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Importance of the limbs in the physiological control of heat exchange in Iguana iguana and Sceloporus undulatus
ARTICLE IN PRESS
Journal of Thermal Biology 29 (2004) 299–305
Importance of the limbs in the physiological control of heat exchange in Iguana iguana and Sceloporus undulatus Edward M. Dzialowski*, Michael P. O’Connor Department of Bioscience and Biotechnology, Drexel University, Philadelphia, PA 19104, USA Received 25 November 2003; accepted 28 April 2004 Available online 1 January 1900
Abstract Lizards use physiological mechanisms to control warming and cooling. Theoretical studies have predicted that the limbs are a major site for the physiological control of heat exchange via changes in blood flow during warming and cooling. To test this we measured thermal time constants in Iguana iguana (1 kg) and Sceloporus undulatus (10 g) warming and cooling in a wind tunnel. We isolated the limbs from the environment by wrapping them in cotton gauze and aluminum foil to test for the importance of the limbs as sites of heat exchange control. We used a physiologically based heat transfer model that included the contribution of blood flow to estimate the thermal time constants. In response to long warming and cooling, the thermal time constants for warming and cooling differed consistently only in I. iguana. Insulating the limbs with cotton gauze abolished the difference between warming and cooling in I. iguana. In S. undulatus, animals with both insulated and uninsulated limbs had warming and cooling thermal time constants that were not significantly different. As predicted, the limbs were a major site for physiological control of heat exchange in 1 kg Iguanas. However, the small lizard was unable to physiologically control warming and cooling and is expected to rely heavily on behavioral thermoregulation. r 2004 Elsevier Ltd. All rights reserved. Keywords: Reptile; Thermal time constant; Thermoregulation; Blood flow; Appendages
1. Introduction Lizards can control rates of heating and cooling by changing peripheral blood flow (Bartholomew, 1982; O’Connor, 1999; Dzialowski and O’Connor, 1999; Seebacher, 2001). The typical pattern for these animals
Abbreviations: t1 ; dominant thermal time constant; t2 ; longest subdominant thermal time constant; t3 ; second longest subdominant thermal time constant; t4 ; shortest subdominant thermal time constant; t1w =t1c ; ratio of the warming dominant thermal time constant over the cooling dominant thermal time constant *Corresponding author. Department of Biological Sciences, University of North Texas, P.O. Box 305220, Denton, TX 76203, USA. Tel.: +1-940-565-3631; fax: +1-940-565-3821. E-mail address: [email protected] (E.M. Dzialowski).
is an increased rate of warming, compared to the rate of cooling (Grigg et al., 1979). A number of theoretical studies have examined specific sites at which changes in blood flow affect heat exchange (Turner and Tracy, 1986; Dzialowski and O’Connor, 1999; O’Connor and Dodson, 1999). Using heat transfer models Turner and Tracy (1986) and Dzialowski and O’Connor (1999) showed that by altering blood flow to the limbs an animal could alter the rates of warming or cooling. In animals up to 1 kg, a 40% increase in cardiac output during warming with the increase in blood flow distributed to the limbs resulted in predicted differences in warming and cooling of similar magnitude to those measured in a number of reptilian species (Grigg et al., 1979). Simulations carried out by O’Connor and Dodson (1999) using a cylindrical model animal further suggested that the limbs in animals of all
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masses should be important sites in the control of heat exchange. In fact, they suggested that in animals less than 1 kg, the limbs should be better heat exchangers than a heat exchange organ such as a sail. Tracy et al., (1986) used a simple model to show that in a 3.2 kg Iguana iguana differences in warming and cooling can be brought about by changes in blood flow at the limbs. At masses larger than about 5 kg changes at both the torso and limbs are required to bring about control of thermoregulation (Dzialowski and O’Connor, 1999). Using a sphere to represent an animal, Turner (1987) predicted that above a body mass is 5 kg an animal’s ability to control heat exchange by changing blood flow at the limbs should diminish. Only one study has been carried out to experimentally examine the role of the limbs in controlling heat exchange (Turner and Tracy, 1983). In juvenile alligators, occlusion of blood flow to the limbs with inflatable cuffs decreased the rate of warming, compared with the rate of warming in the unoccluded animal. The rate of warming upon occlusion was similar to the rate of cooling. During cooling, occlusion had no effect on the rate of temperature change. This suggests that blood flow to the limbs is important in controlling warming and cooling in animals of this size. To understand better, how lizards physiologically control heat exchange, one must know the sites at which the control of heat exchange occurs. To address this problem we experimentally isolated the limbs of I. iguana and Sceloporus undulatus from the environment to determine their contribution to the physiological control of heat exchange. Further knowledge about the sites at which physiological control occurs will allow for greater ability to examine the physiological and ecological importance of heat exchange.
