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Effects of cold acclimation on the standard metabolic rate and osmotic fragility of erythrocytes in an aquatic anura, Xenopus laevis
03(K)-9629:82.110431-06803.00’0 0 1%~ krgamon Press Ltd
Camp. Biochem. Physial. Vol. 73A. No. 3. pp. 431 to 436, 1982 Printed in Great Britain.
EFFECTS OF COLD ACCLIMATION ON THE STANDARD METABOLIC RATE AND OSMOTIC FRAGILITY OF ERYTHROCYTES IN AN AQUATIC ANURA, X~~~PUS LAEVIS KATSUJI TSUGAWA Department
of Biological
Sciences, Osaka Women’s Sakai 590, Japan (Receiwd
19 February
University,
Daisen-cho
2,
1982)
The rates of standard metabolism (CO1 production) were measured in intact Xenopus in&s faecis acclimated to 14 and 25°C using an open-circuit system at IS, 20 and 25°C. 2. The rates were increased by 15-22x 3-8 weeks after acclimation to 14°C at every temperature measured, showing the partial compensation. 3. Erythrocytes from cold-acclimated toads tolerated lower concentration of NaCl solution under physiological conditions (pH 7.4, 25°C) and O’C (pH 6.2 and 7.4) than those from warm-acclimated toads. 4. Incubation of erythrocytes at 37°C abolished the differences between acclimation temperatures, due to more pronounced increase of osmotic sensitivity by effect of high temperature in the cold-acclimated toads. Abstract-l.
INTRODUCTION
The metabolism of amphibians changes during thermal acclimation as in many other poikilotherm~c animals. In anura, some show the reasonable compensation in respiratory rate [B. horeas (Bishop & Gordon, 1967; Carey, 1979), R. temporuria (Jankowsky, 1960; Harri, 197311, while others show the inverse compensation CR. remporaria in summer (Harri, 1973), R. catesheiana (Weathers, 1976), Pseuducris triseriata (Packard, 1972; Dunlap, 1980), Acris crepitans (Dunlap, 1973. 198O)J Thermoregulation of amphibia is mainly behavioral, including selection of microclimates (Brattstrom, 1970). In aquatic anura, however, thermal acclimation seems to be essentially physiological, since they are exposed directly to the environmental temperatures from which they cannot escape. Therefore, thermal acclimation of such forms can be analyzed more easily than those of terrestrial or semi-terrestrial forms. However, despite relatively large number of studies in the latter forms, only a few ,have been done in the former (Tsugawa, 1976, 1980; De Costa et al., 1981). In the previous experiments, isozyme patterns and apparent K, of pyruvate for lactate dehydrogenase of liver tissue were found to change with cold acclimation of Xenopus laevis laevis, a typical aquatic anura (Tsugawa, 1976, 1980). The effect of cold acclimation on the tissue lactate dehydrogenase activities and isozyme patterns were also reported in another aquatic anura, Discoglossus pictus pictus (De Costa et al., 1981). But changes in metabolic rates of the intact animals during thermal acclimation were left unconfirmed. So, firstly, the effect of thermal acclimation on the rate of CO2 production of intact animals was determined. 431
Acclimation temperature affects membrane-associated phenomena and constituents of lipid fractions in many poikilothermic animals (e.g. Martner & Almon, 1981 in R. pipiens). In nurse shark erythrocytes a reduction of the acclimation temperature was reported to increase resistance to hemolysis (Abramowitz et al., 1977). So, secondly, the osmotic fragility of X. Iaecis erythrocytes was determined to demonstrate an example of the effect of thermal acclimation on membrane-associated phenomena in this toad. The data demonstrate that the African clawed toad, X. laeois, can partially compensate the temperatureinduced alteration in respiratory rate by increasing its capacity, and that acclimation temperature also affects a characteristic of erythrocytes in this animal. MATERIALS
AND METHODS
Animals Adult X. IaeGis, hatched from eggs in the laboratory, were routinely stored at 1%2o’C, then divided randomly into two groups and kept at cold (14 i 1’C) and warm (24-25°C) temperatures. Fluorescent lieht orovided a 13L: IlD photoperiod (083G-2130 light) 111 a ‘light-sealed room. They were fed on cattle liver twice a week at 1%25°C or once a week at 14’C.
