two species of dunnart and could therefore be used as a measure of FMR for free-ranging dunnarts, and the significant relationships that we obtained suggest that we can predict FMR of dunnarts by measuring 86Rb kb. However, combining our data with previous comparisons of 86Rb biological turnover to (expressed here as LCO2.day−1; Fig. 3) provide a more general test of 86Rb kb as a measure of FMR. We found that the slope of the relationship was significantly steeper (F2,33 = 31·2, P = 2·0 × 10−8) for ectotherms (9·3 × 10−3) than endotherms (3·4 × 10−3), indicating that ectotherms have a higher turnover of 86Rb per CO2 consumed. Initial examination of the relationship between and 86Rb kb for endotherms suggested a shallower slope (F2,31 = 4·64; P = 0·017) for T. rostratus (Bradshaw & Bradshaw 2007) than the two dunnarts in the present study, with T. rostratus exhibiting smaller increases in 86Rb kb for a given increase in than the dunnarts. Bradshaw & Bradshaw (2007) found that a power curve best fit their data, as a result of four extremely high metabolic rates measured during inclement conditions at their study site that were inconsistent with relationship at the lower DLW and 86Rb kb. Following exclusion of these four unreasonably high points, their data were similar to ours, which are the only other validations conducted for endotherms. In our data, there was a significant regression between 86Rb kb and with a slope of 4·0 × 10−3. As a result, although we do not suggest the use of 86Rb kb as the only or even the best alternative technique to DLW, given the large volume of work exploring heart rate telemetry (see Cooke et al. (2004) and Green (2011) for review) and other techniques (Hambly, Harper & Speakman 2002), our meta-analysis suggests that 86Rb kb has a general relationship with for all of the taxa studied so far.
Figure 3. Comparisons of previous regressions of 86Rb turnover with metabolism (converted here to in L.day−1 from the original published data sets) for the toad Bufo terrestris (∆, r2 = 0·67; Peters 1996), lizard Dipsosaurus dorsalis (□, r2 = 0·93; Peters et al. 1995), Tarsipes rostratus (◊, r2 = 0·74; Bradshaw & Bradshaw 2007), Sminthopsis macroura (•, r2 = 0·73) and Sminthopsis ooldea (■, r2 = 0·46). The overall endothermic relationship is 86Rb = (3·4 × 10−3) × − (4·6 × 10−4) (r2 = 0·76, P = 2·9 × 10−12), and the ectothermic relationship is 86Rb = (9·3 × 10−3) × − (2·1 × 10−4) (r2 = 0·96, P = 4·7 × 10–22). All published regressions used to compile this figure were significant.
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Our understanding of the physiology and biochemistry of 86Rb kb is currently poor. Being an alkali metal of higher molecular weight than potassium, it is thought that Rb+ is substituted for K+ in metabolic processes (Adam & Craik 1989; Bradshaw & Bradshaw 2007). The precise nature of that substitution is not known, and so a sound theoretical basis for why 86Rb kb reflects metabolic rate is lacking. Hence, there is currently an apparent requirement to validate the FMR–86Rb kb relationship for each species in which it might be used. To validate that relationship requires some measure of FMR as a comparator. Flow-through respirometry is recognised as the best method of measuring standardised energy use by animals other than direct heat production (Frappell 2006). Techniques that measure ADMR (such as isotope turnovers) require long washout times, as shown in Fig. 1. Therefore, when respirometry is used to validate isotope methods, there is a requirement to make comparisons over a long period. With this in mind, FTR should provide a reliable avenue to validate 86Rb kb.
The rate of 86Rb washout measured here supports the proposition made by Bradshaw & Bradshaw (2007) that the washout of 86Rb is slower than H and O isotopes, and so it allows a longer period between enrichment and recapture than the DLW technique. In a previous study of FMR of Sminthopsis made using DLW, the data collection period was limited to 3 days because of the rapid washout of DLW isotopes (Nagy et al. 1988). The 86Rb washout rates measured here suggest that FMR could be estimated using 86Rb for as long as 14 days after enrichment for Sminthopsis. Despite the fact that curve splining (Cerling et al. 2007) suggested only a single pool turnover system for the elimination of 86Rb, the significantly higher 86Rb kb in the first 2 days following enrichment may result in noise in those initial days that require a longer washout period to attenuate, suggesting that the longer the animals can be left free ranging, the more reliable will be the estimate of FMR. A longer period between initial enrichment and final recapture reduces the proportion of the washout period that an animal spends in captivity relative to the free ranging.