2. Materials and methods Seven S. undulatus were collected in the Lebonnon State Forest, New Jersey in July 1998. Animals were housed individually in 10–20 gallon aquaria and were provided with shelter, water, and a 75 w lamp for thermoregulation. Animals were fed wax worms dusted with supplemental vitamins and calcium (Rep-cal Herptivite and calcium w/vitamin D3) twice a week. One I. iguana was purchased at a local pet store in 1996 and 5 I. iguana were obtained from an I. iguana farm in Costa Rica and transported to Drexel University in January 1997. Iguanas lived either individually or in pairs in large cages and had access to water and a 75 W lamp for basking. They were fed a mixture of greens, vegetables and fruits supplemented with vitamins and calcium (Rep-cal Herptivite and calcium w/vitamin D3) every other day. Experiments on S. undulatus took place
in September 1998 and on I. iguana in November and early December 1998. We conducted warming and cooling experiments on these animals in a temperature-controlled recirculating wind tunnel using the methods of Dzialowski and O’Connor (2001b). We restrained the animals on a wooden dowel with cloth tape (Durapore, 3 M) and placed a 32 or 36 gauge thermocouple coated in Humiseal (type 1B31 Chase Co., Woodside, NY) in the cloaca to measure body temperature. We secured the limbs by taping them to wooden dowels running perpendicular to the long axis of the body. The tape covered less than 5% of the body and had a width of 1 cm and 0.3 cm for the I. iguana and S. undulates, respectively. The lizard was then placed in the wind tunnel with wind speeds of 1 ms1 and mean air temperature of 22.9770.18 C (m7SD for I. iguana) and 21.2470.34 C (m7SD for S. undulatus). I. iguana were initially cooled for 111727.9 min (m7SD) and the S. undulatus were initially cooled for 3777.8 min (m7SD). Once the body temperature was near ambient temperature, we turned on three infrared heat lamps (250 W), heating the animals from above through a Plexiglass window in the top of the wind tunnel. Air temperature was maintained in the wind tunnel by recirculating chilled water through a heat exchanger in the wind tunnel (Neslab HX-300, Portsmouth, NH). Then we turned off the lights and the animal cooled for a similar time period. To determine the length of time for warming and cooling, time constants were initially predicted for each animal using the model described by O’Connor (1999). These predicted time constants were then used to determine the length of time that an animal was warmed. The length of warming and cooling was four times the predicted time constant for a given animal. This insured that all animals were warmed and cooled for similar mass specific time periods. The temperature of a flat black ping pong ball provided a measure of radiative temperature (Dzialowski and O’Connor, 2001b). Air temperature was measured using a thermocouple painted white and shielded by aluminum foil. During the warming and cooling experiment we measured temperatures every second using LabView (National Instruments). To determine the importance of the limbs in the control of heat exchange, we isolated them from the thermal environment. We wrapped the limbs of the animals in cotton gauze that effectively insulated them from the environment and aluminum foil to reflect radiation. We the repeated the above warming and cooling experiment on the same animals. The order of the experimental condition (insulated or uninsulated) was randomly chosen for each animal. Measurements in uninsulated animals were made in conjunction with another study (Dzialowski and O’Connor, 2001b). The six I. iguana used in the study had a mean mass of
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3. Results Dominant time constants increased with mass in I. iguana. The dominant time constants during warming and during cooling differed significantly (P ¼ 0:02) with the dominant time constant during warming being shorter than that during cooling in a given animal. Insulating the limbs from the environment did not significantly affect the dominant time constants
(P ¼ 0:07). However, there was a significant interaction between the animals warming or cooling state and whether the limbs were insulated or uninsulated (P ¼ 0:004; Fig. 1). Dominant time constants for animals warming with uninsulated limbs were significantly shorter than dominant time constants during cooling with uninsulated limbs or during warming with insulated limbs (Po0:005; Fig. 1A). The dominant time constant during warming in uninsulated animals was faster than during insulated cooling but not significantly (P ¼ 0:068). Warming vs. cooling, insulation status of the limbs, and their interactions had no significant effect
4
dominant time constant
3 Residual τ1-τ1mass
2 1 0 -1 -2 Cooling Warming
-3 -4 -5 uninsulated
(A)
insulated
Status of Limbs subdominant time constant 1.0
0.5
Residual τ2-τ2mass
0.955 kg70.22 SD and the seven S. undulatus had a mean mass of 9.97 g72.8 SD. We calculated time constants for these animals from the warming and cooling experiments using the technique described in Dzialowski and O’Connor (2001a). This technique uses the controlled convective and radiative inputs and air temperature in the wind tunnel to estimate body temperature of the animal using a physiologically based biophysical heat transfer model. A genetic algorithm optimization technique was used to find the best fit between the estimated body temperature of the model and the body temperature of the animal by optimizing both warming and cooling blood flow in the model (see Dzialowski and O’Connor 2001a, b; see Michalewicz (1994) for a good description of genetic algorithms). The heat transfer model provided a dominant time constant (t1 ) and three subdominant time constants (t2 ; t3 ; and t4 ). The dominant time constant is the time constant typically measured by physiologists, which describes how body temperature changes over long time periods (Spotila et al., 1973). The subdominant time constant describes how thermal gradients within the body change, upon a change in environmental temperature. Inclusion of the subdominant time constants provides a model with the dynamics that capture how an animal’s body temperature changes when experiencing thermal transients. Only the dominant and largest subdominant time constants will be reported. Multivariate repeated measures analysis of variance using the Proc MIXED command in SAS 8.0 tested for effects of warming vs. cooling, insulated vs. uninsulated limbs, and their interactions. The residuals of the time constants were used in all statistical analysis of the time constants to remove the effect of mass. We calculated residuals of the time constants by subtracting the time constant from the predicted time constant determined from a regression analysis run on the entire data set. Data for the residuals were plotted as the least square means7S.E.M. Post-hoc tests were preformed using Tukey’s test. Differences in the ratio of warming to cooling for each species was determined using a paired Wilcoxon rank-sign test. The level of significance for all tests was Po0:05: All statistics were carried out using SAS version 8.0.
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0.0
-0.5
-1.0 uninsulated
(B)
insulated
Status of Limbs
Fig. 1. Residuals of the dominant and subdominant thermal time constants for I. iguana during warming and cooling with insulated and uninsulated limbs. Residuals were calculated as the difference between the thermal time constant and the predicted thermal time constant. Predicted thermal time constants were calculated from the regression of all thermal time constants on mass for the entire data set for I. iguana. Values are presented as the least square mean 7 SEM (n ¼ 6). (A) Dominant thermal time constants. Squares are the warming thermal time constants and circles are the cooling thermal time constants. (B) Subdominant thermal time constants with symbols as in A.
ARTICLE IN PRESS E.M. Dzialowski, M.P. O’Connor / Journal of Thermal Biology 29 (2004) 299–305 dominant time constant 0.4 0.3
Residual τ1-τ1mass
on the subdominant time constants, but the pattern of the subdominant time constants was similar to that for the dominant time constants (P > 0:05; Fig 1B). In I. iguana, insulating the limbs decreased the difference between the dominant time constant during warming and during cooling as measured by the ratio of t1w =t1c : The difference between the ratio of the dominant time constant during warming to that during cooling in insulated and uninsulated animals was significant (P ¼ 0:016). Animals with their limbs wrapped had a mean ratio of 0.99 7 0.06, while those without insulation had a mean ratio of 0.92 70.03 (m7SD). This difference was mainly due to a slower dominant time constant during warming when the limbs were insulated (Fig. 1A). The mean difference between the dominant time constant during warming and the dominant time constant during cooling for animals with their limbs insulated was 0.59 min and for those with their limbs uninsulated the difference was 4.17 min. There were no significant differences in the ratio of the subdominant time constants during warming and during cooling for animals with insulated and uninsulated limbs (P ¼ 0:844). The dominant time constants increased with mass in S. undulatus. There were no significant effects of warming vs. cooling, insulated vs. uninsulated animals, or their interaction on the dominant time constants (all P > 0:05; Fig. 2A). A similar pattern was observed in the subdominant time constants where no significant differences were observed during warming vs. cooling, in insulated vs uninsulated animals, or the interaction between the two factors (all P > 0:05; Fig. 2B). Insulating the limbs of S. undulatus had no effect on the ability of these animals to control rates of warming and cooling, as measured by the ratio t1w =t1c (insulated = 0.9970.03; uninsulated = 0.9770.05) and t2w =t2c (insulated = 0.9870.18; uninsulated = 0.9570.42) The ratio of both the dominant and subdominant warming to cooling time constants did not differ in the animals under the two conditions (P > 0:05).