Stundard metabolic rate (SMR) Resting metabolism of intact female toads, 2-4yr old, 17-52 g body wt, was determined by measuring the rate of CO, production using an open-circuit system. Toads were fasted 2-3 days before and during each measurement. Each animal was placed in a 1.5 1 respiratory chamber (aluminium box with plastic ceiling) with 0.3 I of tap water in which the toad was almost completely submerged. Respiratory chamber(s) and spiral ducts for supplying fresh air were kept dark and immersed into a water bath, the temperature of which was changed from the temperature of acclimation to that of measurement at a rate of about
432
KAWIJI TWGAWA
1L2”C every 5 min and controlled within &O.S’C. The Co, concentrations of water CO2 and air phases were equilibrated continuously by blowing bubbles of fresh air from the air bag to the water, with an air flow rate of 0.5-0.6, 0.7 and 0.7-0.8 jimin at 15. 20 and 25”C, respectively, The air leaving the respiratory chamber was channelled to an air dessicating chamber and COz analyzer (Hitachi-Horiba EIA-IA. full scale 600 ppm; Horiba-seisakusho, Kyoto) through a flowmeter and an automatic change-over valve, and the concentration of COZ was recorded at 1 or 3-min intervals repeatedly for 1 hr and 45 min following monitoring of the concentration of the supplied air for IS min. Preliminary experiments indicated that the rate of CO, production occasionally declined, even if the recording chart showed a relatively inactive state (low frequency of expiration), up to 8-10 hr after an animal was placed in the respiratory chamber. Therefore, after a 10 hr or longer eqL]i~ib~tion period. the rate of CO2 production was measured for i@20hr at a temperature of measurement. Often, measurements at other temperatures were followed. After measurements were carried out at an acclimation temperature, toads were transferred to the other acclimation temperature. The rates of CO, production of an animal were calculated for each 1.75-hr period. and the results were sorted using the following criteria: (I) Not more than the two highest values among the 35 observed at 3-min intervals exceeded twice the average for the 7 lowest ones among them and (2) not more than the 7 highest values exceeded 1.5~times the average for the lowest 7. An average of the lowest 1:3 among the values corresponding to the two items was regarded as the standard metabolic rate (SMR) of the measurement. SMR of toads correlated with 3i4-powers of their body wt rather than the weight itself. so that values were expressed as ,J CO, producedimin per 3/4-powers of g body wt. Body wt of each toad fluctuated during the experimental period but the changes were irregular. Over 4-10 months of experiments the curves of body wt of each toad did not exhibit net increase or decrease in weight. Therefore. the mean values for the period of experiment were used as representations of their body wt. Mean SMRs of toads acclimated to either cold or warm temperature were compared with those of the same animals after acclimation to the other temperature using the Student t-tests of paired and unpaired observations.
Male and female toads. 15-44 g body wt. were sacrificed by decapitation at 0900- 1200 in December-February. Blood obtained from the carotid artery or heart was immediately mixed with phosphate buffered saline (PBS; 6.1 g NaCI. 0.15 g KCI. 0.876 g Na,HPO, and 0.153 g KH,PO, in I I of water. pH 7.4) containing 20 units/ml heparin. After the blood suspension was diluted with 3 vol. of heparin-free PBS, a few small aggregates were removed. Then, diluted blood was centrifuged at 700 rev,‘min for 3 min and washed twice with heparin-free PBS at room temperature. Experiments were carried out on blood samples from individual toads without pooling of sample. Twenty 10 50 ~1 of fresh erythrocyte suspension in heparinfree PBS was added to a test tube containing 3 ml of prewarmed or pre-cooled NaCi solution with 2 mM Na-PO,. pH 6.2 or 7.4. After itlcubation at 0, 25 or 37°C for 10 or 30 min. the test tubes were immediately centrifuged at 800 rev;min for 3 min and the resulting supernatant was quickly removed. The absorbance of the supernatant was determined at 577 nm and plotted as a function of the concentration of
NaCl solution. Mean corpuscular fragility (MCF), the concentration of NaCl in which 505; of the erythrocytes are hemolyzed (Catlett & Millich, 1976). was determined the hemolysis curves by interpolation.
