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A bioenergetic budget for overwintering newts (Notophthalmus viridescens) from southern ohio: Their fat reserves and aerobic metabolic rates in water
Camp. Biochem. Physiol.
Vol. 101 A, No. 4, pp. 743-750, 1992
0300-9629/92$5.00+ 0.00 0 1992Pergamon Press plc
Printed in Great Britain
A BIOENERGETIC BUDGET FOR OVERWINTERING NEWTS (NOTOPHTHALMUS VIRIDESCENS) FROM SOUTHERN OHIO: THEIR FAT RESERVES AND AEROBIC METABOLIC RATES IN WATER SUPING JIANG and DENNIS L. CLAUSSEN Department of Zoology, Miami University, Oxford, OH 45056, U.S.A. (Received 22 July 1991) Abstract-l. Red-spotted newts (Notophthalmusuiridescens) remain active throughout the winter. Locomotor costs and fat reserves of newts from southern Ohio were investigated to determine their energy balance under winter food shortage. 2. A magnetic rotating respirometer was devised to exercise the newts, acclimated at 5 or 25”C, at speeds of 0, 30, 60 and 90cm/min and at 1, 5 and 25°C. 3. Our results suggest that this device is useful for studying the locomotor metabolism of small ectotherms. 4. The metabolic rates were incorporated into a total energy budget. 5. Fat reserves were determined for the newts before and after a 55% rednction of their body mass. 6. Oxygen consumption increased with speed at both 1 and 5”C, but differences among treatments were not significant. 7. Oxygen uptake at 25°C was significantly higher than that at 1 or 5°C. 8. Metabolic rates at 25°C increased to a plateau when the newts moved at 30 cm/min or higher speeds. 9. Transport costs for the newts decreased as locomotion speed increased. 10. Newts can store at least 85 mg fat prior to winter. This could support low levels of activity for a minimum of 78 days at low winter temperatures. 11. Red-spotted newts thus appear to be capable of remaining active and surviving winter without ingesting food.
INTRODUCTION Although red-spotted newts (Notophthalmus viridescens), widely distributed in North America, have been intensely studied in many disciplines, little is
known about their winter biology. It is apparent that adult newts have different overwintering patterns in different localities during winter. Gill (1978) and Logier (1952) found that newts in Virginia and parts of Canada migrate to land for hibernation, whereas newts in Ohio (Smith and Pfingsten, 1989) and other states (Mecham, 1967) remain in ponds throughout the winter. The population in New York tends to stay in water permanently, but with some individuals emigrating from water in autumn and hibernating on land (Hurlbert, 1969). Unlike other ectothermic animals which enter dormancy in winter, the newts overwintering in ponds remain active, and have often being found swimming under ice (Conant, 1958). In Ohio, many newts can be found actively swimming in ponds throughout the winter (personal observation), whereas the newts in New York are rarely caught in March and November (Hurlbert, 1969). The physiological mechanisms of the newts for overwintering are poorly known however, and there is an obvious need for more specific information about the winter behavior and physiology of these animals. Primary production is reduced to a minimum as temperatures decrease in the winter and this is a chief factor promoting hibernation in most ectotherms.
Many zooplancters, insects, frogs and salamanders decrease activity and enter torpor because of the low temperatures and the decreased availability of food. Thus, the eggs, larvae, nymphs, pupae or tadpoles of these animals, all of which serve as food for newts (Behre, 1953; Ries and Bellis, 1966; Morin, 1983), are scarce or absent in winter. Generally, animals exposed to cold winters survive by storing fat prior to the onset of winter to increase energy supply and by maintaining low metabolic rates to reduce energy demand. Nevertheless, in spite of the shortage of food, some populations of red-spotted newts clearly maintain their activities in ponds in the cold of winter and complete their reproductive activities, i.e. mating and egg-laying, in early spring at temperatures around 5°C (Conant, 1958; and personal observation). It seems likely that active newts in winter would consume much more stored energy than would hibernating ones. If the newts do not forage at all in winter, it is not clear how their internal reserves could be sufficient to sustain activity for several months. In spite of the extensive distribution of the redspotted newt (Notophthalmus viridescens), its metabolism has received only scattered attention. Verrell (1985) investigated the energetic cost of reproductive behavior in this animal by counting breathing times. The effects of dissolved oxygen on the metabolism of newts were reported by Wakeman and Ultsch (1975), and the maximum activity metabolism of juvenile efts were measured at 15°C in air by Stefanski et al.
743
144
SUPINGJIANGand DENNISL. CLAUSSEN
(1989). Other than these few studies, there is no research focusing on the problem of how much oxygen the newts must consume in order to maintain active locomotion at temperatures just a few degrees above 0°C. The measurement of oxygen consumption during activity in amphibians has been plagued with methodological difficulties (Walsberg, 1986). This has balked extensive investigations of amphibian activity metabolism and estimations of their bioenergetics. The exercise regimens employed generally result in unnatural locomotion. In addition, the stimulation tends to be irregular, incorporating reversals of direction, erratic variation in velocity and vertical motions (Walsberg, 1986). Therefore, the resulting metabolic rates cannot exactly reflect those occurring in nature. The major problem with all previously employed methods is how to normally stimulate animals to move as naturally as possible inside an exercise chamber. In this study, the bioenergetic budgets of redspotted newts (Notophthalmus viridescens) were compared under different degrees of activities at “winter” ambient temperatures of 1 and 5°C and at a “summer” temperature of 25°C as simulated in environmental chambers. A magnetic rotating respirometer was custom-designed, which overcomes most of the difficulties of previous regimens, to exercise the newts. Aerobic metabolic rates of the animal in water were measured in order to calculate the energy demands of newts at different activity levels and temperatures. Also, reserved fat prior to winter, as a major component of energy supply, was examined by measuring triglycerides of the whole body before and after the reduction of body mass. The data on energy demands at different activity levels at low ambient temperatures were evaluated in order to compute if the autumn fat reserves are sufficient to maintain the activities of fasting newts throughout the entire winter. MATERIALS AND METHODS The newts (Notophthalmus uiriakscens) were seined from a pond in Adams County, Ohio in March and August, 1990. All animals were kept in aquariums and acclimated in 5 and 25°C environmental rooms on a L : D 14 : 10 photoperiod for at least 2 weeks before experiments. The newts in each environmental room were fed with blood worms once every week; the ones at 5°C were fed once every 2 weeks. The newts acclimated at 5°C had an average body mass around 3.6g. The body mass of the newts in 25°C acclimation averaged 2.1 g, since these animals rapidly lost weight during the first 3 weeks of acclimation. The magnetic rotating respirometer was utilized to exercise the newts at different locomotor speeds in water. This respirometer consists of several parts including a respiratory chamber, gear motors, magnets and a rotating plate (Fig. la, b). The respiratory chamber was made of 30 mm i.d. glass tubing bent into a doughnut-like chamber with a circumference of 6Ocm. Two such respiratory chambers, with the volume of 325 and 385 ml respectively, were made. A stopcock with a short neck was made on the top of the glass chamber. A small magnetic stirrer tightly wrapped with a few pieces of plastic twist-ties was placed into the chamber and this composite was shaped into a ball through the stopcock by a pair of forceps. A gear motor was mounted beside the chamber and a horse-shoe magnet was