Cooling Warming
0.2 0.1 0.0 -0.1 -0.2 -0.3 -0.4 uninsulated
(A)
insulated
Status of Limbs subdominant time constant 0.10
Residual τ2-τ2mass
302
0.05
0.00
-0.05
-0.10 uninsulated
(B)
insulated
Status of Limbs
Fig. 2. Residuals of the dominant and subdominant thermal time constants for S. undulatus during warming and cooling with insulated and uninsulated limbs. Residuals were calculated as in Fig. 1. Values are presented as the least square mean 7 SEM (n ¼ 7). (A) Dominant thermal time constants. Squares are the warming thermal time constants and circles are the cooling thermal time constants. (B) Subdominant thermal time constants with symbols as in A.
4.1. Utility of limbs in I: iguana
4. Discussion The use of insulation to examine physiological control of body temperature during warming and cooling was previously used by one other investigator. Cowles (1958) was able to create effective insulation around the bodies of Dipsosaurus dorsalis and Sauromalus obesus using mink fur. In both species, Cowles (1958) found that the animals wrapped in fur tended to warm more slowly than those without the insulation. Using a similar technique we found that insulating the limbs in I. iguana effectively isolated the limbs from exchanging heat with the environment.
Insulating the limbs from the environment served to increase the warming time constant to values estimated for cooling in I. iguana (Fig. 1A). Dizalowski and O’Connor (1999) predicted that lizards with a mass of 1 kg and a high baseline blood flow should be able to control warming and cooling by changing blood flow in the limbs. However, at low blood flows, changes at both the limbs and torso may be needed to bring about large differences between warming and cooling (Dzialowski and O’Connor, 1999). The results presented here support the theory that the limbs are a major site for the control of heat exchange in ectotherms of 1 kg. Turner and Tracy (1986) suggested that as mass increases the ability of an animal to control heat
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exchange via the limbs should decrease. This is due to a decrease in external resistance at both the limbs and torso with an increase in mass. However, the iguanas used in this study fell within the range of body sizes that Turner and Tracy (1986; torso length o approx. 1.7 m) suggested will not be dominated by external heat exchange. A simulation of warming and cooling in a 3.2 kg Iguana by Tracy et al. (1986) predicted that animals even this size (3.2 kg) could control warming and cooling solely by changing blood flow in the limbs. The effect of insulating the limbs of I. iguana correlated well with results in juvenile Alligator mississippiensis (Turner and Tracy, 1983). Turner and Tracy (1983) attempted to examine the importance of the limbs in controlling heat exchange by intermittently occluding the limbs in juvenile A. mississippiensis. They measured an equilibration rate for warming and cooling rather than actual time constants during warming and cooling due to the protocol they used. Warming was initiated for 5 min then the limbs were occluded for 5 min. This was repeated four times and from this an equilibration rate was calculated. Equilibration rates from the time periods when the limbs were occluded were not significantly different during warming and cooling. When the limbs were not occluded or were intermittently occluded the equilibration rates for warming and cooling were significantly different (Turner and Tracy, 1983). The importance of the limbs in warming and cooling that Turner and Tracy (1983) observed are very similar to those found in this study. Turner (1982) measured equilibration rates for various surfaces of A. mississippiensis and found at the torso during warming and cooling the rates did not differ, but at the limbs the equilibrium rates during cooling were higher than during warming. Even though equilibrium rates were faster during cooling at the limbs, the energy exchanges across the limbs was greatest during warming (Turner, 1987). 4.2. Subdominant time constants This was one of the first studies to estimate subdominant time constants in warming and cooling lizards. The subdominant time constant is important to consider because it helps to describe the disappearance of thermal gradients within an animal’s body though time (Turner, 1987; Voss and Hainsworth, 2001; Dzialowski and O’Connor, 2001). If one wants to accurately estimate and simulate body temperature for animals moving through complex thermal environments one must include these shorter time constants in any model. The effects of insulating the limbs on the subdominant time constant were not significant. However, there was a definite pattern in which the uninsulated subdominant warming time constant was shorter than all of the other subdominant time
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constants, which followed closely the pattern of the dominant time constant (Fig. 1). Even though the repeated measures ANOVA failed to find a significant interaction effect, comparisons of the differences in the least square means for the subdominant time constants indicated a difference between the subdominant time constant for warming and for cooling in animals without insulation (P ¼ 0:02). The main reason the pattern was not as apparent was due to the high variance in the subdominant time constants. Even though they varied from animal to animal, the pattern was still apparent. Subdominant time constants should be included in any predictive model of body temperature (Dzialowski and O’Connor, 2001a).
4.3. Small animals—S: undulatus Small lizards are predicted to control warming and cooling mainly by changes in blood flow at the limbs (Turner, 1987; Dzialowski and O’Connor, 1999). This is thought to be due to the short conductive pathway of the torso of these animals. Any change in blood flow in the torso will have little effect on the overall heat exchange because conduction within the animal already occurs rapidly across the small torso. Thus, changes in blood flow at the surface of the torso should have only limited effects on an animal’s ability to control heat exchange. Changes in blood flow to the limbs, which act as heat exchangers, should be the major site for the control of heat exchange in small lizards. However, in this study, small lizards (S. undulatus) showed no ability to alter t1 or t2 during warming or cooling. The lack of a discernable pattern in the utility of the limbs in S. undulatus may be due to a number of things. The S. undulatus may be too small and thus their time constants may be too small to measure accurately. Another factor may have been that the limbs were too small to effectively wrap. Insulating the limbs may have had an effect on the heat exchange properties of the whole animal, causing changes in the convective coefficient. Insulated animals had thermal time constants that were larger than the uninsulated animals, which may be due to changes in the convective properties of the insulated animals. Alternatively, the limbs may be important in determining how the animal exchanges heat with the environment, but changes in blood flow at the limbs may be small and ineffective in controlling heat exchange. This may be supported by the effect of insulating the limbs on the dominant time constant. The insulated animals had higher dominant time constants than the uninsulated animals. Thus, although changes in blood flow at the limbs may not play a large role in controlling heat exchange under the conditions used in this study, the limbs may still be a site for heat exchange.
