Effect of Thermal Acclimation on Locomotor Energetics and Locomotor Performance in A Lungless Salamander, Desmognathus Ochrophaeus

Thermal acclimation may markedly alter the rate of oxygen consumption in amphibians, with some salamanders undergoing a 10–35% decrease in the resting or standard during acclimation to a moderately warm temperature (Feder, Gibbs, Griffith & Tsuji, 1984; Feder, 1985). Although such decreases in are often considered adaptive because they reduce the overall energy requirement, a decrease in may also reduce the capacity for physical activity, which may offset the advantage of a reduced energy requirement. The purpose of the present study was to characterize any changes in during exercise that are associated with acclimatory changes in resting ⁠, and to determine how acclimatory changes in during activity affect locomotor performance. Interspecific comparisons at a single temperature suggest that amphibians with a relatively high resting have a relatively high during activity as well, and vice versa (Taigen, 1983). If the during rest and during activity are likewise coupled in thermal acclimation of metabolism in amphibians, a reduced resting due to thermal acclimation should be accompanied by a reduced during activity. Such variation is likely to affect locomotor performance. According to Bennett’s (1982) model, which relates the maximum aerobic speed (which approximates to the maximum sustainable speed) to the maximum during activity, acclimatory reductions in the during activity ought to: (1) reduce the maximum aerobic speed; (2) decrease the speed at which significant anaerobiosis is sustained (Taigen & Beuchat, 1984); and (3) decrease stamina at non-sustainable speeds. Similar logic underlies claims that inverse acclimation of resting at warm temperatures (i.e. increases in resting should potentiate physical activity in amphibians active during the summer (Weathers, 1976).

A test of these expectations requires measurements of during graded exercise at known levels of activity, such as are available for fishes swimming in flow chambers or reptiles and mammals walking on treadmills. To obtain such measurements, previous investigators have used respirometers in which amphibians were prodded, shocked, or overturned to yield locomotor activity that is presumably maximal but is not amenable to quantification (Hillman, Shoemaker, Putnam & Withers, 1979). The present study, by contrast, used an exercise wheel in which small (<3g), lungless salamanders would walk for as much as 2 h at controlled and relatively constant speeds.

Allegheny Mountain salamanders, Desmognathus ochrophaeus, were collected near Highlands, North Carolina, USA. This species experiences temperatures between 1 and 20°C in the field (Feder, Lynch, Shaffer & Wake, 1982) and prefers 17–20°C in laboratory gradients (Spotila, 1972). Salamanders were maintained in the laboratory at 13–14°C on an LD 14:10 photoperiod centred at 13·00 h local time. Animals were fed fruit flies regularly. Body masses of animals ranged between 0·89 and 3·24 g and averaged 2 g.

Preliminary experiments established that body mass was inversely related to both in resting salamanders and in salamanders moving at various speeds. To minimize the effect of body mass, animals were sorted into groups with similar distributions of body masses before experimentation. Groups were then maintained at one of three acclimation temperatures (ATs): 5–6°C (hereafter 5° AT), 13–14°C (13° AT), and 21–22°C (21° AT). Animals were acclimated 2–3 weeks before use. Animals were not fed during the acclimation period; in addition, 5° AT animals were not fed during the week preceding acclimation to allow clearing of the gut (Feder et al. 1984). The photoperiod was unchanged during acclimation.

Each respirometer was essentially a hollow rectangular beam of Plexiglas (inner dimensions: 1·3 cm height X 1·5–1·8cm width × 40cm) bent in a circle to form a wheel. A port allowed access to the inside of the beam. Salamanders could walk inside the beam; i.e. within the wheel’s rim. The top and bottom of the inside of the beam (salamanders would walk on both) were coated with plastic screening or roughened to facilitate traction. Several curved pieces of lead were cast to fit loosely inside the beam; these had the tips of 30-gauge syringe needles mounted in their ends. In use, the wheel respirometer was cleaned thoroughly, the interior of the beam was moistened, a salamander and the pieces of lead were placed in the track, and the port in the wheel’s rim was bolted into place to form an air-tight seal. The wheel was then mounted vertically on a fixed axle connected by a pulley to an electric motor. A Minarik SL15 Motor Speed Controller (Minarik Electric Co., Los Angeles, CA) controlled motor speed and thus rotation of the wheel. As the wheel rotated, the lead pieces slid to the bottom of the wheel. Salamanders would walk forward in the track to avoid the sliding lead pieces. Accordingly, their speed would match that of wheel rotation.