fixed on the motor very close to the chamber to attract the magnetic stirrer inside. A plexiglass plate under the chamber could be revolved through a belt by another gear motor, the speed of which could be varied by a rheostat. Both motors would rotate simultaneously after turning on a switch. The motor driving the plate rotated at any given speed, while the one driving the horse-shoe magnet maintained a constant speed and spun the stirrer inside the chamber, which remained at a fixed position. Thus, relative to the chamber, the stirrer would move forward to chase the animals as the chamber was rotated. Accordingly, the apparent speed of the stirrer matched that of the chamber. Before experiments, a newt was placed into the exercise chamber, without water, through the stopcock for half an hour without disturbance in order to become accustomed to the system. Then, the chamber was filled with water of known dissolved oxygen content and sealed by a stopper. If the newt hesitated to move forward while the exercise chamber was revolving, the spinning stirrer touched its tail and stimulated walking. When the newt finished exercise, the stirrer was spun on a stir plate for agitating the water and the probe of a dissolved oxygen analyser (Beckman 123309) was inserted into the chamber through the stopcock to measure oxygen content after exercise. Also, 10 ml water samples were removed from the chamber before and after the exercise and analyzed by the micro-Winkler titration method (American Public Health Association et al., 1980) to confhm the results from the dissolved oxygen analyser. Approximately 110 newts were used for measuring oxygen consumption under simulated winter temperatures of 1 and 5°C and a summer temperature of 25°C. The experimental animals were randomly selected from aquariums and weighed before the experiments. The newts acclimated at 25°C were only tested at the simulated summer temperature of 25”C, whereas the ones acclimated at 5°C were exercised at the winter temperatures of 1 and 5°C. The animals examined at 1°C were exercised at speeds of 0, 30 and 6Ocm/min since they did not locomote normally at a faster speed. The newts tested at 5 and 25°C were locomoted at speeds of 0, 30, 60 and 90 cm/min. The exercise duration was 30, 30, 20 and 15 mm for the speeds of 0, 30, 60 and 9Ocm/min at the experimental temperatures of 1 and 5°C. and 25, 25, 20 and 15 min for the tests at 25°C (Table 1). The experiments at 1°C were conducted in a low temperature incubator under a dim light. A video camera was mounted inside the incubator in order to monitor the newt’s running behavior during the tests through a video monitor outside the incubator. The exercise at 0 cm/min was accomplished by turning off the gear motors and the newts were allowed to stay in the respiratory chamber without stimulation. The data obtained from both micro-Winkler titration and the dissolved oxygen analyser represented the change in the oxygen concentration of the water in ppm. These were converted into metabolic rates as ~1 oxygen per gram body mass per hour @l/g/hr) under STP by the following equation:
(STP)JVo,-
v
02
VOJ
v,.p,1.51x lo4
T.t,M
where V,,, (STP) = standardized metabolic rate @l/g/hr), V, = initial oxygen content in water (mg/l) before the exercise of newts, V, = final oxygen content after the exercise of newts, V, = volume of the respiratory chamber (325 or 385 ml), T = absolute experimental temperature (“T), P = experimental pressure (mmHg), t = exercising time (min) and M = body mass (g). This equation was derived from the usual equation for correcting data of consumed oxygen, to standard conditions. To measure fat reserves, 12 newts collected in November 1990 were quickly killed by immersion in liquid nitrogen after they were acclimated at 5°C for approximately 5 weeks without food. This relatively long acclimation period was
Bioenergetic budget for newts
745
l-14
-2
Fig. 1. (a) A cross-section of the magnetic rotating respirometer. (b) A top view of the magnetic rotating respirometer (I, gear motor to rotate outer magnet; 2, gear motor to rotate the base of respiratory chamber; 3, inner magnet; 4, outer magnet; 5, power source; 6, wires; 7, rheostat; 8, respiratory chamber; 9, base of respiratory chamber; 10, stopcock; 11, belt; 12, a newt; 13, rubber stopper; 14, platfo~).
necessary to allow the newts to digest and evacuate all food in their digestive system. After this acclimation, the fat measured in the whole body represents the major source of stored energy. The triglyceride content in these animals was regarded as the fat storage for overwintering, since they were caught in late fail. The other newts, with an average body mass of 3.46g, collected in August 1990 were acclimated at 25°C until their body mass reduced to 45% of their original body weight, but they remained alive. These
newts were offered bloodworms once every week, but they usually refused the food, so they were essentially fasted. Twelve of them were used to determine the fat content which should be equivalent to the fat left after winter. An enzymatic kit (32~UV, Sigma Chemical Co.) was employed for the test of triglycerides in the homogenized whole bodies of the newts. The difference in triglyceride concentrations between these two groups was used to estimate the amount of fat the newts could reserve for ove~intering. The
SUPING JIANG
744 Table 1. Experimental
and DENNISL. CLAUSSEN
scheme for red-spotted newts
(NotophHt4nus uiridescens) under different accliiation (AT) and experimental (ET) temperatures and loco-
motor speeds
speed (cm/min)
Test temperature (“C)
Locomotion speed (cm/min) 0 30 60 90
1 5 25 Acclimation temperature (“C) 5 5 25 30 30 20
30 30 20 15
Table 3. Metabolic rates (mean f SD) of red-spotted newts (Norophrhalmus uiridescens) at three experimental temperatures and four exercise speeds
25 25 25 15
The numbers represent the exercising times (min).
reserved fat was finally converted to energy in kilocalories to estimate the overwintering energy supply of the newts. The data for oxygen consumption were analysed by SAS program (SAS, 1985). Microsoft Statview SE was employed to test the significant difference among the data using P = 0.05 as the fiducial limit upon ANOVA. All data are expressed as mean f 1 SD. The data of the metabolic rates of newt activity from both micro-Winkler titration and the oxygen analyser were compared by one-way ANOVA. Since there was no significant difference between each pair of measurements, we took averages by adding the values together from both measuring methods (Table 2).
RESULTS
The newts (iVotophrha~mu.r viridescent) maintained a low level metabolic rate within narrow limits at simulated winter temperatures at all locomotor speeds (Table 3). Thus, there were no significant differences in oxygen uptake between 1 and 5°C at exercise speeds of 0, 30 or 6Ocm/min (all F values below 0.017). Metabolism increased with locomotor speed at both 1 and 5”C, but the differences in oxygen consumption among these speeds are not significant at either simulated winter temperature (F values below 1.223). However, when comparing the metabolic rates at 25°C with the ones at 1 and Y’C, the differences are highly significant, with all F values above 3.932. The newts have much higher oxygen consumption at 25°C than at 1 and 5°C as they exercised at the same locomotor speeds. It is obvious that there are two levels of metabolic rate: one is a very low level for overwintering, whereas the other is a high level for summer activity. There is an apparent separation among Q,,, values from the metabolic rate data. When the active metabolic rates at the simulated winter temperatures
0 30 60 90
Metabolism rate (~1 O,/g/hr) 1°C (5°C) 5°C (5°C) 25°C (25°C) 28.11 k 9.541 44.35 * 17.71 54.31 + 31.69
27.41 + 39.51 * 52.64 k 65.33 f
1.398 10.13 15.25 24.35
0.87
QlO
0 30 60 0 30 60 90 0 30 60 90
Temperature (“C) 1 5 5 5 2: 25 25 25
Sample size (N) 8 7 10 10 9 9 9 10 8 9 9
24.74* 30.71’ 37.75. 26.61’
I .74
The data
from electrode oxygen analyser and micro-Winkler titration shown in Table 1 were combined, but the sample sizes were not changed. The temperatures in parentheses are acclimation temperatures. Q,,, values were computed from the mean metabolic rates at two different acclimation temperatures and the simulated summer and winter test temperatures respectively (5 vs 25°C; 1 and 5 vs 25°C). *Significant at 95%.
(1 and SC) and all exercise speeds were used for a Q10 calculation, the values averaged 0.87. This value is low and suggests that there is no remarkable change of 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 Q10 values averaged 1.74 when the active metabolic rates of the newts from the 5°C acclimation and those of the newts from 25°C acclimation were compared. The newts acclimated and tested at 25°C had a relatively low metabolic rate (about 98 ~1 O,/g/hr) as they were allowed to move about randomly or rest within the static respiratory chamber. Their metabolic rates reached a plateau at about 160 ~1 O,/g/hr when the newts were stimulated to locomote at any given speed (30,60 or 90 cm/min). The resting metabolism is significantly different from the active metabolism at the plateau (F values above 3.091), but the newts acclimated at 5°C displayed a different pattern. Their metabolic rates linearly elevated as their locomotor speeds increased, but the slopes (p l/g/m) are all less than one, i.e. 0.728 for the metabolic rates at 1°C and 0.705 for that at 5°C. No plateau was reached within the testing velocities used in our study (Fig. 2). 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 water. In general, the newts tended to decrease their cost of transport as their exercise speed increased (Fig. 3 and Table 4). The newts tested at 1 and 5°C only slightly showed this trend. Their costs of transport
Table 2. Aerobic metabolic rates (mean f SD) of red-spotted newts (Notophthalmus oiridescens) in Ohio at different exercise steeds and temueratures speed (cm/min)
98.28 k 160.5 + 158.94 f 158.74 k
Electrode
Winkler F-value
22.73 k 40.92 f 48.90 f 24.50 + 42.97 + 52.52 + 60.80 + 113.96 f 196.74 f 165.27 f 166.15 +
8.46 22.07 15.83 8.67 12.13 13.32 18.39 30.66 47.03 40.82 25.09
33.49 +_13.77 47.17 f 24.36 59.72 + 51.00 30.33 + 9.70 36.04 k 11.97 52.76 k 19.13 69.86 f 33.43 82.61 f 21.96 124.26 + 40.26 152.62 f 47.49 151.32 + 33.18
0.027 0.01 0.034 0.01 0.013
All data were obtained from both electrode oxygen analyser and micro-Winkler titration.