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Other investigators found conflicting results in terms of the ratio of dominant thermal time constants for warming and cooling for other small lizards (o30 g). Fraser and Grigg (1983) found that the ratio of the dominant time constant during warming to that during cooling in a number of small skinks (o20 g) ranged from 1.3 to 1.02. However, in that study dead animals had t1w =t1c ratios ranging from 0.92 to 1.14. Sceloperous undulatus and Cnemidophorus sexlineatus heat more rapidly than they cool (McKenna and Packard, 1975). However, in measurements made on S. undulatus, 30% of the animals tested in the spring cooled more rapidly than they warmed (McKenna and Packard, 1975). Kour and Hutchinson (1970) measured warming and cooling in S. occidentalis, Phrynosoma cornutum, Anolis carolinensis, and Xantusia vigilis and found that all animals tended to warm more quickly than they cooled. It is possible that morphological differences in limb and torso proportions may influence the utility of the limbs in these different species and bring about the observed differences. 4.4. Correlation with blood flow To investigate the role of blood flow changes we carried out simulations of a 1 kg and 10 g animal to examine the effect of changing blood flow at the limbs on the thermal time constants (Table 1). The model we used represents the animal as a cylindrical torso with 4 cylindrical limbs and a tail and incorporates blood flow in the layers of the animal. Heat exchange between the torso and the limbs only occurred by advection brought about by the blood flow between the torso and limbs. This allowed us to manipulate the blood flow to the limbs and examine how it affects the thermal time constants (see O’Connor, 1999 for further descriptions of the model). We initially provided the simulated animals with a low blood flow rate during cooling. The thermal time Table 1 Predicted dominant thermal time constants (min) and the ratio t1w =t1c for a 1 kg lizard and a 10 g lizard with various blood flow rates to the limbs. Factorial increase in blood flow
1 kg lizard t1 ðminÞ
t1w =t1c
10 g lizard t1 ðminÞ
t1w =t1c
Cooling 1.5 2 2.5 3
48.3 46.2 44.7 43.7 42.9
1 0.96 0.93 0.91 0.89
5.28 5.14 5.03 4.95 4.87
1 0.97 0.95 0.94 0.92
Time constants were calculated with simulations using a physiologically based heat transfer model described in O’Connor (1999).
constants were then simulated for an animal using a range of blood flows similar to those measured in warming lizards (Table 1). Increasing blood flow to the limbs up to 3 fold to simulate heating resulted in faster time constants in both the 1 kg and 10 g animals (Table 1). Under our simulation conditions, a 1 kg I. iguana would only have to increase blood flow to the limbs by a factor of 2–2.5 to achieve the difference in warming and cooling observed in this study, with a t1w =t1c between 0.93–0.91. Changes in cardiac output during warming and cooling in I. iguana remains to be measured. This simulation suggests that the smaller S. undulatus (10 g) should have some control over warming and cooling by altering blood flow to the limbs. However, this did not bear out in the animals used in this study. The importance of the limbs in controlling warming and cooling correlates well with previous blood flow changes measured at the limbs during warming and cooling (Baker et al., 1972; Dzialowski and O’Connor, 2001b). Using laser doppler surface blood flow probes, blood flow measured at the surface of the limbs during initial warming in I. iguana increased by a factor of 1.3– 3.2. In two animals in which blood flow was measured with implanted laser doppler probes just under the skin, blood flow increased 1.6- and 9- fold during the initial warming. Similar changes have been measured using the 133-Xenon clearance technique. In I. iguana and Tupinambis nigropunctatus blood flow in the limbs responded to warming and cooling (Baker et al., 1972). During both local heating of the tail and hind limb and whole animal heating, cutaneous blood flow increased 1.7-fold, while deep muscle blood flow did not change during warming (Baker et al., 1972). Similar increases in blood flow at the limbs occurred in I. iguana with mean mass of 2.55 kg (Baker et al., 1972). These changes in blood flow during warming and cooling are predicted to be large enough to bring about the differences in thermal time constants observed in the I. iguana (Table 1). As a measure of total blood flow in the limbs, Turner and Tracy (1983) measured brachial artery blood flow in A. mississippiensis. They found that blood flow increased during warming and decreased during cooling (Turner and Tracy, 1983). In the one animal graphically presented by Turner and Tracy (1983) blood flow in the brachial artery increased two fold during warming, which accords well with blood flow measurements of other studies. Turner and Tracy (1983) interpreted this as an indication that blood flow to the limbs changed in the expected directions upon warming and cooling. 4.5. Conclusions There were significant differences in dominant thermal time constants in I. iguana between insulated and uninsulated animals. The limbs appeared to be a major
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site for the physiological control of heat exchange in I. iguana ranging in mass from 0.5 to 1.2 kg. These results support the theoretical predictions that the limbs serve as a major site for the control of heat exchange (Dzialowski and O’Connor, 1999). In the smaller S. undulatus, there was a marginally significant effect of insulating the limbs on heat exchange, suggesting that the limbs were involved in warming and cooling, but that blood flow was not effective in controlling heat exchange. It appeared from this study and a number of others (Dzialowski and O’Connor, 2001b; Fraser and Grigg, 1984) that small lizards should not be able to control rates of warming and cooling to an appreciable level and must rely on behavioral thermoregulation.
Acknowledgements Many of the ecology graduate students at Drexel University helped set up the experiments. J.R. Spotila, S.S. Kilham, and A. Dunham all provided helpful comments on the experimental design and earlier versions of the manuscript. S. undulatus were collected under a New Jersey scientific collecting permit to MPO. This study was funded in part by a Sigma-Xi Grant in Aid of Research to EMD. The animal protocols were approved by the Drexel University IACUC.