from
RESCILTS
Typical records of respiratory activity of a toad at 2S”C are shown in Fig, 1. Rapid increase in CO, concentration followed by rapid decline indicates expiratory movement. The frequency of the expiratory movement was high during active movement of the toad. and therefore used as a criterion to distinguish the resting state from the active state. Figure 2 shows the “physiological” and the “unphysiological” SMRs at different temperatures of measurement in toads acclimated to cold or warm temperature for 3-8 weeks. The “physiological” SMR means the rates measured at the acclimation temperature prior to measurement at other temperatures or those after stepwisc upward or downward change in the measurement temperature from acclimation temperature. The ‘~unphysiolo~jc~I1” SMR meitns the rates measured firstly at the temperature opposed to that of their acclimation or those at other temperatures following such measurement. SMR at 2O”C, irrespective of whether physiological or unphysiological. was higher in cold-acclimation than warm-acclimation phase. Physiological SMR of cold-acclimated toads was higher bv 9”:. than that of
PPM
600
Fig. I. Carbon dioxide production of a toad in active (A) and standard (3) states at 25°C. Air flow rate, 0.80 I/mini body wt of the toad. 43 g. Carbon dioxide concentration above 600 ppm is for reference because it was recorded in a range over full scale of analy7er used.
25
20
15
Temp. CC) F1g ? Standard metabolic rate (SMR) and SEM of female toads accltmated to I? I4.C (open symbols) and to 24 25 C (closed symbols) for 3 8 weeks. “Phystoloplcal” (P: 0) and “unphysiological” (U; Cl) SMRs related to the order of exposure 10 the temperature of measurements. DiR’erences m “physiological” SMRs between the two acclimation temperatures were significant by paired r-test. For details. see text.
Table
1 ShiR
warm-acclimated toads. but comparisons of each toad between the cold and warm-acclimation phases showed a significant difference (P < 0.02) of about 15”;,. Difference in unphysiological SMRs was also significant at this temperature (P < 0.05). SMR at 25‘C was also higher in cold-acclimation phase. Comparisons of physiological SMR of each toad exhibited statistically significant difference (P < 0.05). At 15 C. physiological SMR was increased by 20”;, during cold acclimation as compared with warm-acclimation phase (P < 0.01 by paired t-test). Table 1 shows SMRs after different prrlods of acchmation. Physiological SMR at 20cC cxhibttcd a tendcncy to increase 1.5 2.5 weeks after acclimation to cold. though this was not statistically significant. presumably due to the small number of measurements. Three to 6 weeks after acclimation. SMR of coldacclimated toads was higher by IO”,, than that of warm-acclimated toads (P < 0.05 by palred r-test). SMR at 2o‘C of cold-acclimated toads. and therefore the difference in SMR between the two acclimation temperatures. tended to decrease with duration of acclimation. At 15 and 25’C’. however. differences in physiological SMR between the two acclimation tzmpcratures increased with extending period of acchmation. The order of exposure to the temperature of measurement affected the SMR in some cases. When toads acclimated to 25°C were exposed to 15 C immediately from their acclimation temperature, SMR showed a higher rate compared IO the value when toads were exposed via 20 C. in spite of rhe long
of toads acchmatcd to Lold or uarm temperature for ditferent periods. Vaiucs per g ” of body WI) 2 SEM wtth the number of toads measured in parentheses
arc’ mean SMR (~1 CO,;min
(‘C)
Duration of accl. (weeks)
I5
1.5 2.5
Temp. of measurement
.Acclimation
(14-C) p:
u 36 .:- 6.5
20
I.5 2.5 36 ;I b.5
‘> 6.5
temperature (25’ c‘)
c::w*
c:wt .--
1.71 x 0.07 (8)
P U P I:
1.X0 1.96 1.79 I .x4 1.82
+ + + * f
0.16(4) 0.14(12) 0.23 (6) 0.09 (101 0.15 (5)
P L! P I: P II
2.96 3.43 2.88 2 6X 2.66 3.36
2 & f * + k
0.36 (41 0.08 (3) O.l7(L?I 0.29(2) 0.16(X) 0.14 (6)
P U P t; P U
3.45 4.17 4.25 3.8? 3.69 4.79
+ * + I + ?
