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The resting and active metabolism of red-spotted newts (Notophthalmus viridescens) on land at simulated winter and summer temperatures
Camp. Biwhem. Physioi. Vol. WA, No. 4, pp. 80>81 I, 1993
Printed in
030@9629/93 S6.00 + 0.00 Pcrpmon Press Ltd
Great Britain
THE RESTING AND ACTIVE METABOLISM OF RED-SPOTTED NEWTS (NOTOPHTHALMWS VIRIDESCEALS) ON LAND AT SIMULATED WINTER AND SUMMER TEMPERATURES SUPING JIANG*
and DENNIS L. CLAUSSEN
Department of Zoology, Miami University, Oxford, OH 45056, U.S.A. (Received 8 June 1992; accepted 8 July 1992) Abstract-l. By measuring resting and active oxygen uptake at various locomotor speeds, the aerobic metabolic rates of the newts were determined. 2. Oxygen uptake of the newts slightly increased with locomotor speed in winter, but no significant difference was found between 1 and 5°C. 3. Oxygen uptake at 25°C was significantly higher than that at 1 or 5°C. 4. Transport costs for the newts decreased as locomotion speed increased. 5. The metabolic rates were incorporated into a total energy budget which indicated that the newts could only survive about 30 days on land if they maintained a low level of activity in cold winter. 6. The newts thus do not appear to be capable of remaining active and surviving winter on land without ingesting food. 7. Lactate contents after resting and locomotion were measured to determine the anaerobic metabolism of the newts. 8. Lactate accumulation and formation after locomotion indicated that most of the energy for supporting the movement of newts (about 90% in winter and 75% in summer) was derived from anaerobiosis. 9. The production of lactate by the newts after locomotion was independent of seasonal changes, but the quantity of formed lactic acid increased with increased locomotor speeds.
INTRODUCTION
Previous studies (Jiang and Claussen, 1992) have shown that red-spotted newts (~o~~p~~~al~~ viridescem) from southern Ohio maintain low levels of both resting and active aerobic metabolism in water at winter temperatures. These animals can apparently store sufficient energy prior to the onset of winter to meet the modest energy demands of their winter activity. However, this conclusion was based entirely upon m~surements of the aerobic metabolism of aquatic newts. In fact, populations of newts in some regions [e.g. in Canada (Logier, 1952), Virginia (Gill, 1978) and part of New York (Hurlbert, 1969)] migrate onto land for hibernation. Although several studies have considered the terrestrial metabolism of newts (e.g. Verrell, 1985; Stefanski et al., 1989), no research has focused on energy expenditure of this animal on land at temperatures near 0°C. Accumulation of lactic acid is proportional to the degree of anaerobic metabolism. During intense activity, animals experience hypoxia and much of the energy needed for locomotor function is provided by glycolysis. Consequently, lactate as the end product
*Present Address: Department of Biological Sciences, Rieveschl Hall (ML 006), University of Cincinnati, Cincinnati, OH 45221, U.S.A. CBFA W,4-M
of glycolysis, accumulates in body tissues. Thus, the measurement of lactate content in tissues has long been used as a measure of anaerobic metabolism (Ciatten, 1985). A number of studies have correlated lactate accumulation with animal activity in various amphibians, including plethodontids, hylids and bufonids (Feder, 1986; Stefanski et al., 1989; Full, 1986; Bennett and Licht, 1973; Taigen and Pough, 1981). The association of anaerobic metabolism with the critical thermal maxima in ~ot5p~t~uZ~~ viridescerts was also studied by Burke and Pough (1976). However, there have apparently been no additional studies on anaerobic metabolism in N. viridescens at different levels of activity and temperature, The energy for supporting the activity of newts throughout the winter may be largely provided by aerobic metabolism (Jiang and Claussen, 1992). but it is still unknown whether or not anaerobiosis plays a major supporting role in supplying energy for their winter activity. It would seem to be generally unnecessary for red-spotted newts to require burst activity to escape predators and catch prey during winters, since the predators and prey are mostly in hibernation. Newts in winter might thus have less need for anaerobic metabolism to support their movements. Previous studies on lactate production and accumulation in amphibians were mostly carried out
805
806
SUPING
JIANGand DENNISL. CLAU~~EN
around 20°C. The scope of anaerobic metabolism in amphibians during locomotion at both winter and summer temperatures has not yet been reported. The lack of simultaneous measurements of lactate production in cold and warm seasons in amphibians has prevented further assessment of the energy supply from anaerobic metabolism. So, it is worth knowing to what degree the newt is capable of utilizing glycolysis as an energy source during activity in winter. In this study, therefore, using the population of red-spotted newts in southern Ohio as a model, we measured oxygen consumption at different exercise speeds and temperatures and calculated the energy expenditure assuming that this animal could also remain active on land during winter. In addition, the whole bodies of red-spotted newts were used for determining lactate accumulation after the terrestrial locomotion in the respirometer chamber and the contributions of energy from both anaerobic and aerobic metabolism were estimated. The study was designed to help us understand better the importance of terrestrial aerobic and anaerobic metabolism in different seasons and the energy demand of the red-spotted newts.
MATERIALSAND
METHODS
Newts (N. viridescens) were seined from a pond in Adams County, Ohio in August and November, 1990. All animals were kept in aquaria and acclimated in 5 and 25°C environmental rooms on an LD 14: 10 photoperiod for at least 2 weeks before experiments. The newts were fed with blood worms once weekly (at 25°C) or once every 2 weeks (at 5°C). The newts acclimated at 5°C had an average body mass around 3.6 g, whereas those at 25°C averaged 2.1 g, since these animals rapidly lost weight during the first 3 weeks of acclimation. A custom-designed magnetic rotating respirometer (Jiang and Claussen, 1992) was applied to exercise the newts terrestrially at different speeds and temperatures. The newts were exercised at 0 cm/min for 20 min at 1 and 5°C and for 15 min at 25°C. The newts assayed at the speed of 30cm/min were exercised for 10min at all testing temperatures. At the speed of 60 cm/min, the exercise duration was 5 min for the groups at 1 and 25”C, and 7min at 5°C. Finally, the newts were exercised for 5 min at a speed of 90 cm/min for all testing temperatures. The newts were kept in the respiratory chamber for 30 min prior to testing. Then, a 501~ air sample was removed by means of a syringe after flushing several times from the respiratory chamber which, afterward, was immediately closed. This was designated as the sample before exercise. Another 50 cc air sample was removed from the metabolic chamber after exercise by two 50 cc syringes, one of which was initially full of 5Occ air. Both syringes were flushed alternately
several times to mix the air inside the chamber. Then, the sample was removed as the after exercise sample for oxygen analysis. The air samples before and after exercise were injected into a dual channel oxygen analyser (Ametek S-3A/Ii) through two columns of Drierite and Ascarite (to remove water and carbon dioxide, respectively) with a syringe pump (Model 351, Sage Instruments). The differences of oxygen content (A02%), read from the analyser, were converted and standardized to metabolic rates (~1 O,/g/hr) by the following equation: I’O,(STPD) =
(AO,%)x
Vs x(P-r)x2.16x
lo4 (1)
TxtxM
Where: AO,% = difference of oxygen content between the air samples before and after the exercise from the readout of the oxygen analyser; r = vapor pressure; Vs = the volume of the respiratory chamber (325ml or 385ml) + 50ml of the syringes; T = absolute experimental temperature (To); P = atmospheric pressure (mm Hg); t = exercise duration (min) and M = body mass (g). The anaerobic metabolism of the newts was obtained by measuring the accumulated lactic acid after locomotion. Each newt was weighed before initiation of the experiment. After the measurement of terrestrial oxygen consumption at different velocities including rest and at simulated winter and summer temperatures, newts were immediately frozen in liquid nitrogen. The whole frozen carcass was cut into small pieces which were placed in a large test tube. Approximately 10 ml of chilled distilled water was added to the tube and the contents were homogenized in an ice-bath. The homogenate was then poured into a 25 ml volumetric flask. Chilled distilled water was added to dilute to 25 ml and the contents were mixed thoroughly by inverting the flask several times. Two ml of the homogenate were pipetted into a 10 ml test tube containing 4ml chilled 8% perchloric acid. Afterward, the test tube was shaken vigorously to extract lactate from the homogenized tissue until all large particles descended to the bottom. The shaken solution was then centrifuged at 5000 rpm for 10 min at 3°C and the supernatant was used for lactate determination with an enzymatic kit (826~UV; Sigma, St Louis, MO). A spectrophotometer (Spectronic 601, Milton Roy Company) was used for the determination of absorbance. The data on oxygen consumption were analysed by SAS program (SAS, 1985). Microsoft StatView SE was employed to test for significant differences among the data using P = 0.05 as the fiducial limit upon Anova. Two factor factorial Anova was used to test for significant differences among all groups at different experimental temperatures and speeds. Then, Scheffe’s F-test was used for separation of the means. All data are expressed as mean & SD. A Student t-test was used to compare the significance between the aquatic and terrestrial metabolic rates.