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Journal of Thermal Biology 29 (2004) 299–305
Importance of the limbs in the physiological control of heat exchange in Iguana iguana and Sceloporus undulatus Edward M. Dzialowski*, Michael P. O’Connor Department of Bioscience and Biotechnology, Drexel University, Philadelphia, PA 19104, USA Received 25 November 2003; accepted 28 April 2004 Available online 1 January 1900
Abstract Lizards use physiological mechanisms to control warming and cooling. Theoretical studies have predicted that the limbs are a major site for the physiological control of heat exchange via changes in blood flow during warming and cooling. To test this we measured thermal time constants in Iguana iguana (1 kg) and Sceloporus undulatus (10 g) warming and cooling in a wind tunnel. We isolated the limbs from the environment by wrapping them in cotton gauze and aluminum foil to test for the importance of the limbs as sites of heat exchange control. We used a physiologically based heat transfer model that included the contribution of blood flow to estimate the thermal time constants. In response to long warming and cooling, the thermal time constants for warming and cooling differed consistently only in I. iguana. Insulating the limbs with cotton gauze abolished the difference between warming and cooling in I. iguana. In S. undulatus, animals with both insulated and uninsulated limbs had warming and cooling thermal time constants that were not significantly different. As predicted, the limbs were a major site for physiological control of heat exchange in 1 kg Iguanas. However, the small lizard was unable to physiologically control warming and cooling and is expected to rely heavily on behavioral thermoregulation. r 2004 Elsevier Ltd. All rights reserved. Keywords: Reptile; Thermal time constant; Thermoregulation; Blood flow; Appendages
1. Introduction Lizards can control rates of heating and cooling by changing peripheral blood flow (Bartholomew, 1982; O’Connor, 1999; Dzialowski and O’Connor, 1999; Seebacher, 2001). The typical pattern for these animals
Abbreviations: t1 ; dominant thermal time constant; t2 ; longest subdominant thermal time constant; t3 ; second longest subdominant thermal time constant; t4 ; shortest subdominant thermal time constant; t1w =t1c ; ratio of the warming dominant thermal time constant over the cooling dominant thermal time constant *Corresponding author. Department of Biological Sciences, University of North Texas, P.O. Box 305220, Denton, TX 76203, USA. Tel.: +1-940-565-3631; fax: +1-940-565-3821. E-mail address: [email protected] (E.M. Dzialowski).
is an increased rate of warming, compared to the rate of cooling (Grigg et al., 1979). A number of theoretical studies have examined specific sites at which changes in blood flow affect heat exchange (Turner and Tracy, 1986; Dzialowski and O’Connor, 1999; O’Connor and Dodson, 1999). Using heat transfer models Turner and Tracy (1986) and Dzialowski and O’Connor (1999) showed that by altering blood flow to the limbs an animal could alter the rates of warming or cooling. In animals up to 1 kg, a 40% increase in cardiac output during warming with the increase in blood flow distributed to the limbs resulted in predicted differences in warming and cooling of similar magnitude to those measured in a number of reptilian species (Grigg et al., 1979). Simulations carried out by O’Connor and Dodson (1999) using a cylindrical model animal further suggested that the limbs in animals of all
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masses should be important sites in the control of heat exchange. In fact, they suggested that in animals less than 1 kg, the limbs should be better heat exchangers than a heat exchange organ such as a sail. Tracy et al., (1986) used a simple model to show that in a 3.2 kg Iguana iguana differences in warming and cooling can be brought about by changes in blood flow at the limbs. At masses larger than about 5 kg changes at both the torso and limbs are required to bring about control of thermoregulation (Dzialowski and O’Connor, 1999). Using a sphere to represent an animal, Turner (1987) predicted that above a body mass is 5 kg an animal’s ability to control heat exchange by changing blood flow at the limbs should diminish. Only one study has been carried out to experimentally examine the role of the limbs in controlling heat exchange (Turner and Tracy, 1983). In juvenile alligators, occlusion of blood flow to the limbs with inflatable cuffs decreased the rate of warming, compared with the rate of warming in the unoccluded animal. The rate of warming upon occlusion was similar to the rate of cooling. During cooling, occlusion had no effect on the rate of temperature change. This suggests that blood flow to the limbs is important in controlling warming and cooling in animals of this size. To understand better, how lizards physiologically control heat exchange, one must know the sites at which the control of heat exchange occurs. To address this problem we experimentally isolated the limbs of I. iguana and Sceloporus undulatus from the environment to determine their contribution to the physiological control of heat exchange. Further knowledge about the sites at which physiological control occurs will allow for greater ability to examine the physiological and ecological importance of heat exchange.