Animals from the three acclimation regimes were examined individually at 13°C. After 2–3 h for equilibration, of the inactive animal was determined for three successive 20-min intervals. Thereafter, the animal was exercised for 15 min at each of a series of increasing speeds separated by a 5-min rest. was determined during each 15-min interval. The speeds were 6, 12, 22, 31, 39 and 47 cm min⁻¹. When animals were unable to sustain 15 min activity at a given speed, the run was terminated. Animals were considered exhausted when they could no longer maintain forward movement and instead were pushed continually by the lead pieces within the wheel.

Animals from the three acclimation regimes were examined individually at 21 °C. After 2–3 h for equilibration, an initial gas sample was withdrawn. The animal was then exercised at a single speed, 31cmmin⁻¹, until it became exhausted. was determined during this period. Next, the animal was allowed to recover for 30 min. was determined for the last 20 min of this interval. Finally, the animal was again exercised at 31 cm min⁻¹ until exhaustion, during which was determined.

Animals from the three acclimation regimes were exercised at 21 °C in wheels fashioned from plastic Petri dishes; the track within such wheels resembled that in the respirometer described above. Acclimated animals were weighed the day before measurement, placed in an individual wheel, and then returned to their respective acclimation temperatures overnight. Wheels containing salamanders were then placed at 21 °C for 2–3 h for equilibration. Each wheel was individually affixed to the motor, and the animal within exercised at 31 cm min⁻¹ for either 0, 5, 10, 15, 20 or 30min. Each animal was then immediately frozen in liquid nitrogen; the frozen carcass was homogenized in ice-cold 0·6 mol I⁻¹ perchloric acid. The lactate content of the homogenate was determined enzymatically according to Bennett & Licht (1972) with reagents from Sigma (St Louis, MO).

The standard (i.e. minimum) of acclimated animals was determined with a Gilson respirometer. Methods and complete data have been reported elsewhere (Feder et al. 1984; Feder, 1985). These previous data were adjusted to the body sizes of animals in the present study by assuming that scales as body mass raised to the 0·8 power (Feder, 1976).

In experiments at 13 °C, in which the of individual salamanders was measured repeatedly during or after exercise at several speeds, the data were analysed using analysis of covariance with repeated measures (Dixon, 1977). Body mass was the covariate, speed during exercise (including recovery) was the trial factor, and acclimation temperature was the grouping factor. In experiments 3 and 4, each measurement was analysed individually with analysis of covariance (Dixon, 1977); body mass was the covariate. When body mass significantly affected the analysis programmes ‘adjusted’ for this effect (Dixon, 1977); the adjusted mean is presented in the figures where appropriate. The ‘honestly significant difference’ was computed from the analyses of covariance (Sokal & Rohlf, 1969) and is presented in the figures; means differing by more than this amount differ significantly (P < 0·05). Nonparametric statistics (Kolmogorov-Smirnov test, Spearman’s rank correlation coefficient) are according to Siegel (1956).

When placed in the wheel respirometer, salamanders typically explored the chamber but soon became quiescent. Despite the lack of obvious movement, the of salamanders resting in the wheel respirometer averaged twice that of salamanders measured in the Gilson respirometer chambers. For example, in preliminary measurements at 13 °C, the of eight inactive salamanders in the two respirometer systems was 1·93 ±0·18 (mean ± standard error) and 0·89 ± 0·02/zmolg⁻¹ h⁻¹, respectively. The Gilson chambers were small glass vials containing moist paper towelling, and thus more closely resembled natural retreats of salamanders than did the wheel respirometer. Accordingly, the ⁠, of animals resting in the wheel respirometer is henceforth termed the ‘routine and the lower determined with the Gilson respirometer is termed the ‘standard ⁠.

When rotation of the wheel was begun, salamanders moved forward in response to contact with the lead pieces, and generally matched their forward speed to the rate of wheel rotation. Most often, salamanders walked upward at approximately a 45° gradient. increased with the average forward speed at speeds between 0 and approximately 14cmmin⁻¹ (Fig. 1). At greater average speeds, was independent of speed and averaged 4–6 times the routine This increment corresponds to 9–12 times the standard Because ⁠, did not increase at speeds greater than approximately 14cmmin⁻¹, this speed is the ‘maximum aerobic speed’ (Bennett, 1982). However, all but a single animal were able to sustain activity at greater speeds for at least 30 min.