Bicmnergctic
budget
741
for newts
Table 5. Average fat storage in the whole body of red-spotted newts (Notophrhalmus viridewetu)
Sampk size N=12 Mean weight (g) Fat content (mgjg) Total body fat (mg)
Before reduction of body mass
A&r rednction of body mass
3.8 f 0.361 23.2 f 6.86
1.5*0.17 1.8 * 1.08 2.1
88.5
Reserved fat (mp) Rekased energy (calorks) O2for oxidation of the reserved fat (ml)
between the metabolic rates of the newts and their locomotor speeds at the simulated winter and summer temperatures.
Fig. 2. The relationships
?
20 -
9
,7-
‘, gj
14-
5
11-
8
-1
0
1’C
A 5-C
.
0
259c
\
0.025
0.015
0.022
Locomotor ymd
0.046
0.066
(k&r)
Fig. 3. The cost of transport as a function of speed in the red-spotted newts. did not change very much as their exercise speeds were varied. The costs between the two experimental temperatures were not very different (Table 4); 2.46 and 2.20 ml oxygen/g body mass/km for the speed 30 cm/min and 1.51 and 1.47 ml/g/km for the speed 60 cm/min. However, the active costs at 25°C exhibit an obvious declining trend from a high level of 8.92 ml oxygen/g/km at a speed of 30cm/min to lower levels of 4.42 and 2.94 ml/g/km at speeds of 60 and 90 cm/min, respectively. These costs are also significantly different from the ones at 1 and 5°C. The triglyceride concentration from the newts before the reduction of their body mass averaged
0 30 60 0 30 60 90 0 30 60 90
I I 1 5 5 5 5 25 25 25 25
Sample size 8 7 10 10 9 9 9 IO 8 9 9
Body weight (9) 3.26 f 3.34 f 3.60 f 3.72 f 3.90 f 3.83 f 3.83 f 2.18 + I .79 * 2.02 f 2.52 f
0.343 0.328 0.632 0.451 0.613 0.368 0.634 0.302 0.407 0.378 0.588
171.6
The reserved fat is the difference of total body fat of the newts before and after their body mass redoced to 45% of the original weight. The released energy represents the energy from complete oxidation of the -cd fat. The conversion of the fat, energy and oxygen is based on 9.7 calories/mg fat and 4.7 calorks/ml 0, (from Kleibcr, 1961).
23.2 mg/g body weight, whereas the triglyceride concentration from the newts with their body mass reduced to 45% of their original body weight, averaged 1.8 mg/g (Table 5); the difference is obviously significant. By multiplying the body mass by the concentration of the triglyceride, it was computed that the newts averaged 88.5 mg fat in their bodies before the reduction of body mass, however, the fasted newts had only 2.7 mg fat left in the body. From the point of fat reserves, it appears that at least 85.8 mg fat could be stored before winter, since these newts were collected in late fall. This amount of fat storage represents 806.5 kilocalories of energy, and 171.6 ml oxygen would be needed for the complete oxidation of this much lipid. Based on the metabolic rates measured, the energy (calories) needed by a newt every 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. The duration of survival which could be supported by 85 mg fat can also be calculated (Table 4). Theoretically, based on the data for a speed of 0 cm/mitt, a newt could survive 78 days at 1°C 70 days at 5°C and 33 days at 25°C when resting or randomly moving around by consuming the reserved 85 mg fat without any food. If the newt continually exercised at 30 cm/mm, it could survive 48 days at 1°C 46 days at 5°C and 25 days at 25°C at fasting status. The faster a newt locomotes and the higher the ambient temperatures are, the shorter time it could survive without ingesting any food.
Table 4. Speculated survival duration for red-spotted newts (Nofophthdmus virideseens) under different active levels and temneratures without inaestina food Active levels Temperature (cm/min) (“C)
85.8 806.5
0, consumption for a newt (ml/day)
Transport cost (ml Wg/hm)
Survival (days)
2.20 3.56 4.69 2.45 3.70 4.84 6.01 5.14 6.90 7.71 9.60
2.464 1.509 2.195 1.462 1.210 8.917 4.415 2.940
78 48 37 70 46 36 29 33 25 22 18
The survival times were computed assuming that 171.6 ml 0, would be consumed for the complete oxidation of 85.8 mg of reserve fat
SUPINGJIANGand
748 DISCUSSION
Previous studies of urodele metabolism during exercise were generally a~omplished by one of three regimes: first, a treadmill has often been used (Feder, 1988; Full, 1986; Full et al,, 1988). This method has an advantage of being able to track oxygen consump tion by open-flow respirometry when animals are exercising and recovering. The second method requires a custom-design respirometer which is a sealed metabolic chamber rotated vertically by a small eiectric motor at various rates (Feder, 1986). A few pieces of lead are inside the chamber in order to prod the animals to walk forward. These two approaches are advantageous in allowing exact replication of experimental conditions and avoiding the tedium of manual stimulation (Walsberg, 1986), but the animal, in a vertically rotating chamber, might tend to slide and to be pushed downward by the lead prods instead of walking forward because of the vertical rotation. This could distort the measurement of activity metabolism. In the last method, animals in a sealed chamber are manually stimulated to exercise. The manual stimulations include: (a) turning the chamber, and thus, repeatedly flipping the animal onto its dorsum and forcing it to right itself (Withers, 1980; Stefanski et al., 1989), (b) turning a steel rod outside the chamber to move pieces of wire soldered on the rod inside the chamber, so that the animal could be “chased” (Stefanski et al., 1989), and (c)electric shock to keep the animal vigorously moving (Bennett and Licht, 1973). A Iong-existing problem in respirometer designs for small ectotherms is thus how to keep animals naturally moving forward either in water or on land at a given speed. Custom-designed respirometers, such as those described by Feder (1986), Walsberg (1986) and Stefanski et al. (1989) attempted to solve the problem. Although they obtained a great deal of data from their designs, the difficulty in inducing animals to walk normally at an expected speed was not completely resolved. We applied the magnetic principle to our device and successfully overcame this difficulty. Through more than 300 tests on N. viridescens in water (present study) and in air (unpublished data), most of the newts stimulated by the rotating magnetic stirrer locomoted very well inside the respiratory chamber. Only a small portion of the tested animals did not behave as expected, apparently because they were in poor health or condition. The data from these newts, which were pushed by the stirrer most of the time, were discarded. The stimulation from the touch of the spinning stirrer on an animal’s tail is harmless and not greatly different from that they might naturally encounter if they contacted sticks, logs or rocks in ponds. The newts swam or walked in the chamber at the tested speeds without any apparent stress. They were not required to flip over and they could not slide downward. Their locomotion, we believe, was much closer to that in their natural movement. The doughnut-like glass respiratory chamber of this design could be properly enlarged for accommodating some larger salamanders, lizards and even small fishes, or could be reduced for small ectotherms. Therefore, this apparatus could become an effective tool for studying the metabolism of small ectothermic vertebrates.