2. Materials and methods Seven S. undulatus were collected in the Lebonnon State Forest, New Jersey in July 1998. Animals were housed individually in 10–20 gallon aquaria and were provided with shelter, water, and a 75 w lamp for thermoregulation. Animals were fed wax worms dusted with supplemental vitamins and calcium (Rep-cal Herptivite and calcium w/vitamin D3) twice a week. One I. iguana was purchased at a local pet store in 1996 and 5 I. iguana were obtained from an I. iguana farm in Costa Rica and transported to Drexel University in January 1997. Iguanas lived either individually or in pairs in large cages and had access to water and a 75 W lamp for basking. They were fed a mixture of greens, vegetables and fruits supplemented with vitamins and calcium (Rep-cal Herptivite and calcium w/vitamin D3) every other day. Experiments on S. undulatus took place
in September 1998 and on I. iguana in November and early December 1998. We conducted warming and cooling experiments on these animals in a temperature-controlled recirculating wind tunnel using the methods of Dzialowski and O’Connor (2001b). We restrained the animals on a wooden dowel with cloth tape (Durapore, 3 M) and placed a 32 or 36 gauge thermocouple coated in Humiseal (type 1B31 Chase Co., Woodside, NY) in the cloaca to measure body temperature. We secured the limbs by taping them to wooden dowels running perpendicular to the long axis of the body. The tape covered less than 5% of the body and had a width of 1 cm and 0.3 cm for the I. iguana and S. undulates, respectively. The lizard was then placed in the wind tunnel with wind speeds of 1 ms1 and mean air temperature of 22.9770.18 C (m7SD for I. iguana) and 21.2470.34 C (m7SD for S. undulatus). I. iguana were initially cooled for 111727.9 min (m7SD) and the S. undulatus were initially cooled for 3777.8 min (m7SD). Once the body temperature was near ambient temperature, we turned on three infrared heat lamps (250 W), heating the animals from above through a Plexiglass window in the top of the wind tunnel. Air temperature was maintained in the wind tunnel by recirculating chilled water through a heat exchanger in the wind tunnel (Neslab HX-300, Portsmouth, NH). Then we turned off the lights and the animal cooled for a similar time period. To determine the length of time for warming and cooling, time constants were initially predicted for each animal using the model described by O’Connor (1999). These predicted time constants were then used to determine the length of time that an animal was warmed. The length of warming and cooling was four times the predicted time constant for a given animal. This insured that all animals were warmed and cooled for similar mass specific time periods. The temperature of a flat black ping pong ball provided a measure of radiative temperature (Dzialowski and O’Connor, 2001b). Air temperature was measured using a thermocouple painted white and shielded by aluminum foil. During the warming and cooling experiment we measured temperatures every second using LabView (National Instruments). To determine the importance of the limbs in the control of heat exchange, we isolated them from the thermal environment. We wrapped the limbs of the animals in cotton gauze that effectively insulated them from the environment and aluminum foil to reflect radiation. We the repeated the above warming and cooling experiment on the same animals. The order of the experimental condition (insulated or uninsulated) was randomly chosen for each animal. Measurements in uninsulated animals were made in conjunction with another study (Dzialowski and O’Connor, 2001b). The six I. iguana used in the study had a mean mass of
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3. Results Dominant time constants increased with mass in I. iguana. The dominant time constants during warming and during cooling differed significantly (P ¼ 0:02) with the dominant time constant during warming being shorter than that during cooling in a given animal. Insulating the limbs from the environment did not significantly affect the dominant time constants
(P ¼ 0:07). However, there was a significant interaction between the animals warming or cooling state and whether the limbs were insulated or uninsulated (P ¼ 0:004; Fig. 1). Dominant time constants for animals warming with uninsulated limbs were significantly shorter than dominant time constants during cooling with uninsulated limbs or during warming with insulated limbs (Po0:005; Fig. 1A). The dominant time constant during warming in uninsulated animals was faster than during insulated cooling but not significantly (P ¼ 0:068). Warming vs. cooling, insulation status of the limbs, and their interactions had no significant effect
4
dominant time constant
3 Residual τ1-τ1mass
2 1 0 -1 -2 Cooling Warming
-3 -4 -5 uninsulated
(A)
insulated
Status of Limbs subdominant time constant 1.0
0.5
Residual τ2-τ2mass
0.955 kg70.22 SD and the seven S. undulatus had a mean mass of 9.97 g72.8 SD. We calculated time constants for these animals from the warming and cooling experiments using the technique described in Dzialowski and O’Connor (2001a). This technique uses the controlled convective and radiative inputs and air temperature in the wind tunnel to estimate body temperature of the animal using a physiologically based biophysical heat transfer model. A genetic algorithm optimization technique was used to find the best fit between the estimated body temperature of the model and the body temperature of the animal by optimizing both warming and cooling blood flow in the model (see Dzialowski and O’Connor 2001a, b; see Michalewicz (1994) for a good description of genetic algorithms). The heat transfer model provided a dominant time constant (t1 ) and three subdominant time constants (t2 ; t3 ; and t4 ). The dominant time constant is the time constant typically measured by physiologists, which describes how body temperature changes over long time periods (Spotila et al., 1973). The subdominant time constant describes how thermal gradients within the body change, upon a change in environmental temperature. Inclusion of the subdominant time constants provides a model with the dynamics that capture how an animal’s body temperature changes when experiencing thermal transients. Only the dominant and largest subdominant time constants will be reported. Multivariate repeated measures analysis of variance using the Proc MIXED command in SAS 8.0 tested for effects of warming vs. cooling, insulated vs. uninsulated limbs, and their interactions. The residuals of the time constants were used in all statistical analysis of the time constants to remove the effect of mass. We calculated residuals of the time constants by subtracting the time constant from the predicted time constant determined from a regression analysis run on the entire data set. Data for the residuals were plotted as the least square means7S.E.M. Post-hoc tests were preformed using Tukey’s test. Differences in the ratio of warming to cooling for each species was determined using a paired Wilcoxon rank-sign test. The level of significance for all tests was Po0:05: All statistics were carried out using SAS version 8.0.
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0.0
-0.5
-1.0 uninsulated
(B)
insulated
Status of Limbs
Fig. 1. Residuals of the dominant and subdominant thermal time constants for I. iguana during warming and cooling with insulated and uninsulated limbs. Residuals were calculated as the difference between the thermal time constant and the predicted thermal time constant. Predicted thermal time constants were calculated from the regression of all thermal time constants on mass for the entire data set for I. iguana. Values are presented as the least square mean 7 SEM (n ¼ 6). (A) Dominant thermal time constants. Squares are the warming thermal time constants and circles are the cooling thermal time constants. (B) Subdominant thermal time constants with symbols as in A.
ARTICLE IN PRESS E.M. Dzialowski, M.P. O’Connor / Journal of Thermal Biology 29 (2004) 299–305 dominant time constant 0.4 0.3
Residual τ1-τ1mass
on the subdominant time constants, but the pattern of the subdominant time constants was similar to that for the dominant time constants (P > 0:05; Fig 1B). In I. iguana, insulating the limbs decreased the difference between the dominant time constant during warming and during cooling as measured by the ratio of t1w =t1c : The difference between the ratio of the dominant time constant during warming to that during cooling in insulated and uninsulated animals was significant (P ¼ 0:016). Animals with their limbs wrapped had a mean ratio of 0.99 7 0.06, while those without insulation had a mean ratio of 0.92 70.03 (m7SD). This difference was mainly due to a slower dominant time constant during warming when the limbs were insulated (Fig. 1A). The mean difference between the dominant time constant during warming and the dominant time constant during cooling for animals with their limbs insulated was 0.59 min and for those with their limbs uninsulated the difference was 4.17 min. There were no significant differences in the ratio of the subdominant time constants during warming and during cooling for animals with insulated and uninsulated limbs (P ¼ 0:844). The dominant time constants increased with mass in S. undulatus. There were no significant effects of warming vs. cooling, insulated vs. uninsulated animals, or their interaction on the dominant time constants (all P > 0:05; Fig. 2A). A similar pattern was observed in the subdominant time constants where no significant differences were observed during warming vs. cooling, in insulated vs uninsulated animals, or the interaction between the two factors (all P > 0:05; Fig. 2B). Insulating the limbs of S. undulatus had no effect on the ability of these animals to control rates of warming and cooling, as measured by the ratio t1w =t1c (insulated = 0.9970.03; uninsulated = 0.9770.05) and t2w =t2c (insulated = 0.9870.18; uninsulated = 0.9570.42) The ratio of both the dominant and subdominant warming to cooling time constants did not differ in the animals under the two conditions (P > 0:05).