0.74(3) 0.25(31 0.33 (12) 0.68 (5) 0.22 (7) 0.94 (6)
1.69+ 0.I2 (1 I I I .9J * 0.34 (4) I.46 + 0.07 (8) 1.76 _c 0.17(41
I.16 0.92 1.26, 1.04
1.17”(Y)) 0.93(l) I .‘9‘ (6) 1.69 (2)
2.60 5 0.1317)
II4
l.OP(Z) .
-. 2.62 2.31 2.51 2.61 3.x.3 3.89 3.76 4.08 3.11
+ & * I
0.15(12) 0 IY (3) O.lO(7l 0.20(5)
f 0.26(7) f 0.13(2) f 0.23 (9) 1 0.330) rf- 0.1 I(6l 3.832 1.06(5)
1.10 1.15~(llll I 16 106 1.03(5, 1.19” I.lX’(ZI 0.90
I .07 1.13 l.lh(7l 1.05 1.06(lJ 1.19h 1.19(4) I.25 1.36(4)
* Ratio of mean SMR during cold acclimation to that during warm acchmaiton a. h and c Indicate statistically significant or doubtful drfferences by unpaired r-test (P < 0.01, 0.05 and 0.1. respectively). + Mean of ratios of SMR during cold to rhat durmg warm m the same animals with the level of significance by paired t-rest (symbols as in ‘). : P, physlological SMR; U. unphyslologcal SMR (for detaila see rest)
434
KATSU~
Tsuc.4~~ l?lSCLSSiON
The body wt of 13 toads used for SMR determination ranged from 16.6 to 52,Og with the mean of 33.3 and standard deviation of 9.8. In most cases. SMR has been expressed per g body wt in the study of thermal acclimation. However, metabolic rate is not simply proportional to body wt. but proportional to body mass to the power 0.75 for varieties of vcrtebrates. In this experiment, the regression line for SMR (pi CO,/min) of ZYC-acclimated toads at 2O’C was in SMR = 1.22 + 0.67 In (body wt) r = 0.72. II = I?
Fig. 3. Osmotic
fragility of X. becis erythrocytes. Twenty ~1 of erythrocyte suspensions in PBS were incubated in 3 ml of various concentrations of NaCl solutions containing 2 mM Na-PO,, pH 6.2 (closed symbols) or pH 7.4 (open symbols) at 0°C (A). 25°C (0) or 37’C (a) for 30 min. 0 indicates combined symbols of A. o and D.
equilibrium period (Fig. 2). When SMR of toads acciimated to 14°C for 6.5 weeks or longer was measured at 20°C the unphysiological SMR was significantly higher (P < 0.01) than the physiological SMR (Table 1).
A typical hemolysis curve of a toad acclimated to 25’C is shown in Fig. 3. As complete hemoiysis was achieved with 20-4OmM NaCi solution, the maximum absorbances of su~ernatants obtained from these solutions were considered as 100%. Erythrocytes hemoiyzed in saline-free solution occasionally contained amounts of hemoglobin, presumably due to drastic hemolysis and re-sealing of fragments. Figure 4 shows osmotic fragility measured at three different temperatures. Percent hemolvsis at pH 7.4 increased with increasing duration of-incubation at every temperature examined (statistically significant except for the difference between the values of warmacclimated toads at 37C; Table 2). Erythrocytes of X. laevis were less stable at 37 than at 25’C, irrespective of the acclimation temperature, while there was no difference in stability between the extremely- cold and moderate temperatures except the values of warmacclimated toads for 30min. Incubation at low pH accelerated hemolysis irrespective of acclimation or incubation temperature, but the stimulating effect of low pH was more pronounced with increasing temperature. Erythrocytes from cold-acclimated toads exhibited conditions higher stability under physiological (pH 7.4, 25°C) as well as at extremely cold temperature compared with erythrocytes from warm-acclimated toads (Fig. 4 and Table 2). At 37C, however, the difference in MCFs between the two acclimation temperatures disappeared after incubation for 30 min, due to more pronounced increase in MCF in erythrocytes from cold-acclimated than from warm-acclimated toads (but the difference in increase of MCFs due to elevated temperature of elongated period of incubation between the two acclimation temperatures was not statistically significant).