Metabolism of red spotted newts Table 1. Metabolic rates (mean k SD) of red-spotted newts (Noroph~halmus uiridescens) at three experimental temperatures and four exercise speeds in air. Metabolic
Speed 0 cm/min 30 cm/min 60 cm/min 90 cm/min 0,”
rate (~1 O,/g/hr)
1°C
5°C
38.72 + 9.75 69.51 f 22.17 79.50 + 17.22
38.38 f 13.48 67.21 &-20.91 18.32 rf: 23.12 93.93 + 21.89 0.95
25°C 138.44 275.25 330.87 376.71
+ + f f
30.23’ 87.24. 47.21’ 134*
1.90
Mean Q,,,s were computed from
the mean metabolic rates at two different acclimation temperatures and test temperatures respectively (5°C vs 1°C; 1 and 5°C YS 25°C). Oxygen uptakes of newts in water at each speed and temperature were significantly less than in air (P < 0.05). The metabolic rates of newts at 25°C were significantly higher than those at 5°C (P c 0.05) and indicated by an asterisk.
RESULTS
No significant differences were found among the terrestrial metabolic rates at 1°C and 5°C (P > 0.05) when the newts were tested at either locomotor speed (Table 1). However, all metabolic rates of the newts at the simulated summer temperature of 25°C were significantly higher than the corresponding rates at 1 and 5°C (P < 0.05). The resting metabolic rates of the newts at 25°C were at least three-fold higher than those at the simulated winter temperatures, whereas their active metabolic rates at the summer temperature were about four-fold greater (Table 1). Also, terrestrial metabolism slightly increased with locomotor speed at both 1 and 5°C (Fig. l), but the differences in oxygen uptake among these speeds were not significant at either simulated winter temperature (P > 0.05) (Table 1). This implies that the energy consumed by the newts during winters would not be markedly raised by modest activity. When the animals were tested at 25”C, their resting metabolism in air was significantly lower than their active metabolism (F values above 3.356, P c 0.05), but the newts
Locomotor
807
also increased their oxygen uptakes linearly as their exercise speeds increased, with a slope of 4.28 @l/g/m) (Table 1 and Fig. 1). The newts acclimated at 5°C displayed a pattern similar to that for the terrestrial metabolism at 25°C. Their terrestrial metabolic rates elevated linearly as locomotor speeds increased, but the slopes @l/g/m) for the metabolism in air were all around 1, i.e. 0.921 for the metabolic rates at 1°C and 1.025 for that at 5°C. No plateau was reached within the testing velocities used in our study (Fig. 1). All animals tested at 1 and 5°C could not locomote normally above 90 cm/min when they were touched and stimulated by the turning stirrer in air. There is an apparent separation among Q,, values from the metabolic rate data (Table 1). When the active metabolic rates at the simulated winter temperatures (1 and 5°C) and all exercise speeds were used for a Q,, calculation, the values averaged 0.95 for the newts exercised in air. These values are low and suggest that there is no remarkable change in active metabolic rates when the newts are tested at 5°C as opposed to 1°C even though their exercise. speeds varied from resting to 90 cm/min. On the other hand, the Q,, values averaged 1.9 for the terrestrial metabolism when the active metabolic rates of the newts from the 5°C acclimation and those of the newts from 25°C acclimation were compared. This indicates that the newts acclimated and tested at summer temperatures more than triple their metabolic rates compared to animals at winter temperatures. In general, the newts tended to decrease their cost of transport as their exercise speed increased (Fig. 2 and Table 2). The newts tested at 1 and 5°C only slightly showed this trend. Their costs of transport did not change very much as their exercise speeds
speed
(cmlmln)
Fig. 1. The relationships between the metabolic rates of the newts and their locomotor speeds in air at the simulated winter and summer temperatures. The newts acclimated at 5°C were tested at 1 and 5°C but the ones acclimated at 25°C were only tested at 25°C. The squares, triangles and circles represent the tests at 1, 5 and 25°C respectively. Standard deviations (SD) of the means are shown for the 5 and 25°C symbols at each assay speed. The bars of SD have been omitted from 1°C symbols to avoid excessive overlap. The mean oxygen uptake at 25°C was significantly greater than that at 1 and 5°C (P < 0.05) when newts were tested at each speed.
SUPINGJIANGand DENNISL. CLAUSEN
B
20
I \
i:~~~
T
o--. . 10
I
20
.
.
,
30
.
.
I.
40
.
I
.
50
Locomotor
.
I
.
60
.
I
.
70
.
I
.
80
.,
_
.
90
.
I.
I
.
100
110
speed (cm/mitt)
Fig. 2, The cost of transport as a function of speed when the newts locomoted in air. The newts acclimated at 5°C were tested at 1 and 5°C and the ones acclimated at 25 were only tested at 25’C. The faster newts locomoted, the lower transport cost they had. Newts moving at 25°C had greater cost than at 1 and 5°C.