Acclimation to a warm temperature (21 °C) clearly decreased during locomotion at 13°C (Fig. 1). The of animals acclimated to 21 °C averaged 74% of that of animals acclimated to 5 and 13 °C (combined) at speeds between 6 and 39 cm min⁻¹. Moreover, the routine of the 21° AT animals was just 61% of the routine of the 5° AT and 13° AT animals. Complete data were available for 15 animals at 0, 6, 12, 22 and 31 cm min⁻¹ (many animals became exhausted at greater speeds). These data were subjected to an analysis of variance with repeated measures, which confirmed that the effect of acclimation temperature on was statistically significant (P = 0·0027). Thus, the total aerobic cost of locomotion was lower in warm-acclimated salamanders than in cool-acclimated salamanders.

This acclimatory difference in during activity, however, had no obvious consequences for the maximum speed that salamanders could sustain at 13°C. The maximum aerobic speeds of the two groups were similar (Fig. 1). Of the cold-acclimated animals (N = 10), 90% could sustain 15 min of activity at 31 cm min⁻¹, 60% could sustain 39 cm min⁻¹, 30% could sustain 47 cm min⁻¹ and 10% could sustain 56 cm min⁻¹. Of the warm-acclimated animals (N = 6), 100% could sustain 31 cm min⁻¹, 84% could sustain 39 cm min⁻¹ and none could sustain 47 cm min⁻¹. These two distributions do not differ significantly (Kolmogorov-Smirnov test, P<0·5). Within the cold-acclimated group, by contrast, animals with the greatest also had the greatest sustainable speed (Spearman’s r= 0-623; P= 0·05).

The unexpected finding of a major effect of thermal acclimation on during locomotion without a corresponding effect on the maximum sustainable speed prompted a second series of measurements, in which procedures differed in two respects. First, the experimental temperature was increased to 21 °C in the hope of eliciting greater differences in among acclimation treatments (see Feder, 1978). Second, stamina and recovery from fatigue were used as measures of locomotor performance in the expectation that they might reflect acclimatory differences in better than had the maximum sustainable speed.

Contrary to expectations, acclimation temperature had no significant effect (P = 0·52) on during exercise to exhaustion at the test speed, 31 cm min⁻¹ (Table 1). Moreover, acclimation failed to affect during a 30-min recovery period (P = 0·41) and during a second period of exercise to exhaustion (P = 0·25).

Although during locomotion at 21 °C was unaffected by thermal acclimation, stamina differed markedly among the acclimation treatments during both the first exercise period (P = 0-011) and the second exercise period (P = 0·002) (Table 1). In both exercise periods, 5° AT animals had a significantly lower stamina than either the 13° AT animals or the 21° AT animals, which did not differ from one another significantly. Stamina of animals in the latter two groups was as much as 133 min (<41 m total distance) and averaged more than 1 h. Stamina during the second exercise period, which followed 30min of rest, was 51–55% of the initial stamina; this percentage was not affected by thermal acclimation.

One possible explanation for the rapid fatigue of 5° AT animals, whose was not different from that of the other groups, was that the 5° AT animals accumulated lactate at a greater rate than did the other groups. To test this explanation, whole body lactate concentration was determined in acclimated animals at various times during 30 min of exercise at 31 cm min⁻¹. The experimental temperature was 21 °C. Salamanders acclimated to 13 °C increased lactate concentrations seven-fold during the first 10 min of activity, but accumulated little or no additional lactate during the subsequent 20 min. Salamanders acclimated to 5 °C, by contrast, continued to accumulate lactate for the entire 30-min period (Fig. 2). Salamanders acclimated to 21 °C, which had the greatest average stamina, had relatively low lactate concentrations after 15 min of exercise but unexpectedly high lactate levels after 30 min of exercise. The latter value may have stemmed from exceptionally vigorous activity in two animals in the sample; levels in the three other animals were low.

This study includes the first measurements of during controlled locomotion in a small (<3g) terrestrial ectothermic vertebrate. The of Desmognathus in the exercise wheel increased with speed at low speeds and was independent of speed at high speeds, and thus resembled the pattern of in larger lizards exercising on treadmills (Bennett, 1982) as well as many other terrestrial animals. Also, the mass specific was inversely related to the body size of salamanders both at rest and during activity. In other aspects, however, Desmognathus is unusual. The maximum aerobic speed, approximately 14cmmin⁻¹ or 0·008 k mh⁻¹, was spectacularly low. This speed is three orders of magnitude less than the maximum aerobic speed predicted for mammals by Garland’s (1983) equation 5 and thus two orders of magnitude less than the maximum aerobic speed expected for lizards (Garland, 1982). This last comparison is confounded by the gross extrapolation that is required, the use of an exercise wheel rather than a treadmill in the present study, and the low body temperatures at which salamanders are active. Nonetheless, Desmognathus, which is by no means sedentary among salamanders, clearly is exceptionally slow.