and coefficients of variation (CV; $13 of SMR per g body wt were higher than those per g3’4 or g2 3 (e.g. for SMRs at 20°C; 25, 20 and 20%. respectively), indicating that it is better to express the SMR as ~1 CO, producedjmin per 0.75 power of the body wt. For a hypothetical individual whose body wt corresponded with the mean body wt of 33.3g, SMRs expressed classically as ~1 C02/min per g body wt are given by multiplying the values shown in this paper by 0.416. Carey (1979) reported that the rates of oxygen consumption of B. boreus and R. pip&s were lower between 0300 and 0600 than at any other time. X. inevis examined in the present experiment. however, were occasionally active before dawn and least active at mid-day, even after one day in the dark respiratory chamber: so that the time of measurement was neglected.
loor
Fig. 4. Mean corpuscular fragility {MCF) wrth SEM of erythrocytes from toads acclimated to 14°C (open symbols) and to 2s”C (closed ones) for 411 weeks. MCF, the concentration of NaCl where 5046 of erythrocytes are hemoivzed, was determined at pH 6.2 (Cl) or pH 7.4 (A and 01. I&ubation time; at pH 6.2 for 30 min, at pH 7.4 for 10 (G) or 30min 40). lvtCF at 37’C, nH 6.2 were higher than f 10 mM irrespective of the &chmation te&erature. Number of animals examined: ZYC-acclimated, 10: 14’C-acclimated, 8 (pH 7.4 at OT for 30min and at 37°C). 9 (pH 7.4 at 0°C for IOmin and at 25°C) or 10 (pH 6.2).
435
Thermal acclimation of Xenopus laeuis Table 2. Statistical analysis of MCF shown in Fig. 4 Experimental conditions pH 7.4 10 min 30min pH 6.2 30 min
Between incubation temperatures* 14”Cacclimated ZSC-acclimated 0 vs 25 25 vs 37 0 vs 31 0 vs 25 25 vs 37 0 vs 31 c(NS) NS(NS) a(a)
b(a) a(a) _(a)$
b(a) b(b) _(a)’
NS(NS) b(b) a(a)
b(b) a(a) (b)’
c(NS) NS(NS) +a)’
Between incubation conditions* 14”C-acclimated 25”Cacclimated (0°C) (25°C) (37°C) (0°C) (25°C) (37°C) 10’ vs 30’ (pH 7.4) pH 6.2 vs 7.4 (30’)
a(a) a(a)
a(a) a(a)
b(b) ga)’
a(a) a(b)
Between acclimation temperaturest (25°C) (0°C) pH 7.4 10 min 30 min pH 6.2 30 min
b(b) b(b) b(c)
c(c) c(b) NS(NS)
a(b) a(a)
C(NS) 4aY
(37°C) NS(NS) NS(NS) -(-)
* Paired r-test and Wilcoxon’s signed rank test or sign test where indicated by s (in parentheses). t Unpaired r-test and Mann-Whitney’s U-test (in parentheses). a = significant at P < 0.01. b = significant at P < 0.05. c = not sienificant but doubtful (P < 0.1) for t- and U-tests. NS = not-significant.