were varied. The costs of walking at these two assay temperatures were at 3.86 and 3.74ml O,/g/km for the speed 30 cm/mm and 2.21 and 2.18 ml Q/g/km for the speed 60cm/min. However, their walking costs steeply decreased from 15.29 ml O,/g/km at 30cm/min to 9.19 and 6.98 ml O,/g/km at 60 and 90 cm/min respectively as these animals locomoted at 25°C. Based on the metabolic rates we measured, the energy (calories) needed by a newt per day at both the simulated summer (25°C) and winter (1 and 5°C) temperatures when locomoting at different speeds can be compared with the reserved energy released by the stored fat. Based on these data, we calculated the survival period which could be supported by 85 mg fat (average reserved fat of the red-spotted newts; Jiang and Claussen, 1992), and we obtained estimated durations ranging from 8 to 59 days, dependent upon temperature and locomotor velocity (Table 2). The lactate accumulation of the newts after exercise displayed a complex picture since their locomotor times were different. But, a general trend was evident (Fig. 3 and Table 3); the whole body content of lactic Table 2. Estimated survival durationsand
acid increased as locomotor speeds and exercise time increased. As expected, the newts had a tow level of lactate accumulation when they were allowed to rest or randomly move about in the respiratory chamber at any testing temperatures, even though the testing duration was much longer. Once they were stimulated to move forward, their lactate content greatly increased. At the speed of 30 cm/min, the newts were all assayed for 10 min so that a comp~son among three groups of experimental temperatures could be made. There were no statistically significant differences among them. Likewise, at the speed of 90cm/min, there was also no significant variation between the newts exercised at 5 and 25°C (Fe,,, =0.544, P > 0.05). When exercised at 60 cm/mm, newts tested at 5 and 25°C accumulated similar amounts of lactate despite the fact that the group at 5°C had 3 more min of movement. Lactate formation by the newts evidently increased with the exercise speed, but did not vary strikingly with temperature (Table 3; Fig. 3). When tested at l”C, newts produced a very small amount of lactate at resting status. The formed lactate level was much transport costs in air for red-spotted newts
(Norophthulmus viridescens) under different activity levels and temperatures without
ingesting food Speed (cm/min) and tampcrature 0 @ 1°C
30 60 0 30 60 90 0 30 60 90
@ 1°C @ 1°C @ 5°C @ 5°C @ 5°C @ 5°C @ 2S”C GI 25°C @ 25°C @ 25°C
Body mass (g) 3.40 3.4 3.31 3.82 4.05 3.61 3.60 3.23 3.09 2.83 2.66
0, Uptake for a newt Wday) 3.18 5.66 6.40 3.55 6.46 6.82 8.13 10.60 20.14 22.42 23.19
Survival Transport cost Wglhm)
3.86 2.21 3.74 2.18 1.74 15.29 9.19 6.98
length (days) 59 33 28 ;: 27 23 17 9 8 8
The survival times were computed assuming that 171.6 ml Or would be consumed for the complete oxidation of 85.8 mg of reserve fat.
Metabolism of red spotted newts
0
60
40
20
Locomotor
speed
809
100
60
(cm/mln)
Fig. 3. Accumulation of lactate with locomotor speeds in Norophthalmus oiridescens, showing little impact of temperature on lactate formation (P c 0.05). The newts acclimated at 5°C were tested at 1 and 5°C and the ones acclimated at 25°C were tested only at 25°C. The squares, circles and triangles represented the tests at 1, 5 and 25°C respectively. Standard deviations (SD) of the means are shown for the symbols of 5 and 25°C tests at each assay speed. T’he bars of SD have been omitted from 1°C tests to avoid excessive overlap.
higher as the newts locomoted, but there was no significant difference between the data at 30 cm/min and the resting. The newts exercised at 60 cm/min had a significantly higher lactate formation than did the resting animals (F,,, = 2.505, P < 0.05). Similarly, at SC, newts walking at speeds of 60 cm/min and above produced significantly more lactate than did those moving below 30cm/min (F values above 2.025, P c 0.05). The data from 25°C experiments were somewhat different. Only the lactate formation of newts exercised at 90 cm/mm was significantly higher than that of those walking at other speeds (F(,,,, = 5.136, P < 0.05). The lactate produced by newts at 30 and 60 cm/min was very high compared to that of resting animals, but the differences were not statistically significant. No significant difference was found when comparing the lactate formation at different assay temperatures at a given speed. DISCUSSION
Generally, terrestrial animals tend to consume more energy for their locomotion on land than do
those swimming in water (Hainsworth, 1981). Our results are consistent with this trend. Compared to the aquatic metabolism at each assay temperature and locomotor speed (Jiang and Claussen, 1992), the terrestrial metabolism was significantly higher (all t values below 2.116, P < 0.05). Red-spotted newts locomoting on land consumed much more oxygen than when they swam at an equivalent speed in water either in winter or summer. At 1 and 5°C the terrestrial metabolic rates averaged about 30% higher than the equivalent aquatic metabolic rates, and newts walking in the respiratory chamber at 25°C consumed about twice the oxygen of those swimming in water. The transport costs for aquatic and terrestrial locomotion are thus very different. Red-spotted newts may reserve at least 85 mg fat prior to winter (Jiang and Claussen, 1992). This amount of fat could support resting newts for around 80 days in water at simulated winter temperatures. In contrast, this amount of stored fat could support the newts for less than 60 days if they remained resting on land during winter (Table 2). If they locomoted on land at a very low speed (30 cm/min) in winter, they
Table 3. Lactate formation and accumulation by red-spotted newts (Norophlholmus viridescens~ after terrestrial exercise at simulated winter (1 and 4°C) and summer (25°C) temperatures ~ -’
Speed (cm/min) and temperate
Exercise length (min)
Sample size 0’)
20 IO
7 7
5 20 10 7 5 15 10 IO 5
6
0 @ 1°C 30 @ 1°C 606 1°C 0 @ 5°C 30 @ 5°C 60 @ 5°C 90 @ 5°C 0 @ 25°C 30 @ 25°C 60 @ 25°C 90 @ 25°C The data are represented
as the means +
Lactate accumulation
Lactate formation
(Wg) 1.11 I .75 I .33 1.15 1.30 2.31 1.87 1.47 1.89 2.18 2.19
I SD.