Advantages of the 86Rb technique to infer FMR, at least for small animals, include lower cost, lower equipment requirements and technical expertise, reduced animal stress during measurement and the longer time span during which effective measurements can be made (Bradshaw & Bradshaw 2007). Our results provide much to support some of these advantages for the 86Rb method. Our 86Rb turnover measurement cost was c. 12% of our DLW measurement. Given that isotope and analysis costs are cited as prohibitive restrictions to measures of FMR in large species (Westerterp & Speakman 2008; Zub et al. 2009), 86Rb potentially provides a very useful tool for investigations in large species. The further complications of correct and complete sample distillation and sometimes complex calculations required to correct for changes in body mass, body water content and dilution spaces, make DLW the more complicated of the two methods, even in a controlled laboratory validation. Where the 86Rb technique is to be used in the field, costs and ethical limitations of any study can be much reduced, and NaI crystal gamma counters are portable, allowing measurements to be made in the field. A practical limitation at present is the necessity to validate the 86Rb kb to relationship, which can be achieved using FTR in the laboratory, but this is not straightforward for large species.
There are several limitations currently associated with 86Rb kb, not least of which are the indirect method of estimation, the paucity of comparative validation for the technique and a poor understanding of the mechanism(s) of turnover. Even the generalised regression lines for ectotherms and endotherms may not provide a sufficiently specific correlation between and 86Rb kb for a particular species, because very little is known about the comparative physiology of normal potassium metabolism other than for pathological disorders (Greenberg et al. 1938; English 1966). Without a general understanding of the role of K+ in metabolism, and whether it correlates with metabolism under all conditions, any taxonomic generalities about 86Rb kb remain difficult to interpret. Furthermore, if 86Rb is a proxy for K+, then it will be distributed mainly in the intracellular space, and using blood to estimate total body burden of 86Rb will vary with haematocrit. A second difficulty that remains to be overcome for the 86Rb technique is a limitation of size. Whole-body counting of animals much larger than the dunnarts studied here becomes impossible, or at the very least expensive, and the equipment involved becomes nonportable. Subsampling of a body pool, such as blood from larger animals, may provide an avenue to measure gamma emissions and infer the total body burden of 86Rb, but this has not been validated. The 86Rb isotope required also has substantial safety and environmental concerns associated with the control of radioactive substances. It must be explicitly noted; however, that the levels of enrichment required for small animals constitute a fraction of the internationally recognised safe limits of exposure of 1 mGyd−1 (International Atomic Energy Agency 1992; Peters et al. 1995). Radiation exposure levels were monitored during this study by the Australian Radiation Council for all participants, and dangerous exposure was never recorded, even during high-intensity laboratory validation programmes. The risks of working with smaller animals are negligible providing that proper equipment and training are available. This risk is likely to increase with the levels of enrichments required for larger animals and may become a radiation hazard to the experimenters and to the animals themselves. To avoid this, the dosage requirements to optimise the signal to noise ratio need to be further investigated. Finally, DLW gives the advantage of measuring both and water flux (Nagy 1983; Speakman 1997), but 86Rb kb cannot presumably provide any information on water use. There are also some practical requirements for the use of radioisotopes to measure metabolism (compared to the stable isotopes used for DLW), including the need to have current radiation licences and appropriate training for the use of unsealed radioisotopes, and the need for appropriate decontamination and waste disposal.
We have established that there is a good correlation between the directly measured and 86Rb kb for dunnarts, and general correlations for ectotherms and endotherms. Validation studies on more taxa are still required to establish whether correlations between 86Rb kb and metabolic rate can be generalised or whether validation is required on a species-by-species basis. It remains to be established whether the same relationship holds for all endotherms (e.g. eutherian mammals, passerine and nonpasserine birds). While there remain questions regarding the generality of the 86Rb kb technique as a measure of FMR, validation of the method will permit measurements for problematic species with a high water turnover relative to , such as in nectarivores (Williams 1985; Weathers & Stiles 1989; Powers & Conley 1994; Geiser & Coburn 1999; Voigt, Kelm & Visser 2006; Bradshaw & Bradshaw 2007), diving species (Bevan, Speakman & Butler 1995b; Bevan et al. 1995a; Sparling et al. 2008; Jones et al. 2009) and amphibians (Hillman et al. 2009). Similarly, FMR could be measured for fossorial animals, where the atmosphere within a burrow may become DLW enriched and the isotopes re-enter the animal (Nagy 1980). This is because the 86Rb isotope is not volatile (i.e. it cannot enrich the atmosphere of the burrow) and is unlikely to re-enter the animal passively by respiration in the same way that DLW may do. Problematic taxa such as these should be studied to determine if 86Rb kb does provide a meaningful measure of FMR. The assumption that 86Rb is a K+ analogue should be tested, and appropriate dosimetry of 86Rb enrichment to body mass should be optimised to make the method safer and more efficient. This would go some way to informing how the assimilation of 86Rb into the metabolic pathways reflects metabolic processes.