In this experiment, an adaptive metabolic adjustment in response to thermal acclimation has been documented at the level of intact animal (Fig. 2 and Table 1). The same type of acclimation was reported in R. temporaria (Jankowsky, 1960; Harri & Hedenstam, 1972; Harri, 1973) and B. boreas (Bishop & Gordon, 1967; Carey, 1979). On the other hand, the inverse compensation was reported in R. catesbeiana (Weathers, 1976) summer R. temporaria (Harri, 1973), P. triseriata (Packard, 1972; Dunlap, 1980) and A. crepitans (Dunlap, 1973, 1980). Unlike most common anuran amphibians (Brattstrom, 1970) thermal acclimation of X. laeuis is expected to be physiological rather than behavioral, because their aquatic environment imposes a relatively narrow range of temperatures on them. Therefore, it is reasonable that the type of thermal acclimation of this aquatic toad is compensative (Precht’s type 3; see Precht et al., 1973). A slight degree of thermal compensation observed may be due to the stenothermal nature of the toad. In nature, the species is found throughout the temperate regions of South and West Africa (Deuchar, 1975). Callaghan (1953) reported that ion transport of isolated skin of X. laeuis was reduced at temperatures above 25°C and autolysis and death of the skin were caused at even higher temperatures. Deuchar (1975) noted that 25°C was the maximum temperature that intact animals will tolerate, although according to our exprience adult intact toads can tolerate higher temperatures (about 30°C). Cullen & Webster (1977) found that exposure of swimming tadpoles of this toad to 10°C induced abnormalities in optic nerves. In this experiment, SMR was measured during April 1979-February 1980. In amphibia, interactions of season and temperature acclimation were observed (Harri, 1973; Lagerspetz, 1977; Pasanen, 1977). But
frogs used by Harri (1973), for example, were collected from field in each season prior to experiments and kept in laboratory for a period without food. X. laeuis used in this experiment, however, were hatched from eggs in the laboratory and reared under constant temperature and light conditions for many years. In nature, X. laeuis aestivates during summer when pools dry up and breeding usually takes place during a period of 3-5 months per year when the pools are full, while under laboratory conditions, the seasonal pattern of gonadal growth and breeding activity is usually disturbed (Deuchar, 1975). X. laeuis, reared in the same way in our laboratory, could also produce fertile eggs after administration of an adequate dose of luteinizing hormone in any season of the year. Therefore, seasonal effect on thermal acclimation can be expected to be less in this toad. The data did not exhibit a contradiction to this expectation. The order of exposure to the temperature of measurement affected the SMR (Fig. 2 and Table 1). Direct exposure to extreme temperature may induce some disturbance in metabolism and even after half a day at the temperature the “stress effect” may partially remain. Pruitt & Dimock (1979) reported the different effects of the order of exposure to experimental temperatures on metabolic rate in differently acclimated crayfish. As in nature a sudden change in the pool water temperature over 10°C never seems to happen, the “physiological” SMR is expected to be more adequate to estimate the capacity of thermal acclimation of the toad than the “unphysiological” SMR. In this experiment, high ability of Xenopus erythrocytes to tolerate low osmotic solution (Zeidler, 1979) was confirmed. Half the erythrocytes hemolyzed at around 40 mM concentration at physiological pH and
KATSUII TSUGAWA
436
temperature, although Zeidler (1979) reported that MCF of unwashed erythrocytes of X. laeris hemolyzed in 20mM NaCl solution when they were incubated at 22°C. Aging of erythrocytes in citro decreased osmotic resistance, but erythrocytes of X. laer*is were stable. MCF of erythrocytes kept in PBS for 30 hr at room temperature was 47.2 mM (at 2S”C, pH 7.4 for 30min) which was comparable to that of fresh erythrocytes (35.3 mM). Storage for 7 hr did not affect MCF. Cold
acclimation
of toads
stabilized
their
erythro-
cytes (Fig. 4 and Table 2). Abramowitz et ui. (1977) reported that the stability of erythrocytes at 15 C in isotonic urea solution was increased by cold acclimation of nurse shark, Ginglymostoma cirratum. In R. pipierzs an alteration of membrane fluidity by thermal acclimation was suggested by a change in the afinity of the muscarinic cholinergic antogonist for its receptor (Martner & Almon, 1981). Increased stabitity toads
r&u&ion
bf
plasma
osmolality.