According to Bennett’s (1982) characterization of the maximum aerobic speed, activity at greater speeds is supported increasingly by anaerobic metabolism, whose end-products gradually accumulate and result in fatigue; thus, activity at greater speeds should not be sustainable. Some aspects of locomotion in Desmognathus are obviously not in accord with this scheme. For example, Desmognathus can sustain speeds greater than the maximum aerobic speed for considerable periods. At 13°C, most animals could move at a series of greater speeds for at least 45 min. At 21 °C, some animals walked at 31 cm min⁻¹ for nearly 2h. In a different apparatus at 20°C (Feder & Londos, 1984), the same species could maintain 150 cm min⁻¹ for 10 min before fatiguing. Although all animals accumulated lactate during the first 10 min of activity at 31 cm min⁻¹, most of the warm-acclimated animals accumulated little or no additional lactate when active at this speed for up to 20 min more (Fig. 2). Moreover, because did not increase at speeds greater than the maximum aerobic speed, animals could be active at increasing speeds without a concomitant increase in (and, apparently, lactate production). Increases in the speed of sustainable locomotion without parallel increases in although uncommon, have been reported and generally involve specializations such as elastic storage (Dawson & Taylor, 1973) or ram ventilation (Freadman, 1981). One possible explanation for this pattern in Desmognathus is that salamanders change to a more efficient gait at higher speeds (Edwards, 1976). Another (but unlikely) explanation is that the time course of lactate production at 21 °C is greatly different from the time course of lactate production at 13 °C, at which the maximum aerobic speed was determined. Both of these explanations are currently under investigation.

The slopes of the ascending lines relating to speed in Fig. 1 express the net cost of transport (Taylor, Schmidt-Nielsen & Raab, 1970) in Desmognathus. These are 17·2mlO₂g⁻¹km⁻¹ for the cold-acclimated animals and 14·5 m 10 ₂g⁻¹ km⁻¹ in the warm-acclimated animals. Comparable values have been obtained for another species of similar size, Bolitoglossa subpalmata, in the wheel respirometer (M. E. Feder, unpublished data). The net costs of transport for Desmognathus and Bolitoglossa thus differ dramatically from the net cost of transport predicted for 2-g animals by the summary equation 8 of Taylor, Heglund & Maloiy (1982), 3·8 ml O₂g⁻¹ h⁻¹. Although the equation of Taylor et al. (1982) is based exclusively on data for mammals, it satisfactorily describes the cost of transport in a variety of invertebrates (0·01–100g) and lizards (Full, 1984; Schmidt-Nielsen, 1984). By contrast, the net cost of transport in a third, larger species (Plethodon jordani, 4 g) walking on a treadmill is only 2·2mlO₂g⁻¹ km⁻¹ (Full, 1985), which is consistent with the equation of Taylor et al. (1982). Accordingly, it is not clear whether the remarkably high net cost of transport in Desmognathus and Bolitoglossa reflect their peculiar mode of sporadic movement within the respirometer (see above), their small size, the use of an exercise wheel instead of a treadmill, or an unusual case of costly locomotion. Additional studies are under way to clarify this point.

A final comparison concerns the respiratory gas exchange of Desmognathus, which lacks lungs and gills and thus breathes exclusively through its skin and bucco-pharynx. Cutaneous gas exchange has often been considered inadequate or limited because of the large diffusive resistance of skin and inadequate regulatory mech anisms (Feder & Burggren, 1985). Despite their reliance upon cutaneous respiration, the animals showed a considerable ability to increase during activity. The factorial aerobic scope (maximum standard was 9·66 in experiment 1 and 11·7 for the cold-acclimated animals in experiment 2. The maximum of the 21° AT animals in experiment 3 was 17 ¼molg⁻¹ h⁻¹. This corresponds to an O₂ flux of 2· 5 μmolcm⁻²h⁻¹ across the 13·6 cm² of skin surface (Whitford & Hutchison, 1967) of a 2-g salamander; fluxes are even greater at warmer body temperatures (Feder, 1977; Hillman et al. 1979). An average 10 kg mammal, with a pulmonary surface area of 2·96×10⁵cm² (Gehr et al. 1981) and a maximum oxygen consumption of 1·88X 10⁶μmol h⁻¹ (Taylor et al. 1981), has an O₂ flux of 6·3 μmol cm⁻²h⁻¹. Thus, despite a vastly ‘improved’ cardiovascular system, presence of a ventilatory system, and a thinner diffusion barrier (Feder & Burggren, 1985), each cm² of lung surface area in an average mammal sustains little more than twice the O2 flux of a similar area of salamander skin.