50.26 * 0.34 * 0.33 it 0.29 f 0.14 + 0.17 f 0.28 f 0.27 + 0.29 f 0.63 ; 0.32
55.51 174.77 261.66 57.41 130.37 330.60 374.17 97.71 188.64 221.84 444.21
f f f f k + f + f f +
13.08 34.15 72.89 14.40 13.58 23.78 52.91 18.07 29.44 65.37 69.62
SUPING JIANGand DENNIS L. CLAUSSEN
810
could survive only about 30 days by consuming the reserved energy. Evidently, red-spotted newts may not actively overwinter on land without eating since they could not reserve sufficient energy to support winter terrestrial activity. Therefore, the populations of the red-spotted newts hibernating on land probably remain inactive during winters. Red-spotted newts (N. viridescens), like other amphibians, rely partially on anaerobic metabolism for their energy supply during locomotion. This salamander produces limited amount of lactic acid from anaerobic metabolism during exercise. After 5-10 min of exercise at the speeds of 60 or 90 cm/min, the testing newts fatigued and hesitated to move forward. At this point, their whole body lactate ranged approximately from 1.3 to 2.3 mg per gram body mass (Table 3). This amount of lactate might represent their maximum anaerobic capacity, since the newts were not able to locomote further while fatigued. The percentage contributions of aerobic and anaerobic energy sources can be estimated from consumed oxygen and formed lactate by the following formulas (Bennett and Licht, 1973): ATP 1.Omg lactate formed = 0.0167 mmol (adenosine triphosphate). 1.0 ml O2 (STPD) consumed = 0.29 mmol ATP. The converted ATP data from aerobic and anaerobic metabolism of the red-spotted newts reveal that anaerobiosis is the major energy supply for activity (Table 4). In the winter, it appears that at least 83% of the total energy utilized by N. viridescens is anaerobically derived. The percentage of the energy contributed by anaerobic metabolism rises as the locomotor speeds of the newts increase, up to 94%. In the summer, however, anaerobiosis appears to account for only 70% of the energy needed for the activity of the newts. This percentage may reach 80% if the newts locomote at a speed of 90 cm/min. These observation are consistent with the results of Bennett and Licht (1973) on lizards (Anolis carolinensis and Dipsosaurus dorsalis), which further indicated that anaerobic metabolism constitutes the ideal solution Table
4. The
energy
contributions
of aerobic
to the energetic problems posed by the activity of small ectothetms, particularly in winters. Aerobic metabolism involves a tedious series of biochemical reactions, such as Kreb’s cycle and the electron transport pathway, and the complexity of oxygen transport, all of which function sluggishly at low winter temperatures. It would be almost impossible for an aerobic system to be mobilized rapidly when the newts are active in winters. Anaerobiosis, therefore, is an important auxillary system of energy generation which enables the red-spotted newts to remain active throughout the winter. Compared to the results of Burke and Pough (1976) on the same species, we obtained much higher values of lactate accumulation. This is probably due to the different approaches of these two studies. Burke and Pough (1976) measured the accumulation of lactic acid after the critical thermal maximum (CTM) of newts. The lactate produced by the newts after their CTM reflects the degree of struggling to escape from the stress instead of moving in their normal environments. The newts under the CTM test might not reach fatigue before the appearance of the loss of righting response (LRR). At the point of LRR, which is above 35”C, their enzyme system might be unable to continue lactate formation at a high rate. Thus, the production of lactate could perhaps not reach a maximum during a CTM test. The newts in the present study were required to locomote inside the respiratory chamber under natural conditions until they were fatigued. The accumulation of lactate by these exercising newts may represent the true anaerobic capacity of these amphibians. Also, red-spotted newts appear to have a greater anaerobic metabolic scope than do other species of amphibians. In other words, they are able to accumulate and tolerate a very high level of lactate in their body during locomotion. In contrast, a lungless salamander (Batrachoseps attenuarus) accumulates only about 1.4mg lactate per gram body mass after two minutes electrical or manual stimulation (Bennett and Licht, 1973). The lactate production and
anaerobic
red-sootted Locomotor
Converted
ATP
metabolisms
for
the activity
Energy
(a M/g/hr)
contributed
speed Aerobiosis
(cm/min)
Anaerobiosis
Aerobiosis in air
in air
8.15
II.23
55.62
83
30 @I 1°C
12.86
20.16
175.12
90
60 @
1°C
15.75
23.06
262. I8
92
0 @ 5°C
7.95
II.13
57.52
84
19.49
130.63
87
in water
temperate
0 @
II.46
30 @ 5°C
W)
60 @ 5°C
15.27
22.71
33 I .03
94
90 @ 5°C
18.95
21.24
374.92
93
0 @ 25°C
28.50
40.15
97.91
71
30 @ 25°C
46.55
79.82
189.02
70
60 @ 25°C
46.09
95.95
222.28
70
90 @ 25°C
46.03
109.25
445.10
80
The
ATP
(admosine
triphosphate)
is converted
the tested newts based on the formula contributed
by anaerobiosis
from
of Bennett
represents
the consumed
and Licht
the estimate
oxygen
(1972).
of energy
by
anaerobiosis
1°C
and
of the
newts
and formed
The percentage
allocation
in air.
lactate
by
of total energy
811
Metabolism of red spotted newts
of both Desmognathus ochrophaeus and Plethodon is below 1 mg/g body mass after more than 10 min exercise at either low or high speeds (Full, 1986; Stefanski et al., 1989; Feder, 1986). The lactate accumulation of anurans is also markedly lower than that of the newts. Bufo boreas sustains very little anaerobiosis since it accumulated only 0.23 mg lactate/g during exercise, whereas Hyla regilla produced 0.61 mg lactate/g (Bennett and Licht, 1973). Generally, slow-moving ectotherms such as toads have low rates of lactate production; in contrast, amphibians engaging in rapid bursts of activity, such as frogs, have high anaerobic metabolism (Seymour, 1973; Bennett and Licht, 1973, 1974). Therefore, redspotted newts may be adapted for occasional fast swimming in ponds. Their high anaerobic metabolism might have evolved to support this type of burst activity. The notable finding in this study is that the formation of lactate in red-spotted newts is essentially independent of acclimation and assay temperatures. The rates of anaerobic metabolism in the newts increased with the exercising speeds, but remained nearly consistent with changing temperatures and acclimations. Therefore temperature does not seem to impact the anaerobic metabolism of red-spotted newts. They must partially depend on anaerobic metabolism for energy supply while active either in winter or in summer. This suggests that the newts need a constant anaerobic scope to support their activity in all seasons. During warm weather, this animal like most ectotherms has a relatively high body temperature and fast metabolic rate. Since most biochemical reactions are strongly temperature dependent, energy can be rapidly supplied through anaerobiosis. Thus, it is reasonable for the newts to have a high anaerobic metabolism in summer. However, if the newts maintained the same physiological mechanisms in winter, they might not have sufficient energy from anaerobiosis for their locomotion, because of the adverse effect of those low winter temperatures on biochemical reactions. Obviously, the newts must have a compensating mechanism in winter in order to maintain a constant anaerobic scope. This raises the question of how lactate can be formed in the red-spotted newts during winter to a concentration similar to that of summer. Possibly the newts have evolved a mechanism to regulate the functioning of enzymes, such as lactate dehydrogenase, which could compensate for the slow rates of biochemical reactions at low temperatures. In conclusion, red-spotted newts on land linearly increased their aerobic metabolic rates with locomotor speed and ambient temperature. The terrestrial transport costs of the newts also increased with testing temperatures but declined with increased locomotor speed. The newts could not maintain their activity throughout cold winter because their terrestrial metabolism is much greater than their aquatic metabolism. Anaerobic metabolism in red-spotted
jordani
newts plays an equally important role in all seasons. The energy from anaerobiosis for supporting the movements of newts remains steady no matter at what temperatures the newts locomote. Therefore, the production of lactate by the newts after locomotion is independent of seasonal changes, but the quantity of lactic acid formed increases with an increase in locomotor speeds. Acknowledgements-This study was supported by the Theodore Roosevelt Memorial Fund of the American Museum of Natural History (S.J.) and the Department of Zoology at Miami University. We are grateful to Pete Zani and Jiazhu Zhang for their help in our field trips. Thanks also go to S. I. Guttman and R. R. Nielson for providing other equipment.
REFERENCES
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271-283. Full R. J. (1986) Locomotion without lungs: energetics and performance of a lungless salamander. Am. J. Physiol. 251, R775-780.
Gatten R. E. Jr (1985) The uses of anaerobiosis by amphibians and reptiles. Am. Zool. 25, 945-954. Gill D. E. (1978) The metapopulation ecology of the redspotted newt, Notophthalmus viridescens (Rafinesque). Ecol. Monogr. 48, 145-166. Hainsworth F. R. (1981) Animal Physiology: Adaptafions in Function. Addison-Wesley Publishing Company. Hurlbert S. H. (1969) The breeding migrations and interhabitat wandering of the vermilion-spotted newt Notophthalmus viridescens (Rafinesque). Ecol. Monogr. 39, 465488.