DENNEL. CLAUS!EN This study was designed to understand how the newts actively overwinter with regard to their bioenergetic budget. Under usual circumstance, redspotted newts do not encounter very warm weather in winter or very cold conditions in summer. Testing newts, acclimated to simulated winter, at summer temperatures provides valuable data on the impact of acclimation. However, these data mostly reflect the situation in the laboratory instead of in nature. In this study, newts were acclimated to both simulated summer (25°C) and winter (S’C) temperatures. Winter water temperatures in ponds in southern Ohio are often around 5”C, and sometimes at 1°C or below when ice is formed. We measured temperatures in our CoIlecting pond of 8°C at noon in early November and in middle March. During the coldest months of January and February, the temperatures in that pond would be substantially lower than those in November and March. Therefore, for the newts in winter acclimation, we only examined their metabolism at I and 5°C. On the other hand, we measured a summer temperature in that pond approaching 24°C in midAugust at noon. We accordingly tested the newts in summer acclimation at 25°C. The locomotor behavior of the red-spotted newts greatly depends upon both temperature and speed. The newts tested at winter temperatures could easily complete their exercise at 30 and 60 cm/min during the tests. They did not display fatigue after finishing their locomotion within the given exercise time. It seems likely that they were capable of swimming in the chamber for an even longer time. But, the newts were nearly exhausted and reluctant to move forward after 15 min exercise at 90 cm/min. When the respiratory chamber rotated at 30 cm/min, the newts often swam a little bit further in the front of the stirrer after the stirrer touched their tails. Then, they stopped moving until the stirrer caught them again and repeated this cycle. At 60 cm/min, they continually swam forward while they were being chased by the stirrer. The newts struggled moving forward and quickly exhausted under the rotation of the chamber at 90 cm/min. The newts display similar behavior when exercised at 25”C, but they did not fatigue even at the velocity of 90 cm/min within the experimental times. This suggests that the optimal speed for red-spotted newts to locomote in water, either in cold winter or in summer, is around 60 cm/min. This optimal speed is similar to the walking speed (6~l~~/min) of Plethodon jordani on land in the field observed by Stefanski et al. (1989). These two species have a similar body mass, but differ in that P. jordani are lungless. Additionally, P. jordani moves on land during nonwinter seasons, whereas N. viride~ce~ adults are generally aquatic in all seasons. Possibly, urodeles with similar body size have similar optimal locomoting speed regardless of their locomotor patterns. This could be attributed to the fact that they have similar stride length. These urodeles could reduce their energy cost if they move at an optimal speed. The metabolism of our red-spotted newts at summer temperatures were generally nearly identical with that of other populations and with other species of lunged salamanders, in spite of different locomotor patterns. Wakeman and Ultsch (1975) reported that red-spotted newts (N. viridescens) from Florida had
Bioenergetic budget for newts an aquatic oxygen uptake value of 90pl/g/hr when resting at 160 mmHg pOz and 25”C, which is very similar to our data at this temperature (Table 3). The maximum oxygen consumption of the red-spotted newts during vigorous movement induced by electric shock at 25°C was measured by Feder (1977). His results were very similar to our exercise data which were around 160 ~1 O,/g/hr at 25”C, even though Feder’s measure was from newts breathing air. The red efts, juvenile newts, have much lower basal metabolism (61 ~1 O,/g/hr) in air at 25°C (Stefanski et al., 1989) than do adult newts. But, the active metabolism of the red efts in air from Stefanski’s study resembles that of adults in water in the present study. No other detailed comparisons can be made for this species. However, the red-spotted newts have a resting metabolic rate in water twice as great as their relatives, Turicha grunulosu, a rough-skinned newt (Feder, 1983) and T. torosu, a California newt (Harlow, 1977; Ultsch, 1976) under comparable experimental conditions. This is probably because the red-spotted newts are smaller than the rough-skinned and California newts. Nevertheless, T. torosa has an unexpectedly high maximum active metabolism of 194 ~1 O,/g/hr with regard to its body mass (Feder, 1977). N. uiridescens are fully aquatic animals as adults. They have adapted to locomote efficiently in water. It is likely that red-spotted newts walking under water tend to consume less energy in locomotion than when walking on land. Also, the newts tested by Feder (1977) were stimulated by electric shock which may have produced unnaturally elevated metabolic rates. Nevertheless, all of these newts display very similar metabolic responses at simulated summer temperatures. The newts tested at 25°C displayed a plateau at speeds of 30 cm/min or higher, which suggests that the energy for supporting the locomotion in summer is supplemented by anaerobiosis. This plateau was also found in the lungless salamander, Desmognuthus ochropheuus (Feder, 1986). This species reached the plateau at a speed around 15 cm/min. The redspotted newts might have a low limit of dissolved oxygen for their activity. Once this limit is surpassed, the newts are forced to rely on anaerobic metabolism. The low oxygen concentration in water in summer could be very close to the limit and there might not be a sufficient oxygen supply for sustained aerobic locomotion. Fortunately, newts can repay this high cost from their abundant food resources in summer. On the other hand, the N. viridescens in our study exhibited no statistically significant differences among metabolic rates between simulated winter temperatures of 1 and 5°C. This implies that the newts maintain metabolism within a narrow range in winter. Maintaining low-level activity in winter probably does not constitute a major increased energy cost over that of inactive hibernation for the newts in Ohio. It thus seems quite possible that the newts could actively survive the cold of winter without major problems of energy shortage, since their overwintering costs do not change drastically with modest changes in temperatures or with slow movements. Also, winter newts are less likely to require anaerobic metabolism since the high dissolved oxygen content in winter may be well above the aerobic limit. Their overwintering activities
149
can be maintained by aerobic metabolism which may economically produce much more energy. Consequently, two different active metabolic regimens seem to exist in red-spotted newts. One is the combination of a high level of aerobic metabolism and anaerobic metabolism, evolved for summer activities. The other is a relatively constant low level of aerobic metabolism which is adapted for overwintering activities. Our results reveal that red-spotted newts may reserve at least 85 mg fat prior to winter. This amount of fat may support the newts surviving 78 days when experimentally resting at simulated winter temperatures. The newts were tested in the respiratory chamber without rotation. We designated this situation as resting metabolism. In fact, the newts were allowed random movement inside the static chamber without any stimulation. Thus, the fat reserves may be a supply for the newts to perform low level activity for nearly 3 months in winter. In addition, our newts for determining fat reserves had been fasted for about 40 days at 5°C before they were placed in liquid nitrogen, because the newts would need 3 weeks to evacuate all food from their digestive system (unpublished data) at such a low temperature of 5°C. The total duration for the newts to last in winter by consuming their final meal in the fall plus their reserved fat would be approximately 4 months, which is the usual winter length in southern Ohio. Furthermore, in nature, movements at relative high speeds would represent only a small portion of the total movements. Such high-speed locomotion also has a low cost of transport (Fig. 3). Thus, the reserved energy may also be sufficient for the newts to survive throughout winter if they are occasionally active at higher levels. Finally, other newts collected at the same time and location as the ones used in this study were maintained in 5°C environmental room. We did not feed them during acclimation. These newts survived for 4 months with only a few casualties. The dead newts appeared to have succumbed to disease rather than to inanition. Therefore, red-spotted newts are able to maintain their activities and survive long and cold winters without ingesting food. In conclusion, the red-spotted newts from southern Ohio during winter could maintain a low level active metabolism within a narrow range. The energy for their winter movements could be mostly accomplished through the processes of aerobiosis which has advantages of economically consuming stored energy. Therefore, the energy reserved by the newts prior to winter is sufficient to support their overwinter activity even in the absence of available food. Acknowledgements-We are grateful to John L. Morrow for building our respirometer and to S. I. Guttman and R. R. Nielson for providing other equipment. Thanks also go to M. F. Wright and J. P. Constanzo for providing specimens. This study was supported by the department of zoology at Miami University and the Theodore Roosevelt Memorial Fund of the American Museum of Natural History (S.J.). REFERENCES
American Public Health Association, American Water Works Association and Water Pollution Control Federation (1980) Standard methods for the examination of water and wastewater, pp. 388-399.
750
SUPINGJIANGand DENNISL. CLAWEN
Behre E. H. (1952) Food of the salamander Triturus uiridescens. Copeia 1953, 160. Bennett A. F. and Licht P. (1973) Relative ~on~ibutions of anaerobic and aerobic energy production during activity in Amphibia. J. camp. Pi@& 87, 351-360. Conant R. (1958) A Field Guide to Reptiles and Amphibians. Houghton MiWin Co., Boston. Feder M. (1977) Oxygen consumption and activity in salamanders: effect of body size and lunglessness. J. exp. ZooI. uI2,403-414. Feder M. (1983) Metabolic and biochemical correlation of thermal acclimation in the rough-skinned newt Turicbu gr~~losa. Physiol. Zoo/. 56, 513-521. Feder M. (1986) Effect of thermal acclimation on locomotor energetics and locomotor performance in a lungless salamander, Desmogtzathws ochrophaeus. J. exp. Biol. 121, 271-283. Feder M. (1988) Exercising with and without lungs. II. Experimental elimination of pulmonary and buccopharyngeal gas exchange in individual salamanders (Ambystoma fig&urn). J. exp. Biol. 138, 487-491. Full R. J. (1986) Locomotion without lungs: energetics and performance of a lungless salamander. Am. J. PbysioI. 251, R775-780. Full R, J., Anderson B. D., Finnerty C. M. and Feder M. (1988) Exercising with and without lungs. I. The effects of metabolic cost, maximal oxygen transport and body size on terrestrial locomotion in salamander species, J. exp. Biol. 138, 471-485. Gill D. E. (1978) The metapopulation ecology of the redspotted newt, Notophthalmus viridescens (Rafinesque). Ecol. Motzogr. 48, 145-166. Harlow H. J. (1977) Seasonal oxygen metabolism and cutaneous osmo~g~ation in the California newts, Taricha torosa. Physiol. ZooI. So, 231-236. Hurlbert S. H. (1969) The breeding migrations and interhabitat wandering of the vermilion-spotted newt Notophthalmus viridescens (Ratinesque). Ecol. Monogr. 29, 465-488.