Cooling Warming
0.2 0.1 0.0 -0.1 -0.2 -0.3 -0.4 uninsulated
(A)
insulated
Status of Limbs subdominant time constant 0.10
Residual τ2-τ2mass
302
0.05
0.00
-0.05
-0.10 uninsulated
(B)
insulated
Status of Limbs
Fig. 2. Residuals of the dominant and subdominant thermal time constants for S. undulatus during warming and cooling with insulated and uninsulated limbs. Residuals were calculated as in Fig. 1. Values are presented as the least square mean 7 SEM (n ¼ 7). (A) Dominant thermal time constants. Squares are the warming thermal time constants and circles are the cooling thermal time constants. (B) Subdominant thermal time constants with symbols as in A.
4.1. Utility of limbs in I: iguana
4. Discussion The use of insulation to examine physiological control of body temperature during warming and cooling was previously used by one other investigator. Cowles (1958) was able to create effective insulation around the bodies of Dipsosaurus dorsalis and Sauromalus obesus using mink fur. In both species, Cowles (1958) found that the animals wrapped in fur tended to warm more slowly than those without the insulation. Using a similar technique we found that insulating the limbs in I. iguana effectively isolated the limbs from exchanging heat with the environment.
Insulating the limbs from the environment served to increase the warming time constant to values estimated for cooling in I. iguana (Fig. 1A). Dizalowski and O’Connor (1999) predicted that lizards with a mass of 1 kg and a high baseline blood flow should be able to control warming and cooling by changing blood flow in the limbs. However, at low blood flows, changes at both the limbs and torso may be needed to bring about large differences between warming and cooling (Dzialowski and O’Connor, 1999). The results presented here support the theory that the limbs are a major site for the control of heat exchange in ectotherms of 1 kg. Turner and Tracy (1986) suggested that as mass increases the ability of an animal to control heat
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exchange via the limbs should decrease. This is due to a decrease in external resistance at both the limbs and torso with an increase in mass. However, the iguanas used in this study fell within the range of body sizes that Turner and Tracy (1986; torso length o approx. 1.7 m) suggested will not be dominated by external heat exchange. A simulation of warming and cooling in a 3.2 kg Iguana by Tracy et al. (1986) predicted that animals even this size (3.2 kg) could control warming and cooling solely by changing blood flow in the limbs. The effect of insulating the limbs of I. iguana correlated well with results in juvenile Alligator mississippiensis (Turner and Tracy, 1983). Turner and Tracy (1983) attempted to examine the importance of the limbs in controlling heat exchange by intermittently occluding the limbs in juvenile A. mississippiensis. They measured an equilibration rate for warming and cooling rather than actual time constants during warming and cooling due to the protocol they used. Warming was initiated for 5 min then the limbs were occluded for 5 min. This was repeated four times and from this an equilibration rate was calculated. Equilibration rates from the time periods when the limbs were occluded were not significantly different during warming and cooling. When the limbs were not occluded or were intermittently occluded the equilibration rates for warming and cooling were significantly different (Turner and Tracy, 1983). The importance of the limbs in warming and cooling that Turner and Tracy (1983) observed are very similar to those found in this study. Turner (1982) measured equilibration rates for various surfaces of A. mississippiensis and found at the torso during warming and cooling the rates did not differ, but at the limbs the equilibrium rates during cooling were higher than during warming. Even though equilibrium rates were faster during cooling at the limbs, the energy exchanges across the limbs was greatest during warming (Turner, 1987). 4.2. Subdominant time constants This was one of the first studies to estimate subdominant time constants in warming and cooling lizards. The subdominant time constant is important to consider because it helps to describe the disappearance of thermal gradients within an animal’s body though time (Turner, 1987; Voss and Hainsworth, 2001; Dzialowski and O’Connor, 2001). If one wants to accurately estimate and simulate body temperature for animals moving through complex thermal environments one must include these shorter time constants in any model. The effects of insulating the limbs on the subdominant time constant were not significant. However, there was a definite pattern in which the uninsulated subdominant warming time constant was shorter than all of the other subdominant time
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constants, which followed closely the pattern of the dominant time constant (Fig. 1). Even though the repeated measures ANOVA failed to find a significant interaction effect, comparisons of the differences in the least square means for the subdominant time constants indicated a difference between the subdominant time constant for warming and for cooling in animals without insulation (P ¼ 0:02). The main reason the pattern was not as apparent was due to the high variance in the subdominant time constants. Even though they varied from animal to animal, the pattern was still apparent. Subdominant time constants should be included in any predictive model of body temperature (Dzialowski and O’Connor, 2001a).
4.3. Small animals—S: undulatus Small lizards are predicted to control warming and cooling mainly by changes in blood flow at the limbs (Turner, 1987; Dzialowski and O’Connor, 1999). This is thought to be due to the short conductive pathway of the torso of these animals. Any change in blood flow in the torso will have little effect on the overall heat exchange because conduction within the animal already occurs rapidly across the small torso. Thus, changes in blood flow at the surface of the torso should have only limited effects on an animal’s ability to control heat exchange. Changes in blood flow to the limbs, which act as heat exchangers, should be the major site for the control of heat exchange in small lizards. However, in this study, small lizards (S. undulatus) showed no ability to alter t1 or t2 during warming or cooling. The lack of a discernable pattern in the utility of the limbs in S. undulatus may be due to a number of things. The S. undulatus may be too small and thus their time constants may be too small to measure accurately. Another factor may have been that the limbs were too small to effectively wrap. Insulating the limbs may have had an effect on the heat exchange properties of the whole animal, causing changes in the convective coefficient. Insulated animals had thermal time constants that were larger than the uninsulated animals, which may be due to changes in the convective properties of the insulated animals. Alternatively, the limbs may be important in determining how the animal exchanges heat with the environment, but changes in blood flow at the limbs may be small and ineffective in controlling heat exchange. This may be supported by the effect of insulating the limbs on the dominant time constant. The insulated animals had higher dominant time constants than the uninsulated animals. Thus, although changes in blood flow at the limbs may not play a large role in controlling heat exchange under the conditions used in this study, the limbs may still be a site for heat exchange.