Jiang S. and Claussen D. L. (1992) A bionergetic budget for overwintering newts (Norophthalmus viridescens) from southern Ohio: their fat reserves and aerobic metabolic rates in water. Comp. Biochem. Physiol. 101A, 743-750. Logier E. B. S. (1952) The Frogs, Toads and Salamanders of Eastern Canada. Clarke, Irwin and Co., Toronto, Canada. Seymour R. S. (1973) Physiological correlates of forced activity and burrowing in the spadefoot toad, Schaphiopus hammondii. Copeia 1973, 103-I 15.
Stefanski M., Gatten R. E. Jr and Pough F. H. (1989) Activity metabolism of salamanders: tolerance to dehydration. J. Herpetol. 23, 45-50. Taigen T. L. and Pough F. H. (1981) Activity metabolism of the toad (Bufo americanus): ecological consequences of ontogenetic change. J. camp. Physiol. 144, 247-252. Verrell P. A. (1985) Is there an energetic cost to sex? Activity, courtship mode and breathing in the red-spotted newt, Notophthalmus viridescens (Rafinesque). Monitore Zool. Dal (N.S.).
19, 121-127.
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030@9629/93 S6.00 + 0.00 Pcrpmon Press Ltd
Great Britain
THE RESTING AND ACTIVE METABOLISM OF RED-SPOTTED NEWTS (NOTOPHTHALMWS VIRIDESCEALS) ON LAND AT SIMULATED WINTER AND SUMMER TEMPERATURES SUPING JIANG*
and DENNIS L. CLAUSSEN
Department of Zoology, Miami University, Oxford, OH 45056, U.S.A. (Received 8 June 1992; accepted 8 July 1992) Abstract-l. By measuring resting and active oxygen uptake at various locomotor speeds, the aerobic metabolic rates of the newts were determined. 2. Oxygen uptake of the newts slightly increased with locomotor speed in winter, but no significant difference was found between 1 and 5°C. 3. Oxygen uptake at 25°C was significantly higher than that at 1 or 5°C. 4. Transport costs for the newts decreased as locomotion speed increased. 5. The metabolic rates were incorporated into a total energy budget which indicated that the newts could only survive about 30 days on land if they maintained a low level of activity in cold winter. 6. The newts thus do not appear to be capable of remaining active and surviving winter on land without ingesting food. 7. Lactate contents after resting and locomotion were measured to determine the anaerobic metabolism of the newts. 8. Lactate accumulation and formation after locomotion indicated that most of the energy for supporting the movement of newts (about 90% in winter and 75% in summer) was derived from anaerobiosis. 9. The production of lactate by the newts after locomotion was independent of seasonal changes, but the quantity of formed lactic acid increased with increased locomotor speeds.
INTRODUCTION
Previous studies (Jiang and Claussen, 1992) have shown that red-spotted newts (~o~~p~~~al~~ viridescem) from southern Ohio maintain low levels of both resting and active aerobic metabolism in water at winter temperatures. These animals can apparently store sufficient energy prior to the onset of winter to meet the modest energy demands of their winter activity. However, this conclusion was based entirely upon m~surements of the aerobic metabolism of aquatic newts. In fact, populations of newts in some regions [e.g. in Canada (Logier, 1952), Virginia (Gill, 1978) and part of New York (Hurlbert, 1969)] migrate onto land for hibernation. Although several studies have considered the terrestrial metabolism of newts (e.g. Verrell, 1985; Stefanski et al., 1989), no research has focused on energy expenditure of this animal on land at temperatures near 0°C. Accumulation of lactic acid is proportional to the degree of anaerobic metabolism. During intense activity, animals experience hypoxia and much of the energy needed for locomotor function is provided by glycolysis. Consequently, lactate as the end product
*Present Address: Department of Biological Sciences, Rieveschl Hall (ML 006), University of Cincinnati, Cincinnati, OH 45221, U.S.A. CBFA W,4-M
of glycolysis, accumulates in body tissues. Thus, the measurement of lactate content in tissues has long been used as a measure of anaerobic metabolism (Ciatten, 1985). A number of studies have correlated lactate accumulation with animal activity in various amphibians, including plethodontids, hylids and bufonids (Feder, 1986; Stefanski et al., 1989; Full, 1986; Bennett and Licht, 1973; Taigen and Pough, 1981). The association of anaerobic metabolism with the critical thermal maxima in ~ot5p~t~uZ~~ viridescerts was also studied by Burke and Pough (1976). However, there have apparently been no additional studies on anaerobic metabolism in N. viridescens at different levels of activity and temperature, The energy for supporting the activity of newts throughout the winter may be largely provided by aerobic metabolism (Jiang and Claussen, 1992). but it is still unknown whether or not anaerobiosis plays a major supporting role in supplying energy for their winter activity. It would seem to be generally unnecessary for red-spotted newts to require burst activity to escape predators and catch prey during winters, since the predators and prey are mostly in hibernation. Newts in winter might thus have less need for anaerobic metabolism to support their movements. Previous studies on lactate production and accumulation in amphibians were mostly carried out
805
806
SUPING
JIANGand DENNISL. CLAU~~EN
around 20°C. The scope of anaerobic metabolism in amphibians during locomotion at both winter and summer temperatures has not yet been reported. The lack of simultaneous measurements of lactate production in cold and warm seasons in amphibians has prevented further assessment of the energy supply from anaerobic metabolism. So, it is worth knowing to what degree the newt is capable of utilizing glycolysis as an energy source during activity in winter. In this study, therefore, using the population of red-spotted newts in southern Ohio as a model, we measured oxygen consumption at different exercise speeds and temperatures and calculated the energy expenditure assuming that this animal could also remain active on land during winter. In addition, the whole bodies of red-spotted newts were used for determining lactate accumulation after the terrestrial locomotion in the respirometer chamber and the contributions of energy from both anaerobic and aerobic metabolism were estimated. The study was designed to help us understand better the importance of terrestrial aerobic and anaerobic metabolism in different seasons and the energy demand of the red-spotted newts.