Kleiber M. (1961) The Fire of I&e. Wiley, New York. Logier E. B. S. (1952) The Frogs, To& and Salamanders of &stern Cam&z. Clarke, In&, Toronto. Me&am J. S. (19671 Notooht~I~ viria’escens.Cat. Am. Amphib. Reptiles 43, 1-i. Morin P. J. (1983) Competitive and predatory interactions in natural and experimental populations of Notophthalmus viriakscens dorsalis and Ambystoma tigriman. Copeia 1983, 628-639. Ries K. M. and Bellis E. D. (1966) Spring food habits of the red-spotted newt in Pennsylvania. Herpitologica 22, 152-155. SAS Institute Inc. (1985) SAS user’s guide: statistics. SAS Institute Inc. Gary, NC Smith C. J. and Pflnasten R. A. (19891 Salamumiers of Ohio (Edited by I%gsten R. A. and Downs F. L.), pp. 7987. College of Biological Sciences, The Ohio State University. Stefanski M., Gatten R. E. Jr and Pough F. H. (1989) Activity metabolism of salamanders: tolerance to dehydration. J. Herpetol. 23, 45-50. Ultsch 6. R. (1976) Respiratory surface area as a factor con~olling the standard rate of Or consumption of aquatic salamanders. Resp. Physiol. 26, 357-369. Verrell P. A. (1985) Is there an energetic cost to sex? Activity, courtship mode and breathing in the red-spotted newt, Notophthalmw viridescens (Rafinesque). Monitore Zool. Ital. (N.S.) 19, 121-127. ” Wakeman J. M. and Ultsch G. R. (1975) The effects of dissolved oxygen on metabolism ‘and ‘gas-exchange partitioning in aquatic salamanders. Physiol. ZooI. 48, 348-359. Walsberg G, E. (1986) Comparison of two techniques for estimating the maximum aerobic capacity of amphibians. Heroetoloaica 42, 389-394. Withers P. c. (1986) Oxygen consumption of plethodontid salamanders during rest, activity and recovery. Copeia 19s0, 781-787.
Vol. 101 A, No. 4, pp. 743-750, 1992
0300-9629/92$5.00+ 0.00 0 1992Pergamon Press plc
Printed in Great Britain
A BIOENERGETIC BUDGET FOR OVERWINTERING NEWTS (NOTOPHTHALMUS VIRIDESCENS) FROM SOUTHERN OHIO: THEIR FAT RESERVES AND AEROBIC METABOLIC RATES IN WATER SUPING JIANG and DENNIS L. CLAUSSEN Department of Zoology, Miami University, Oxford, OH 45056, U.S.A. (Received 22 July 1991) Abstract-l. Red-spotted newts (Notophthalmusuiridescens) remain active throughout the winter. Locomotor costs and fat reserves of newts from southern Ohio were investigated to determine their energy balance under winter food shortage. 2. A magnetic rotating respirometer was devised to exercise the newts, acclimated at 5 or 25”C, at speeds of 0, 30, 60 and 90cm/min and at 1, 5 and 25°C. 3. Our results suggest that this device is useful for studying the locomotor metabolism of small ectotherms. 4. The metabolic rates were incorporated into a total energy budget. 5. Fat reserves were determined for the newts before and after a 55% rednction of their body mass. 6. Oxygen consumption increased with speed at both 1 and 5”C, but differences among treatments were not significant. 7. Oxygen uptake at 25°C was significantly higher than that at 1 or 5°C. 8. Metabolic rates at 25°C increased to a plateau when the newts moved at 30 cm/min or higher speeds. 9. Transport costs for the newts decreased as locomotion speed increased. 10. Newts can store at least 85 mg fat prior to winter. This could support low levels of activity for a minimum of 78 days at low winter temperatures. 11. Red-spotted newts thus appear to be capable of remaining active and surviving winter without ingesting food.
INTRODUCTION Although red-spotted newts (Notophthalmus viridescens), widely distributed in North America, have been intensely studied in many disciplines, little is
known about their winter biology. It is apparent that adult newts have different overwintering patterns in different localities during winter. Gill (1978) and Logier (1952) found that newts in Virginia and parts of Canada migrate to land for hibernation, whereas newts in Ohio (Smith and Pfingsten, 1989) and other states (Mecham, 1967) remain in ponds throughout the winter. The population in New York tends to stay in water permanently, but with some individuals emigrating from water in autumn and hibernating on land (Hurlbert, 1969). Unlike other ectothermic animals which enter dormancy in winter, the newts overwintering in ponds remain active, and have often being found swimming under ice (Conant, 1958). In Ohio, many newts can be found actively swimming in ponds throughout the winter (personal observation), whereas the newts in New York are rarely caught in March and November (Hurlbert, 1969). The physiological mechanisms of the newts for overwintering are poorly known however, and there is an obvious need for more specific information about the winter behavior and physiology of these animals. Primary production is reduced to a minimum as temperatures decrease in the winter and this is a chief factor promoting hibernation in most ectotherms.
Many zooplancters, insects, frogs and salamanders decrease activity and enter torpor because of the low temperatures and the decreased availability of food. Thus, the eggs, larvae, nymphs, pupae or tadpoles of these animals, all of which serve as food for newts (Behre, 1953; Ries and Bellis, 1966; Morin, 1983), are scarce or absent in winter. Generally, animals exposed to cold winters survive by storing fat prior to the onset of winter to increase energy supply and by maintaining low metabolic rates to reduce energy demand. Nevertheless, in spite of the shortage of food, some populations of red-spotted newts clearly maintain their activities in ponds in the cold of winter and complete their reproductive activities, i.e. mating and egg-laying, in early spring at temperatures around 5°C (Conant, 1958; and personal observation). It seems likely that active newts in winter would consume much more stored energy than would hibernating ones. If the newts do not forage at all in winter, it is not clear how their internal reserves could be sufficient to sustain activity for several months. In spite of the extensive distribution of the redspotted newt (Notophthalmus viridescens), its metabolism has received only scattered attention. Verrell (1985) investigated the energetic cost of reproductive behavior in this animal by counting breathing times. The effects of dissolved oxygen on the metabolism of newts were reported by Wakeman and Ultsch (1975), and the maximum activity metabolism of juvenile efts were measured at 15°C in air by Stefanski et al.