A major purpose of this study was to determine whether acclimatory changes in resting were reflected in the during activity, and in turn whether acclimatory changes in the during activity were reflected in locomotor performance (e.g. stamina, maximum sustainable speed). Although acclimatory changes in resting ⁠, during activity and locomotor performance all occurred in Desmognathus, these changes individually bore little resemblance to one another. Thus, while acclimation to warm temperatures improved locomotor stamina at warm temperatures in Desmognathus, this improvement was not mediated by acclimatory changes in aerobic metabolism. These findings parallel the conclusions of Feder (1978), Carey (1979) and Miller & Zoghby (1983), who likewise reported a lack of concordance among acclimatory patterns in resting ⁠, during activity and stamina.

Several major discrepancies between aerobic metabolism and performance appear in the data. Acclimation to 21 °C reduced the routine and maximum of salamanders walking at 13°C; the standard is reduced as well in similar circumstances (Fitzpatrick, Bristol & Stokes, 1971; Feder et al. 1984). These changes presumably should have reduced the maximum sustainable speed of these animals. However, 21° AT animals and those acclimated to cooler temperatures did not differ in maximum sustainable speed. In contrast to this pattern of acclimation of without acclimation of the maximum sustainable speed, salamanders measured at 21 °C showed acclimation of locomotor stamina without acclimation of At this temperature, warm acclimation improved stamina but did not affect during activity. Significant acclimation of the standard occurs at this temperature (Fitzpatrick et al. 1971; Feder et al. 1984; Feder, 1985), which was not reflected in the during activity.

One pronounced effect of thermal acclimation in the present study was a reduction in the rate of lactate accumulation during sustained activity at 21 °C. The increased reliance upon anaerobiosis in the 5°C-acclimated animals is consistent with the reduced stamina in this group, both of which are seemingly independent of ⁠. Indeed, several recent studies suggest that changes in neuromuscular phenomena and anaerobic metabolism rather than in aerobic metabolism underlie acclimatory changes in locomotor performance. For example, changes in myofibrillar ATPase, muscle volume and the speed at which white muscle was recruited were all associated with acclimatory differences in sustainable activity in carp (Rome, Loughna & Goldspink, 1985); carp not acclimated to the cool experimental temperature accumulated lactate more readily than acclimated animals. Also, frogs acclimated to cool temperatures fail to jump well at warm temperatures, apparently because of failures in neural control of jumping (Renaud & Stevens, 1983, 1984; Rome, 1983; Hirano & Rome, 1984; Rome et al. 1985). Prosser and his colleagues (e.g. Prosser & Nelson, 1981) have long maintained that improvements in neuromuscular control (rather than in aerobic metabolism) underlie the improved locomotor performance that may result from thermal acclimation.

The present study does not compellingly demonstrate that acclimatory changes in aerobic metabolism in any way potentiate changes in locomotor performance in Desmognathus. Lack of acclimation of locomotor performance has also been reported for both anurans and urodeles that undergo thermal acclimation of metabolism (Putnam & Bennett, 1981; P. Else & A. Bennett, personal communication). A parallel study of salamanders suggests that acclimation of aerobic metabolism provides at best a trivial saving in long-term energy expenditure (Feder, 1985). Thus, at least in salamanders, the ‘adaptive significance’ of thermal acclimation remains to be demonstrated.

I thank Catherina Perkins and John Troyer for technical assistance, John Gilpin for constructing the wheel respirometers, Lynne Houck for supply of experimental animals, and Albert Bennett, Robert Full, Robert Gatten and Johannes Piiper for their constructive criticism of the manuscript. Norbert Heisler suggested the comparison of oxygen flux across salamander skin and mammalian lungs. Research was supported by NSF Grants BSR83-07089 and PCM84-16121, NIH Biomedical Research Support Grant RR-05367-22 and the Andrew Mellon Foundation.