MATERIALSAND
METHODS
Newts (N. viridescens) were seined from a pond in Adams County, Ohio in August and November, 1990. All animals were kept in aquaria and acclimated in 5 and 25°C environmental rooms on an LD 14: 10 photoperiod for at least 2 weeks before experiments. The newts were fed with blood worms once weekly (at 25°C) or once every 2 weeks (at 5°C). The newts acclimated at 5°C had an average body mass around 3.6 g, whereas those at 25°C averaged 2.1 g, since these animals rapidly lost weight during the first 3 weeks of acclimation. A custom-designed magnetic rotating respirometer (Jiang and Claussen, 1992) was applied to exercise the newts terrestrially at different speeds and temperatures. The newts were exercised at 0 cm/min for 20 min at 1 and 5°C and for 15 min at 25°C. The newts assayed at the speed of 30cm/min were exercised for 10min at all testing temperatures. At the speed of 60 cm/min, the exercise duration was 5 min for the groups at 1 and 25”C, and 7min at 5°C. Finally, the newts were exercised for 5 min at a speed of 90 cm/min for all testing temperatures. The newts were kept in the respiratory chamber for 30 min prior to testing. Then, a 501~ air sample was removed by means of a syringe after flushing several times from the respiratory chamber which, afterward, was immediately closed. This was designated as the sample before exercise. Another 50 cc air sample was removed from the metabolic chamber after exercise by two 50 cc syringes, one of which was initially full of 5Occ air. Both syringes were flushed alternately
several times to mix the air inside the chamber. Then, the sample was removed as the after exercise sample for oxygen analysis. The air samples before and after exercise were injected into a dual channel oxygen analyser (Ametek S-3A/Ii) through two columns of Drierite and Ascarite (to remove water and carbon dioxide, respectively) with a syringe pump (Model 351, Sage Instruments). The differences of oxygen content (A02%), read from the analyser, were converted and standardized to metabolic rates (~1 O,/g/hr) by the following equation: I’O,(STPD) =
(AO,%)x
Vs x(P-r)x2.16x
lo4 (1)
TxtxM
Where: AO,% = difference of oxygen content between the air samples before and after the exercise from the readout of the oxygen analyser; r = vapor pressure; Vs = the volume of the respiratory chamber (325ml or 385ml) + 50ml of the syringes; T = absolute experimental temperature (To); P = atmospheric pressure (mm Hg); t = exercise duration (min) and M = body mass (g). The anaerobic metabolism of the newts was obtained by measuring the accumulated lactic acid after locomotion. Each newt was weighed before initiation of the experiment. After the measurement of terrestrial oxygen consumption at different velocities including rest and at simulated winter and summer temperatures, newts were immediately frozen in liquid nitrogen. The whole frozen carcass was cut into small pieces which were placed in a large test tube. Approximately 10 ml of chilled distilled water was added to the tube and the contents were homogenized in an ice-bath. The homogenate was then poured into a 25 ml volumetric flask. Chilled distilled water was added to dilute to 25 ml and the contents were mixed thoroughly by inverting the flask several times. Two ml of the homogenate were pipetted into a 10 ml test tube containing 4ml chilled 8% perchloric acid. Afterward, the test tube was shaken vigorously to extract lactate from the homogenized tissue until all large particles descended to the bottom. The shaken solution was then centrifuged at 5000 rpm for 10 min at 3°C and the supernatant was used for lactate determination with an enzymatic kit (826~UV; Sigma, St Louis, MO). A spectrophotometer (Spectronic 601, Milton Roy Company) was used for the determination of absorbance. The data on oxygen consumption were analysed by SAS program (SAS, 1985). Microsoft StatView SE was employed to test for significant differences among the data using P = 0.05 as the fiducial limit upon Anova. Two factor factorial Anova was used to test for significant differences among all groups at different experimental temperatures and speeds. Then, Scheffe’s F-test was used for separation of the means. All data are expressed as mean & SD. A Student t-test was used to compare the significance between the aquatic and terrestrial metabolic rates.
Metabolism of red spotted newts Table 1. Metabolic rates (mean k SD) of red-spotted newts (Noroph~halmus uiridescens) at three experimental temperatures and four exercise speeds in air. Metabolic
Speed 0 cm/min 30 cm/min 60 cm/min 90 cm/min 0,”
rate (~1 O,/g/hr)
1°C
5°C
38.72 + 9.75 69.51 f 22.17 79.50 + 17.22
38.38 f 13.48 67.21 &-20.91 18.32 rf: 23.12 93.93 + 21.89 0.95
25°C 138.44 275.25 330.87 376.71
+ + f f
30.23’ 87.24. 47.21’ 134*
1.90
Mean Q,,,s were computed from
the mean metabolic rates at two different acclimation temperatures and test temperatures respectively (5°C vs 1°C; 1 and 5°C YS 25°C). Oxygen uptakes of newts in water at each speed and temperature were significantly less than in air (P < 0.05). The metabolic rates of newts at 25°C were significantly higher than those at 5°C (P c 0.05) and indicated by an asterisk.
RESULTS
No significant differences were found among the terrestrial metabolic rates at 1°C and 5°C (P > 0.05) when the newts were tested at either locomotor speed (Table 1). However, all metabolic rates of the newts at the simulated summer temperature of 25°C were significantly higher than the corresponding rates at 1 and 5°C (P < 0.05). The resting metabolic rates of the newts at 25°C were at least three-fold higher than those at the simulated winter temperatures, whereas their active metabolic rates at the summer temperature were about four-fold greater (Table 1). Also, terrestrial metabolism slightly increased with locomotor speed at both 1 and 5°C (Fig. l), but the differences in oxygen uptake among these speeds were not significant at either simulated winter temperature (P > 0.05) (Table 1). This implies that the energy consumed by the newts during winters would not be markedly raised by modest activity. When the animals were tested at 25”C, their resting metabolism in air was significantly lower than their active metabolism (F values above 3.356, P c 0.05), but the newts
Locomotor
807
also increased their oxygen uptakes linearly as their exercise speeds increased, with a slope of 4.28 @l/g/m) (Table 1 and Fig. 1). The newts acclimated at 5°C displayed a pattern similar to that for the terrestrial metabolism at 25°C. Their terrestrial metabolic rates elevated linearly as locomotor speeds increased, but the slopes @l/g/m) for the metabolism in air were all around 1, i.e. 0.921 for the metabolic rates at 1°C and 1.025 for that at 5°C. No plateau was reached within the testing velocities used in our study (Fig. 1). All animals tested at 1 and 5°C could not locomote normally above 90 cm/min when they were touched and stimulated by the turning stirrer in air. There is an apparent separation among Q,, values from the metabolic rate data (Table 1). When the active metabolic rates at the simulated winter temperatures (1 and 5°C) and all exercise speeds were used for a Q,, calculation, the values averaged 0.95 for the newts exercised in air. These values are low and suggest that there is no remarkable change in active metabolic rates when the newts are tested at 5°C as opposed to 1°C even though their exercise. speeds varied from resting to 90 cm/min. On the other hand, the Q,, values averaged 1.9 for the terrestrial metabolism when the active metabolic rates of the newts from the 5°C acclimation and those of the newts from 25°C acclimation were compared. This indicates that the newts acclimated and tested at summer temperatures more than triple their metabolic rates compared to animals at winter temperatures. In general, the newts tended to decrease their cost of transport as their exercise speed increased (Fig. 2 and Table 2). The newts tested at 1 and 5°C only slightly showed this trend. Their costs of transport did not change very much as their exercise speeds
speed
(cmlmln)
Fig. 1. The relationships between the metabolic rates of the newts and their locomotor speeds in air at the simulated winter and summer temperatures. The newts acclimated at 5°C were tested at 1 and 5°C but the ones acclimated at 25°C were only tested at 25°C. The squares, triangles and circles represent the tests at 1, 5 and 25°C respectively. Standard deviations (SD) of the means are shown for the 5 and 25°C symbols at each assay speed. The bars of SD have been omitted from 1°C symbols to avoid excessive overlap. The mean oxygen uptake at 25°C was significantly greater than that at 1 and 5°C (P < 0.05) when newts were tested at each speed.
SUPINGJIANGand DENNISL. CLAUSEN
B
20
I \
i:~~~
T
o--. . 10
I
20
.
.
,
30
.
.
I.
40
.
I
.
50
Locomotor
.
I
.
60
.
I
.
70
.
I
.
80
.,
_
.
90
.
I.
I
.