743
144
SUPINGJIANGand DENNISL. CLAUSSEN
(1989). Other than these few studies, there is no research focusing on the problem of how much oxygen the newts must consume in order to maintain active locomotion at temperatures just a few degrees above 0°C. The measurement of oxygen consumption during activity in amphibians has been plagued with methodological difficulties (Walsberg, 1986). This has balked extensive investigations of amphibian activity metabolism and estimations of their bioenergetics. The exercise regimens employed generally result in unnatural locomotion. In addition, the stimulation tends to be irregular, incorporating reversals of direction, erratic variation in velocity and vertical motions (Walsberg, 1986). Therefore, the resulting metabolic rates cannot exactly reflect those occurring in nature. The major problem with all previously employed methods is how to normally stimulate animals to move as naturally as possible inside an exercise chamber. In this study, the bioenergetic budgets of redspotted newts (Notophthalmus viridescens) were compared under different degrees of activities at “winter” ambient temperatures of 1 and 5°C and at a “summer” temperature of 25°C as simulated in environmental chambers. A magnetic rotating respirometer was custom-designed, which overcomes most of the difficulties of previous regimens, to exercise the newts. Aerobic metabolic rates of the animal in water were measured in order to calculate the energy demands of newts at different activity levels and temperatures. Also, reserved fat prior to winter, as a major component of energy supply, was examined by measuring triglycerides of the whole body before and after the reduction of body mass. The data on energy demands at different activity levels at low ambient temperatures were evaluated in order to compute if the autumn fat reserves are sufficient to maintain the activities of fasting newts throughout the entire winter. MATERIALS AND METHODS The newts (Notophthalmus uiriakscens) were seined from a pond in Adams County, Ohio in March and August, 1990. All animals were kept in aquariums and acclimated in 5 and 25°C environmental rooms on a L : D 14 : 10 photoperiod for at least 2 weeks before experiments. The newts in each environmental room were fed with blood worms once every week; the ones at 5°C were fed once every 2 weeks. The newts acclimated at 5°C had an average body mass around 3.6g. The body mass of the newts in 25°C acclimation averaged 2.1 g, since these animals rapidly lost weight during the first 3 weeks of acclimation. The magnetic rotating respirometer was utilized to exercise the newts at different locomotor speeds in water. This respirometer consists of several parts including a respiratory chamber, gear motors, magnets and a rotating plate (Fig. la, b). The respiratory chamber was made of 30 mm i.d. glass tubing bent into a doughnut-like chamber with a circumference of 6Ocm. Two such respiratory chambers, with the volume of 325 and 385 ml respectively, were made. A stopcock with a short neck was made on the top of the glass chamber. A small magnetic stirrer tightly wrapped with a few pieces of plastic twist-ties was placed into the chamber and this composite was shaped into a ball through the stopcock by a pair of forceps. A gear motor was mounted beside the chamber and a horse-shoe magnet was
fixed on the motor very close to the chamber to attract the magnetic stirrer inside. A plexiglass plate under the chamber could be revolved through a belt by another gear motor, the speed of which could be varied by a rheostat. Both motors would rotate simultaneously after turning on a switch. The motor driving the plate rotated at any given speed, while the one driving the horse-shoe magnet maintained a constant speed and spun the stirrer inside the chamber, which remained at a fixed position. Thus, relative to the chamber, the stirrer would move forward to chase the animals as the chamber was rotated. Accordingly, the apparent speed of the stirrer matched that of the chamber. Before experiments, a newt was placed into the exercise chamber, without water, through the stopcock for half an hour without disturbance in order to become accustomed to the system. Then, the chamber was filled with water of known dissolved oxygen content and sealed by a stopper. If the newt hesitated to move forward while the exercise chamber was revolving, the spinning stirrer touched its tail and stimulated walking. When the newt finished exercise, the stirrer was spun on a stir plate for agitating the water and the probe of a dissolved oxygen analyser (Beckman 123309) was inserted into the chamber through the stopcock to measure oxygen content after exercise. Also, 10 ml water samples were removed from the chamber before and after the exercise and analyzed by the micro-Winkler titration method (American Public Health Association et al., 1980) to confhm the results from the dissolved oxygen analyser. Approximately 110 newts were used for measuring oxygen consumption under simulated winter temperatures of 1 and 5°C and a summer temperature of 25°C. The experimental animals were randomly selected from aquariums and weighed before the experiments. The newts acclimated at 25°C were only tested at the simulated summer temperature of 25”C, whereas the ones acclimated at 5°C were exercised at the winter temperatures of 1 and 5°C. The animals examined at 1°C were exercised at speeds of 0, 30 and 6Ocm/min since they did not locomote normally at a faster speed. The newts tested at 5 and 25°C were locomoted at speeds of 0, 30, 60 and 90 cm/min. The exercise duration was 30, 30, 20 and 15 mm for the speeds of 0, 30, 60 and 9Ocm/min at the experimental temperatures of 1 and 5°C. and 25, 25, 20 and 15 min for the tests at 25°C (Table 1). The experiments at 1°C were conducted in a low temperature incubator under a dim light. A video camera was mounted inside the incubator in order to monitor the newt’s running behavior during the tests through a video monitor outside the incubator. The exercise at 0 cm/min was accomplished by turning off the gear motors and the newts were allowed to stay in the respiratory chamber without stimulation. The data obtained from both micro-Winkler titration and the dissolved oxygen analyser represented the change in the oxygen concentration of the water in ppm. These were converted into metabolic rates as ~1 oxygen per gram body mass per hour @l/g/hr) under STP by the following equation:
(STP)JVo,-
v
02
VOJ
v,.p,1.51x lo4
T.t,M
where V,,, (STP) = standardized metabolic rate @l/g/hr), V, = initial oxygen content in water (mg/l) before the exercise of newts, V, = final oxygen content after the exercise of newts, V, = volume of the respiratory chamber (325 or 385 ml), T = absolute experimental temperature (“T), P = experimental pressure (mmHg), t = exercising time (min) and M = body mass (g). This equation was derived from the usual equation for correcting data of consumed oxygen, to standard conditions. To measure fat reserves, 12 newts collected in November 1990 were quickly killed by immersion in liquid nitrogen after they were acclimated at 5°C for approximately 5 weeks without food. This relatively long acclimation period was
Bioenergetic budget for newts
745
l-14
-2
Fig. 1. (a) A cross-section of the magnetic rotating respirometer. (b) A top view of the magnetic rotating respirometer (I, gear motor to rotate outer magnet; 2, gear motor to rotate the base of respiratory chamber; 3, inner magnet; 4, outer magnet; 5, power source; 6, wires; 7, rheostat; 8, respiratory chamber; 9, base of respiratory chamber; 10, stopcock; 11, belt; 12, a newt; 13, rubber stopper; 14, platfo~).
necessary to allow the newts to digest and evacuate all food in their digestive system. After this acclimation, the fat measured in the whole body represents the major source of stored energy. The triglyceride content in these animals was regarded as the fat storage for overwintering, since they were caught in late fail. The other newts, with an average body mass of 3.46g, collected in August 1990 were acclimated at 25°C until their body mass reduced to 45% of their original body weight, but they remained alive. These
newts were offered bloodworms once every week, but they usually refused the food, so they were essentially fasted. Twelve of them were used to determine the fat content which should be equivalent to the fat left after winter. An enzymatic kit (32~UV, Sigma Chemical Co.) was employed for the test of triglycerides in the homogenized whole bodies of the newts. The difference in triglyceride concentrations between these two groups was used to estimate the amount of fat the newts could reserve for ove~intering. The
SUPING JIANG
744 Table 1. Experimental
and DENNISL. CLAUSSEN
scheme for red-spotted newts
(NotophHt4nus uiridescens) under different accliiation (AT) and experimental (ET) temperatures and loco-
motor speeds
speed (cm/min)
Test temperature (“C)
Locomotion speed (cm/min) 0 30 60 90
1 5 25 Acclimation temperature (“C) 5 5 25 30 30 20
30 30 20 15
Table 3. Metabolic rates (mean f SD) of red-spotted newts (Norophrhalmus uiridescens) at three experimental temperatures and four exercise speeds
25 25 25 15
The numbers represent the exercising times (min).
reserved fat was finally converted to energy in kilocalories to estimate the overwintering energy supply of the newts. The data for oxygen consumption were analysed by SAS program (SAS, 1985). Microsoft Statview SE was employed to test the significant difference among the data using P = 0.05 as the fiducial limit upon ANOVA. All data are expressed as mean f 1 SD. The data of the metabolic rates of newt activity from both micro-Winkler titration and the oxygen analyser were compared by one-way ANOVA. Since there was no significant difference between each pair of measurements, we took averages by adding the values together from both measuring methods (Table 2).
RESULTS
The newts (iVotophrha~mu.r viridescent) maintained a low level metabolic rate within narrow limits at simulated winter temperatures at all locomotor speeds (Table 3). Thus, there were no significant differences in oxygen uptake between 1 and 5°C at exercise speeds of 0, 30 or 6Ocm/min (all F values below 0.017). Metabolism increased with locomotor speed at both 1 and 5”C, but the differences in oxygen consumption among these speeds are not significant at either simulated winter temperature (F values below 1.223). However, when comparing the metabolic rates at 25°C with the ones at 1 and Y’C, the differences are highly significant, with all F values above 3.932. The newts have much higher oxygen consumption at 25°C than at 1 and 5°C as they exercised at the same locomotor speeds. It is obvious that there are two levels of metabolic rate: one is a very low level for overwintering, whereas the other is a high level for summer activity. There is an apparent separation among Q,,, values from the metabolic rate data. When the active metabolic rates at the simulated winter temperatures
0 30 60 90
Metabolism rate (~1 O,/g/hr) 1°C (5°C) 5°C (5°C) 25°C (25°C) 28.11 k 9.541 44.35 * 17.71 54.31 + 31.69
27.41 + 39.51 * 52.64 k 65.33 f
1.398 10.13 15.25 24.35
0.87
QlO
0 30 60 0 30 60 90 0 30 60 90
Temperature (“C) 1 5 5 5 2: 25 25 25
Sample size (N) 8 7 10 10 9 9 9 10 8 9 9
24.74* 30.71’ 37.75. 26.61’
I .74
The data
from electrode oxygen analyser and micro-Winkler titration shown in Table 1 were combined, but the sample sizes were not changed. The temperatures in parentheses are acclimation temperatures. Q,,, values were computed from the mean metabolic rates at two different acclimation temperatures and the simulated summer and winter test temperatures respectively (5 vs 25°C; 1 and 5 vs 25°C). *Significant at 95%.