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Other investigators found conflicting results in terms of the ratio of dominant thermal time constants for warming and cooling for other small lizards (o30 g). Fraser and Grigg (1983) found that the ratio of the dominant time constant during warming to that during cooling in a number of small skinks (o20 g) ranged from 1.3 to 1.02. However, in that study dead animals had t1w =t1c ratios ranging from 0.92 to 1.14. Sceloperous undulatus and Cnemidophorus sexlineatus heat more rapidly than they cool (McKenna and Packard, 1975). However, in measurements made on S. undulatus, 30% of the animals tested in the spring cooled more rapidly than they warmed (McKenna and Packard, 1975). Kour and Hutchinson (1970) measured warming and cooling in S. occidentalis, Phrynosoma cornutum, Anolis carolinensis, and Xantusia vigilis and found that all animals tended to warm more quickly than they cooled. It is possible that morphological differences in limb and torso proportions may influence the utility of the limbs in these different species and bring about the observed differences. 4.4. Correlation with blood flow To investigate the role of blood flow changes we carried out simulations of a 1 kg and 10 g animal to examine the effect of changing blood flow at the limbs on the thermal time constants (Table 1). The model we used represents the animal as a cylindrical torso with 4 cylindrical limbs and a tail and incorporates blood flow in the layers of the animal. Heat exchange between the torso and the limbs only occurred by advection brought about by the blood flow between the torso and limbs. This allowed us to manipulate the blood flow to the limbs and examine how it affects the thermal time constants (see O’Connor, 1999 for further descriptions of the model). We initially provided the simulated animals with a low blood flow rate during cooling. The thermal time Table 1 Predicted dominant thermal time constants (min) and the ratio t1w =t1c for a 1 kg lizard and a 10 g lizard with various blood flow rates to the limbs. Factorial increase in blood flow
1 kg lizard t1 ðminÞ
t1w =t1c
10 g lizard t1 ðminÞ
t1w =t1c
Cooling 1.5 2 2.5 3
48.3 46.2 44.7 43.7 42.9
1 0.96 0.93 0.91 0.89
5.28 5.14 5.03 4.95 4.87
1 0.97 0.95 0.94 0.92
Time constants were calculated with simulations using a physiologically based heat transfer model described in O’Connor (1999).
constants were then simulated for an animal using a range of blood flows similar to those measured in warming lizards (Table 1). Increasing blood flow to the limbs up to 3 fold to simulate heating resulted in faster time constants in both the 1 kg and 10 g animals (Table 1). Under our simulation conditions, a 1 kg I. iguana would only have to increase blood flow to the limbs by a factor of 2–2.5 to achieve the difference in warming and cooling observed in this study, with a t1w =t1c between 0.93–0.91. Changes in cardiac output during warming and cooling in I. iguana remains to be measured. This simulation suggests that the smaller S. undulatus (10 g) should have some control over warming and cooling by altering blood flow to the limbs. However, this did not bear out in the animals used in this study. The importance of the limbs in controlling warming and cooling correlates well with previous blood flow changes measured at the limbs during warming and cooling (Baker et al., 1972; Dzialowski and O’Connor, 2001b). Using laser doppler surface blood flow probes, blood flow measured at the surface of the limbs during initial warming in I. iguana increased by a factor of 1.3– 3.2. In two animals in which blood flow was measured with implanted laser doppler probes just under the skin, blood flow increased 1.6- and 9- fold during the initial warming. Similar changes have been measured using the 133-Xenon clearance technique. In I. iguana and Tupinambis nigropunctatus blood flow in the limbs responded to warming and cooling (Baker et al., 1972). During both local heating of the tail and hind limb and whole animal heating, cutaneous blood flow increased 1.7-fold, while deep muscle blood flow did not change during warming (Baker et al., 1972). Similar increases in blood flow at the limbs occurred in I. iguana with mean mass of 2.55 kg (Baker et al., 1972). These changes in blood flow during warming and cooling are predicted to be large enough to bring about the differences in thermal time constants observed in the I. iguana (Table 1). As a measure of total blood flow in the limbs, Turner and Tracy (1983) measured brachial artery blood flow in A. mississippiensis. They found that blood flow increased during warming and decreased during cooling (Turner and Tracy, 1983). In the one animal graphically presented by Turner and Tracy (1983) blood flow in the brachial artery increased two fold during warming, which accords well with blood flow measurements of other studies. Turner and Tracy (1983) interpreted this as an indication that blood flow to the limbs changed in the expected directions upon warming and cooling. 4.5. Conclusions There were significant differences in dominant thermal time constants in I. iguana between insulated and uninsulated animals. The limbs appeared to be a major
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site for the physiological control of heat exchange in I. iguana ranging in mass from 0.5 to 1.2 kg. These results support the theoretical predictions that the limbs serve as a major site for the control of heat exchange (Dzialowski and O’Connor, 1999). In the smaller S. undulatus, there was a marginally significant effect of insulating the limbs on heat exchange, suggesting that the limbs were involved in warming and cooling, but that blood flow was not effective in controlling heat exchange. It appeared from this study and a number of others (Dzialowski and O’Connor, 2001b; Fraser and Grigg, 1984) that small lizards should not be able to control rates of warming and cooling to an appreciable level and must rely on behavioral thermoregulation.
Acknowledgements Many of the ecology graduate students at Drexel University helped set up the experiments. J.R. Spotila, S.S. Kilham, and A. Dunham all provided helpful comments on the experimental design and earlier versions of the manuscript. S. undulatus were collected under a New Jersey scientific collecting permit to MPO. This study was funded in part by a Sigma-Xi Grant in Aid of Research to EMD. The animal protocols were approved by the Drexel University IACUC.
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