100
110
speed (cm/mitt)
Fig. 2, The cost of transport as a function of speed when the newts locomoted in air. The newts acclimated at 5°C were tested at 1 and 5°C and the ones acclimated at 25 were only tested at 25’C. The faster newts locomoted, the lower transport cost they had. Newts moving at 25°C had greater cost than at 1 and 5°C.
were varied. The costs of walking at these two assay temperatures were at 3.86 and 3.74ml O,/g/km for the speed 30 cm/mm and 2.21 and 2.18 ml Q/g/km for the speed 60cm/min. However, their walking costs steeply decreased from 15.29 ml O,/g/km at 30cm/min to 9.19 and 6.98 ml O,/g/km at 60 and 90 cm/min respectively as these animals locomoted at 25°C. Based on the metabolic rates we measured, the energy (calories) needed by a newt per day at both the simulated summer (25°C) and winter (1 and 5°C) temperatures when locomoting at different speeds can be compared with the reserved energy released by the stored fat. Based on these data, we calculated the survival period which could be supported by 85 mg fat (average reserved fat of the red-spotted newts; Jiang and Claussen, 1992), and we obtained estimated durations ranging from 8 to 59 days, dependent upon temperature and locomotor velocity (Table 2). The lactate accumulation of the newts after exercise displayed a complex picture since their locomotor times were different. But, a general trend was evident (Fig. 3 and Table 3); the whole body content of lactic Table 2. Estimated survival durationsand
acid increased as locomotor speeds and exercise time increased. As expected, the newts had a tow level of lactate accumulation when they were allowed to rest or randomly move about in the respiratory chamber at any testing temperatures, even though the testing duration was much longer. Once they were stimulated to move forward, their lactate content greatly increased. At the speed of 30 cm/min, the newts were all assayed for 10 min so that a comp~son among three groups of experimental temperatures could be made. There were no statistically significant differences among them. Likewise, at the speed of 90cm/min, there was also no significant variation between the newts exercised at 5 and 25°C (Fe,,, =0.544, P > 0.05). When exercised at 60 cm/mm, newts tested at 5 and 25°C accumulated similar amounts of lactate despite the fact that the group at 5°C had 3 more min of movement. Lactate formation by the newts evidently increased with the exercise speed, but did not vary strikingly with temperature (Table 3; Fig. 3). When tested at l”C, newts produced a very small amount of lactate at resting status. The formed lactate level was much transport costs in air for red-spotted newts
(Norophthulmus viridescens) under different activity levels and temperatures without
ingesting food Speed (cm/min) and tampcrature 0 @ 1°C
30 60 0 30 60 90 0 30 60 90
@ 1°C @ 1°C @ 5°C @ 5°C @ 5°C @ 5°C @ 2S”C GI 25°C @ 25°C @ 25°C
Body mass (g) 3.40 3.4 3.31 3.82 4.05 3.61 3.60 3.23 3.09 2.83 2.66
0, Uptake for a newt Wday) 3.18 5.66 6.40 3.55 6.46 6.82 8.13 10.60 20.14 22.42 23.19
Survival Transport cost Wglhm)
3.86 2.21 3.74 2.18 1.74 15.29 9.19 6.98
length (days) 59 33 28 ;: 27 23 17 9 8 8
The survival times were computed assuming that 171.6 ml Or would be consumed for the complete oxidation of 85.8 mg of reserve fat.
Metabolism of red spotted newts
0
60
40
20
Locomotor
speed
809
100
60
(cm/mln)
Fig. 3. Accumulation of lactate with locomotor speeds in Norophthalmus oiridescens, showing little impact of temperature on lactate formation (P c 0.05). The newts acclimated at 5°C were tested at 1 and 5°C and the ones acclimated at 25°C were tested only at 25°C. The squares, circles and triangles represented the tests at 1, 5 and 25°C respectively. Standard deviations (SD) of the means are shown for the symbols of 5 and 25°C tests at each assay speed. T’he bars of SD have been omitted from 1°C tests to avoid excessive overlap.
higher as the newts locomoted, but there was no significant difference between the data at 30 cm/min and the resting. The newts exercised at 60 cm/min had a significantly higher lactate formation than did the resting animals (F,,, = 2.505, P < 0.05). Similarly, at SC, newts walking at speeds of 60 cm/min and above produced significantly more lactate than did those moving below 30cm/min (F values above 2.025, P c 0.05). The data from 25°C experiments were somewhat different. Only the lactate formation of newts exercised at 90 cm/mm was significantly higher than that of those walking at other speeds (F(,,,, = 5.136, P < 0.05). The lactate produced by newts at 30 and 60 cm/min was very high compared to that of resting animals, but the differences were not statistically significant. No significant difference was found when comparing the lactate formation at different assay temperatures at a given speed. DISCUSSION
Generally, terrestrial animals tend to consume more energy for their locomotion on land than do
those swimming in water (Hainsworth, 1981). Our results are consistent with this trend. Compared to the aquatic metabolism at each assay temperature and locomotor speed (Jiang and Claussen, 1992), the terrestrial metabolism was significantly higher (all t values below 2.116, P < 0.05). Red-spotted newts locomoting on land consumed much more oxygen than when they swam at an equivalent speed in water either in winter or summer. At 1 and 5°C the terrestrial metabolic rates averaged about 30% higher than the equivalent aquatic metabolic rates, and newts walking in the respiratory chamber at 25°C consumed about twice the oxygen of those swimming in water. The transport costs for aquatic and terrestrial locomotion are thus very different. Red-spotted newts may reserve at least 85 mg fat prior to winter (Jiang and Claussen, 1992). This amount of fat could support resting newts for around 80 days in water at simulated winter temperatures. In contrast, this amount of stored fat could support the newts for less than 60 days if they remained resting on land during winter (Table 2). If they locomoted on land at a very low speed (30 cm/min) in winter, they
Table 3. Lactate formation and accumulation by red-spotted newts (Norophlholmus viridescens~ after terrestrial exercise at simulated winter (1 and 4°C) and summer (25°C) temperatures ~ -’
Speed (cm/min) and temperate
Exercise length (min)
Sample size 0’)
20 IO
7 7
5 20 10 7 5 15 10 IO 5
6
0 @ 1°C 30 @ 1°C 606 1°C 0 @ 5°C 30 @ 5°C 60 @ 5°C 90 @ 5°C 0 @ 25°C 30 @ 25°C 60 @ 25°C 90 @ 25°C The data are represented
as the means +
Lactate accumulation
Lactate formation
(Wg) 1.11 I .75 I .33 1.15 1.30 2.31 1.87 1.47 1.89 2.18 2.19
I SD.