(1 and SC) and all exercise speeds were used for a Q10 calculation, the values averaged 0.87. This value is low and suggests that there is no remarkable change of 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 Q10 values averaged 1.74 when the active metabolic rates of the newts from the 5°C acclimation and those of the newts from 25°C acclimation were compared. The newts acclimated and tested at 25°C had a relatively low metabolic rate (about 98 ~1 O,/g/hr) as they were allowed to move about randomly or rest within the static respiratory chamber. Their metabolic rates reached a plateau at about 160 ~1 O,/g/hr when the newts were stimulated to locomote at any given speed (30,60 or 90 cm/min). The resting metabolism is significantly different from the active metabolism at the plateau (F values above 3.091), but the newts acclimated at 5°C displayed a different pattern. Their metabolic rates linearly elevated as their locomotor speeds increased, but the slopes (p l/g/m) are all less than one, i.e. 0.728 for the metabolic rates at 1°C and 0.705 for that at 5°C. No plateau was reached within the testing velocities used in our study (Fig. 2). 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 water. In general, the newts tended to decrease their cost of transport as their exercise speed increased (Fig. 3 and Table 4). The newts tested at 1 and 5°C only slightly showed this trend. Their costs of transport
Table 2. Aerobic metabolic rates (mean f SD) of red-spotted newts (Notophthalmus oiridescens) in Ohio at different exercise steeds and temueratures speed (cm/min)
98.28 k 160.5 + 158.94 f 158.74 k
Electrode
Winkler F-value
22.73 k 40.92 f 48.90 f 24.50 + 42.97 + 52.52 + 60.80 + 113.96 f 196.74 f 165.27 f 166.15 +
8.46 22.07 15.83 8.67 12.13 13.32 18.39 30.66 47.03 40.82 25.09
33.49 +_13.77 47.17 f 24.36 59.72 + 51.00 30.33 + 9.70 36.04 k 11.97 52.76 k 19.13 69.86 f 33.43 82.61 f 21.96 124.26 + 40.26 152.62 f 47.49 151.32 + 33.18
0.027 0.01 0.034 0.01 0.013
All data were obtained from both electrode oxygen analyser and micro-Winkler titration.
Bicmnergctic
budget
741
for newts
Table 5. Average fat storage in the whole body of red-spotted newts (Notophrhalmus viridewetu)
Sampk size N=12 Mean weight (g) Fat content (mgjg) Total body fat (mg)
Before reduction of body mass
A&r rednction of body mass
3.8 f 0.361 23.2 f 6.86
1.5*0.17 1.8 * 1.08 2.1
88.5
Reserved fat (mp) Rekased energy (calorks) O2for oxidation of the reserved fat (ml)
between the metabolic rates of the newts and their locomotor speeds at the simulated winter and summer temperatures.
Fig. 2. The relationships
?
20 -
9
,7-
‘, gj
14-
5
11-
8
-1
0
1’C
A 5-C
.
0
259c
\
0.025
0.015
0.022
Locomotor ymd
0.046
0.066
(k&r)
Fig. 3. The cost of transport as a function of speed in the red-spotted newts. did not change very much as their exercise speeds were varied. The costs between the two experimental temperatures were not very different (Table 4); 2.46 and 2.20 ml oxygen/g body mass/km for the speed 30 cm/min and 1.51 and 1.47 ml/g/km for the speed 60 cm/min. However, the active costs at 25°C exhibit an obvious declining trend from a high level of 8.92 ml oxygen/g/km at a speed of 30cm/min to lower levels of 4.42 and 2.94 ml/g/km at speeds of 60 and 90 cm/min, respectively. These costs are also significantly different from the ones at 1 and 5°C. The triglyceride concentration from the newts before the reduction of their body mass averaged
0 30 60 0 30 60 90 0 30 60 90
I I 1 5 5 5 5 25 25 25 25
Sample size 8 7 10 10 9 9 9 IO 8 9 9
Body weight (9) 3.26 f 3.34 f 3.60 f 3.72 f 3.90 f 3.83 f 3.83 f 2.18 + I .79 * 2.02 f 2.52 f
0.343 0.328 0.632 0.451 0.613 0.368 0.634 0.302 0.407 0.378 0.588
171.6
The reserved fat is the difference of total body fat of the newts before and after their body mass redoced to 45% of the original weight. The released energy represents the energy from complete oxidation of the -cd fat. The conversion of the fat, energy and oxygen is based on 9.7 calories/mg fat and 4.7 calorks/ml 0, (from Kleibcr, 1961).
23.2 mg/g body weight, whereas the triglyceride concentration from the newts with their body mass reduced to 45% of their original body weight, averaged 1.8 mg/g (Table 5); the difference is obviously significant. By multiplying the body mass by the concentration of the triglyceride, it was computed that the newts averaged 88.5 mg fat in their bodies before the reduction of body mass, however, the fasted newts had only 2.7 mg fat left in the body. From the point of fat reserves, it appears that at least 85.8 mg fat could be stored before winter, since these newts were collected in late fall. This amount of fat storage represents 806.5 kilocalories of energy, and 171.6 ml oxygen would be needed for the complete oxidation of this much lipid. Based on the metabolic rates measured, the energy (calories) needed by a newt every 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. The duration of survival which could be supported by 85 mg fat can also be calculated (Table 4). Theoretically, based on the data for a speed of 0 cm/mitt, a newt could survive 78 days at 1°C 70 days at 5°C and 33 days at 25°C when resting or randomly moving around by consuming the reserved 85 mg fat without any food. If the newt continually exercised at 30 cm/mm, it could survive 48 days at 1°C 46 days at 5°C and 25 days at 25°C at fasting status. The faster a newt locomotes and the higher the ambient temperatures are, the shorter time it could survive without ingesting any food.
Table 4. Speculated survival duration for red-spotted newts (Nofophthdmus virideseens) under different active levels and temneratures without inaestina food Active levels Temperature (cm/min) (“C)
85.8 806.5
0, consumption for a newt (ml/day)
Transport cost (ml Wg/hm)
Survival (days)
2.20 3.56 4.69 2.45 3.70 4.84 6.01 5.14 6.90 7.71 9.60
2.464 1.509 2.195 1.462 1.210 8.917 4.415 2.940
78 48 37 70 46 36 29 33 25 22 18
The survival times were computed assuming that 171.6 ml 0, would be consumed for the complete oxidation of 85.8 mg of reserve fat
SUPINGJIANGand
748 DISCUSSION
Previous studies of urodele metabolism during exercise were generally a~omplished by one of three regimes: first, a treadmill has often been used (Feder, 1988; Full, 1986; Full et al,, 1988). This method has an advantage of being able to track oxygen consump tion by open-flow respirometry when animals are exercising and recovering. The second method requires a custom-design respirometer which is a sealed metabolic chamber rotated vertically by a small eiectric motor at various rates (Feder, 1986). A few pieces of lead are inside the chamber in order to prod the animals to walk forward. These two approaches are advantageous in allowing exact replication of experimental conditions and avoiding the tedium of manual stimulation (Walsberg, 1986), but the animal, in a vertically rotating chamber, might tend to slide and to be pushed downward by the lead prods instead of walking forward because of the vertical rotation. This could distort the measurement of activity metabolism. In the last method, animals in a sealed chamber are manually stimulated to exercise. The manual stimulations include: (a) turning the chamber, and thus, repeatedly flipping the animal onto its dorsum and forcing it to right itself (Withers, 1980; Stefanski et al., 1989), (b) turning a steel rod outside the chamber to move pieces of wire soldered on the rod inside the chamber, so that the animal could be “chased” (Stefanski et al., 1989), and (c)electric shock to keep the animal vigorously moving (Bennett and Licht, 1973). A Iong-existing problem in respirometer designs for small ectotherms is thus how to keep animals naturally moving forward either in water or on land at a given speed. Custom-designed respirometers, such as those described by Feder (1986), Walsberg (1986) and Stefanski et al. (1989) attempted to solve the problem. Although they obtained a great deal of data from their designs, the difficulty in inducing animals to walk normally at an expected speed was not completely resolved. We applied the magnetic principle to our device and successfully overcame this difficulty. Through more than 300 tests on N. viridescens in water (present study) and in air (unpublished data), most of the newts stimulated by the rotating magnetic stirrer locomoted very well inside the respiratory chamber. Only a small portion of the tested animals did not behave as expected, apparently because they were in poor health or condition. The data from these newts, which were pushed by the stirrer most of the time, were discarded. The stimulation from the touch of the spinning stirrer on an animal’s tail is harmless and not greatly different from that they might naturally encounter if they contacted sticks, logs or rocks in ponds. The newts swam or walked in the chamber at the tested speeds without any apparent stress. They were not required to flip over and they could not slide downward. Their locomotion, we believe, was much closer to that in their natural movement. The doughnut-like glass respiratory chamber of this design could be properly enlarged for accommodating some larger salamanders, lizards and even small fishes, or could be reduced for small ectotherms. Therefore, this apparatus could become an effective tool for studying the metabolism of small ectothermic vertebrates.
DENNEL. CLAUS!EN This study was designed to understand how the newts actively overwinter with regard to their bioenergetic budget. Under usual circumstance, redspotted newts do not encounter very warm weather in winter or very cold conditions in summer. Testing newts, acclimated to simulated winter, at summer temperatures provides valuable data on the impact of acclimation. However, these data mostly reflect the situation in the laboratory instead of in nature. In this study, newts were acclimated to both simulated summer (25°C) and winter (S’C) temperatures. Winter water temperatures in ponds in southern Ohio are often around 5”C, and sometimes at 1°C or below when ice is formed. We measured temperatures in our CoIlecting pond of 8°C at noon in early November and in middle March. During the coldest months of January and February, the temperatures in that pond would be substantially lower than those in November and March. Therefore, for the newts in winter acclimation, we only examined their metabolism at I and 5°C. On the other hand, we measured a summer temperature in that pond approaching 24°C in midAugust at noon. We accordingly tested the newts in summer acclimation at 25°C. The locomotor behavior of the red-spotted newts greatly depends upon both temperature and speed. The newts tested at winter temperatures could easily complete their exercise at 30 and 60 cm/min during the tests. They did not display fatigue after finishing their locomotion within the given exercise time. It seems likely that they were capable of swimming in the chamber for an even longer time. But, the newts were nearly exhausted and reluctant to move forward after 15 min exercise at 90 cm/min. When the respiratory chamber rotated at 30 cm/min, the newts often swam a little bit further in the front of the stirrer after the stirrer touched their tails. Then, they stopped moving until the stirrer caught them again and repeated this cycle. At 60 cm/min, they continually swam forward while they were being chased by the stirrer. The newts struggled moving forward and quickly exhausted under the rotation of the chamber at 90 cm/min. The newts display similar behavior when exercised at 25”C, but they did not fatigue even at the velocity of 90 cm/min within the experimental times. This suggests that the optimal speed for red-spotted newts to locomote in water, either in cold winter or in summer, is around 60 cm/min. This optimal speed is similar to the walking speed (6~l~~/min) of Plethodon jordani on land in the field observed by Stefanski et al. (1989). These two species have a similar body mass, but differ in that P. jordani are lungless. Additionally, P. jordani moves on land during nonwinter seasons, whereas N. viride~ce~ adults are generally aquatic in all seasons. Possibly, urodeles with similar body size have similar optimal locomoting speed regardless of their locomotor patterns. This could be attributed to the fact that they have similar stride length. These urodeles could reduce their energy cost if they move at an optimal speed. The metabolism of our red-spotted newts at summer temperatures were generally nearly identical with that of other populations and with other species of lunged salamanders, in spite of different locomotor patterns. Wakeman and Ultsch (1975) reported that red-spotted newts (N. viridescens) from Florida had
Bioenergetic budget for newts an aquatic oxygen uptake value of 90pl/g/hr when resting at 160 mmHg pOz and 25”C, which is very similar to our data at this temperature (Table 3). The maximum oxygen consumption of the red-spotted newts during vigorous movement induced by electric shock at 25°C was measured by Feder (1977). His results were very similar to our exercise data which were around 160 ~1 O,/g/hr at 25”C, even though Feder’s measure was from newts breathing air. The red efts, juvenile newts, have much lower basal metabolism (61 ~1 O,/g/hr) in air at 25°C (Stefanski et al., 1989) than do adult newts. But, the active metabolism of the red efts in air from Stefanski’s study resembles that of adults in water in the present study. No other detailed comparisons can be made for this species. However, the red-spotted newts have a resting metabolic rate in water twice as great as their relatives, Turicha grunulosu, a rough-skinned newt (Feder, 1983) and T. torosu, a California newt (Harlow, 1977; Ultsch, 1976) under comparable experimental conditions. This is probably because the red-spotted newts are smaller than the rough-skinned and California newts. Nevertheless, T. torosa has an unexpectedly high maximum active metabolism of 194 ~1 O,/g/hr with regard to its body mass (Feder, 1977). N. uiridescens are fully aquatic animals as adults. They have adapted to locomote efficiently in water. It is likely that red-spotted newts walking under water tend to consume less energy in locomotion than when walking on land. Also, the newts tested by Feder (1977) were stimulated by electric shock which may have produced unnaturally elevated metabolic rates. Nevertheless, all of these newts display very similar metabolic responses at simulated summer temperatures. The newts tested at 25°C displayed a plateau at speeds of 30 cm/min or higher, which suggests that the energy for supporting the locomotion in summer is supplemented by anaerobiosis. This plateau was also found in the lungless salamander, Desmognuthus ochropheuus (Feder, 1986). This species reached the plateau at a speed around 15 cm/min. The redspotted newts might have a low limit of dissolved oxygen for their activity. Once this limit is surpassed, the newts are forced to rely on anaerobic metabolism. The low oxygen concentration in water in summer could be very close to the limit and there might not be a sufficient oxygen supply for sustained aerobic locomotion. Fortunately, newts can repay this high cost from their abundant food resources in summer. On the other hand, the N. viridescens in our study exhibited no statistically significant differences among metabolic rates between simulated winter temperatures of 1 and 5°C. This implies that the newts maintain metabolism within a narrow range in winter. Maintaining low-level activity in winter probably does not constitute a major increased energy cost over that of inactive hibernation for the newts in Ohio. It thus seems quite possible that the newts could actively survive the cold of winter without major problems of energy shortage, since their overwintering costs do not change drastically with modest changes in temperatures or with slow movements. Also, winter newts are less likely to require anaerobic metabolism since the high dissolved oxygen content in winter may be well above the aerobic limit. Their overwintering activities
149
can be maintained by aerobic metabolism which may economically produce much more energy. Consequently, two different active metabolic regimens seem to exist in red-spotted newts. One is the combination of a high level of aerobic metabolism and anaerobic metabolism, evolved for summer activities. The other is a relatively constant low level of aerobic metabolism which is adapted for overwintering activities. Our results reveal that red-spotted newts may reserve at least 85 mg fat prior to winter. This amount of fat may support the newts surviving 78 days when experimentally resting at simulated winter temperatures. The newts were tested in the respiratory chamber without rotation. We designated this situation as resting metabolism. In fact, the newts were allowed random movement inside the static chamber without any stimulation. Thus, the fat reserves may be a supply for the newts to perform low level activity for nearly 3 months in winter. In addition, our newts for determining fat reserves had been fasted for about 40 days at 5°C before they were placed in liquid nitrogen, because the newts would need 3 weeks to evacuate all food from their digestive system (unpublished data) at such a low temperature of 5°C. The total duration for the newts to last in winter by consuming their final meal in the fall plus their reserved fat would be approximately 4 months, which is the usual winter length in southern Ohio. Furthermore, in nature, movements at relative high speeds would represent only a small portion of the total movements. Such high-speed locomotion also has a low cost of transport (Fig. 3). Thus, the reserved energy may also be sufficient for the newts to survive throughout winter if they are occasionally active at higher levels. Finally, other newts collected at the same time and location as the ones used in this study were maintained in 5°C environmental room. We did not feed them during acclimation. These newts survived for 4 months with only a few casualties. The dead newts appeared to have succumbed to disease rather than to inanition. Therefore, red-spotted newts are able to maintain their activities and survive long and cold winters without ingesting food. In conclusion, the red-spotted newts from southern Ohio during winter could maintain a low level active metabolism within a narrow range. The energy for their winter movements could be mostly accomplished through the processes of aerobiosis which has advantages of economically consuming stored energy. Therefore, the energy reserved by the newts prior to winter is sufficient to support their overwinter activity even in the absence of available food. Acknowledgements-We are grateful to John L. Morrow for building our respirometer and to S. I. Guttman and R. R. Nielson for providing other equipment. Thanks also go to M. F. Wright and J. P. Constanzo for providing specimens. This study was supported by the department of zoology at Miami University and the Theodore Roosevelt Memorial Fund of the American Museum of Natural History (S.J.). REFERENCES
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