50.26 * 0.34 * 0.33 it 0.29 f 0.14 + 0.17 f 0.28 f 0.27 + 0.29 f 0.63 ; 0.32
55.51 174.77 261.66 57.41 130.37 330.60 374.17 97.71 188.64 221.84 444.21
f f f f k + f + f f +
13.08 34.15 72.89 14.40 13.58 23.78 52.91 18.07 29.44 65.37 69.62
SUPING JIANGand DENNIS L. CLAUSSEN
810
could survive only about 30 days by consuming the reserved energy. Evidently, red-spotted newts may not actively overwinter on land without eating since they could not reserve sufficient energy to support winter terrestrial activity. Therefore, the populations of the red-spotted newts hibernating on land probably remain inactive during winters. Red-spotted newts (N. viridescens), like other amphibians, rely partially on anaerobic metabolism for their energy supply during locomotion. This salamander produces limited amount of lactic acid from anaerobic metabolism during exercise. After 5-10 min of exercise at the speeds of 60 or 90 cm/min, the testing newts fatigued and hesitated to move forward. At this point, their whole body lactate ranged approximately from 1.3 to 2.3 mg per gram body mass (Table 3). This amount of lactate might represent their maximum anaerobic capacity, since the newts were not able to locomote further while fatigued. The percentage contributions of aerobic and anaerobic energy sources can be estimated from consumed oxygen and formed lactate by the following formulas (Bennett and Licht, 1973): ATP 1.Omg lactate formed = 0.0167 mmol (adenosine triphosphate). 1.0 ml O2 (STPD) consumed = 0.29 mmol ATP. The converted ATP data from aerobic and anaerobic metabolism of the red-spotted newts reveal that anaerobiosis is the major energy supply for activity (Table 4). In the winter, it appears that at least 83% of the total energy utilized by N. viridescens is anaerobically derived. The percentage of the energy contributed by anaerobic metabolism rises as the locomotor speeds of the newts increase, up to 94%. In the summer, however, anaerobiosis appears to account for only 70% of the energy needed for the activity of the newts. This percentage may reach 80% if the newts locomote at a speed of 90 cm/min. These observation are consistent with the results of Bennett and Licht (1973) on lizards (Anolis carolinensis and Dipsosaurus dorsalis), which further indicated that anaerobic metabolism constitutes the ideal solution Table
4. The
energy
contributions
of aerobic
to the energetic problems posed by the activity of small ectothetms, particularly in winters. Aerobic metabolism involves a tedious series of biochemical reactions, such as Kreb’s cycle and the electron transport pathway, and the complexity of oxygen transport, all of which function sluggishly at low winter temperatures. It would be almost impossible for an aerobic system to be mobilized rapidly when the newts are active in winters. Anaerobiosis, therefore, is an important auxillary system of energy generation which enables the red-spotted newts to remain active throughout the winter. Compared to the results of Burke and Pough (1976) on the same species, we obtained much higher values of lactate accumulation. This is probably due to the different approaches of these two studies. Burke and Pough (1976) measured the accumulation of lactic acid after the critical thermal maximum (CTM) of newts. The lactate produced by the newts after their CTM reflects the degree of struggling to escape from the stress instead of moving in their normal environments. The newts under the CTM test might not reach fatigue before the appearance of the loss of righting response (LRR). At the point of LRR, which is above 35”C, their enzyme system might be unable to continue lactate formation at a high rate. Thus, the production of lactate could perhaps not reach a maximum during a CTM test. The newts in the present study were required to locomote inside the respiratory chamber under natural conditions until they were fatigued. The accumulation of lactate by these exercising newts may represent the true anaerobic capacity of these amphibians. Also, red-spotted newts appear to have a greater anaerobic metabolic scope than do other species of amphibians. In other words, they are able to accumulate and tolerate a very high level of lactate in their body during locomotion. In contrast, a lungless salamander (Batrachoseps attenuarus) accumulates only about 1.4mg lactate per gram body mass after two minutes electrical or manual stimulation (Bennett and Licht, 1973). The lactate production and
anaerobic
red-sootted Locomotor
Converted
ATP
metabolisms
for
the activity
Energy
(a M/g/hr)
contributed
speed Aerobiosis
(cm/min)
Anaerobiosis
Aerobiosis in air
in air
8.15
II.23
55.62
83
30 @I 1°C
12.86
20.16
175.12
90
60 @
1°C
15.75
23.06
262. I8
92
0 @ 5°C
7.95
II.13
57.52
84
19.49
130.63
87
in water
temperate
0 @
II.46
30 @ 5°C
W)
60 @ 5°C
15.27
22.71
33 I .03
94
90 @ 5°C
18.95
21.24
374.92
93
0 @ 25°C
28.50
40.15
97.91
71
30 @ 25°C
46.55
79.82
189.02
70
60 @ 25°C
46.09
95.95
222.28
70
90 @ 25°C
46.03
109.25
445.10
80
The
ATP
(admosine
triphosphate)
is converted
the tested newts based on the formula contributed
by anaerobiosis
from
of Bennett
represents
the consumed
and Licht
the estimate
oxygen
(1972).
of energy
by
anaerobiosis
1°C
and
of the
newts
and formed
The percentage
allocation
in air.
lactate
by
of total energy
811
Metabolism of red spotted newts
of both Desmognathus ochrophaeus and Plethodon is below 1 mg/g body mass after more than 10 min exercise at either low or high speeds (Full, 1986; Stefanski et al., 1989; Feder, 1986). The lactate accumulation of anurans is also markedly lower than that of the newts. Bufo boreas sustains very little anaerobiosis since it accumulated only 0.23 mg lactate/g during exercise, whereas Hyla regilla produced 0.61 mg lactate/g (Bennett and Licht, 1973). Generally, slow-moving ectotherms such as toads have low rates of lactate production; in contrast, amphibians engaging in rapid bursts of activity, such as frogs, have high anaerobic metabolism (Seymour, 1973; Bennett and Licht, 1973, 1974). Therefore, redspotted newts may be adapted for occasional fast swimming in ponds. Their high anaerobic metabolism might have evolved to support this type of burst activity. The notable finding in this study is that the formation of lactate in red-spotted newts is essentially independent of acclimation and assay temperatures. The rates of anaerobic metabolism in the newts increased with the exercising speeds, but remained nearly consistent with changing temperatures and acclimations. Therefore temperature does not seem to impact the anaerobic metabolism of red-spotted newts. They must partially depend on anaerobic metabolism for energy supply while active either in winter or in summer. This suggests that the newts need a constant anaerobic scope to support their activity in all seasons. During warm weather, this animal like most ectotherms has a relatively high body temperature and fast metabolic rate. Since most biochemical reactions are strongly temperature dependent, energy can be rapidly supplied through anaerobiosis. Thus, it is reasonable for the newts to have a high anaerobic metabolism in summer. However, if the newts maintained the same physiological mechanisms in winter, they might not have sufficient energy from anaerobiosis for their locomotion, because of the adverse effect of those low winter temperatures on biochemical reactions. Obviously, the newts must have a compensating mechanism in winter in order to maintain a constant anaerobic scope. This raises the question of how lactate can be formed in the red-spotted newts during winter to a concentration similar to that of summer. Possibly the newts have evolved a mechanism to regulate the functioning of enzymes, such as lactate dehydrogenase, which could compensate for the slow rates of biochemical reactions at low temperatures. In conclusion, red-spotted newts on land linearly increased their aerobic metabolic rates with locomotor speed and ambient temperature. The terrestrial transport costs of the newts also increased with testing temperatures but declined with increased locomotor speed. The newts could not maintain their activity throughout cold winter because their terrestrial metabolism is much greater than their aquatic metabolism. Anaerobic metabolism in red-spotted
jordani
newts plays an equally important role in all seasons. The energy from anaerobiosis for supporting the movements of newts remains steady no matter at what temperatures the newts locomote. Therefore, the production of lactate by the newts after locomotion is independent of seasonal changes, but the quantity of lactic acid formed increases with an increase in locomotor speeds. Acknowledgements-This study was supported by the Theodore Roosevelt Memorial Fund of the American Museum of Natural History (S.J.) and the Department of Zoology at Miami University. We are grateful to Pete Zani and Jiazhu Zhang for their help in our field trips. Thanks also go to S. I. Guttman and R. R. Nielson for providing other equipment.
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