Catlett
may
&
relate
a
Millich
(1976) found decreased osmolality in goldfish. while Bourne & Cossins (I 981) reported an increased osmolality in carp. MCF at high temperature suggested higher thermolability in erythrocytes from cold acclimated than from warm-acclimated toads (Fig. 4). These results confirmed
that
thermal
acclimation
also
phenomena in this of the difference remains
membrane-associated although the nature
affected
toad, to be
clarified. ,4clinawledgements-The author wishes to thank Miss R. Tomitani for her assistance in determining SMR. Mr T. Kimura for taking photographs and Dr fi. Tsukuda for critical reading of the manuscript. REFERENCES ABRAMOWITZ J., HONN K. V, & CHAVIN W. (1977) Effect of environmental temperature upon rates and duration of hemolysis of squaliform erythrocytes. Camp. B~uc~z~rn. Physiol.
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CULLEN
temperature on myelin formation in optic nerves of Xenopus tadpoles. Tiss~r Cell 9, 1~ 10. DE COSTA J., ALONSO-SEDATE M. & FRAU A. (19X1) Temperature acciimation in amphibians: Changes in lactate dehydrogenase activities and isoenzyme patterns in sevcral tissues from adult Discuylo~s.~~.s pictrrs picru< (Otth.). Corn{‘. Biochem. Phpsiol. 708, 33 l-339. DEUCHAR E, M. (1975) Xvnop~.s: Thr Soz~lk A/T~UUI CIukt& Frog. John Wiley. London. DUNLAP D. G. (1973) Latitudinal effects on metabohc rates in the frog, itcrris crupituns: Seasonal comparisons. Bioi. Bull. 145, 103-I 18. DUNLAP D. G. (1980) Comparative effects of thermal acciimation and season on metabolic compensation to temperature in the hylid frogs, Psrudtrcris triswiarcl and Awis crrpirtrns. Camp. Biochem. Physioi. 66A, 243 249. HARRI M. N. E, (1973) The rate of metabolic temperature acclimation in the frog. Raw f~,~7~p~~r~~ri~~, ?‘h~x&i. Zniii. 46, 14% 156. HAKRI M. & HEDEUSTAM R. (1972) Calorigemc etrect of adrenaline and noradrenaline in the frog, Rurw twnportrriu. Comp. Biochem.
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TSUGAWA K. (1976) Direct adaptation of cells to temperature: Similar changes of LDH isozyme patterns by in rirro and itz siru adaptations in d’utrt,ptcs luer:i.c. Comi>. Biocham. Physioi. 55B, 259-263. TSUGAWA K. (1980) Thermal dependence in kinetic properties of lactate dehydrogenase from the African clawed toad, Xenopus lowis. Comp. Bioc,hcvn. Ph~sioi. 66B. 459-466. WEATHERS W. W. (1976) Influence of temperature acclimation on oxygen ~ollsumption, haemodyila-rni~ and oxygen transport in bullfrogs. Ausr. J. Zooi. 24, 321 330. ZE~DLER R. B. (1979) Hemolysis and transport by red blood cells from the South African clawed toad, Xrnopus Iwri\. Camp. Biochum.
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63A, 223-228.
Camp. Biochem. Physial. Vol. 73A. No. 3. pp. 431 to 436, 1982 Printed in Great Britain.
EFFECTS OF COLD ACCLIMATION ON THE STANDARD METABOLIC RATE AND OSMOTIC FRAGILITY OF ERYTHROCYTES IN AN AQUATIC ANURA, X~~~PUS LAEVIS KATSUJI TSUGAWA Department
of Biological
Sciences, Osaka Women’s Sakai 590, Japan (Receiwd
19 February
University,
Daisen-cho
2,
1982)
The rates of standard metabolism (CO1 production) were measured in intact Xenopus in&s faecis acclimated to 14 and 25°C using an open-circuit system at IS, 20 and 25°C. 2. The rates were increased by 15-22x 3-8 weeks after acclimation to 14°C at every temperature measured, showing the partial compensation. 3. Erythrocytes from cold-acclimated toads tolerated lower concentration of NaCl solution under physiological conditions (pH 7.4, 25°C) and O’C (pH 6.2 and 7.4) than those from warm-acclimated toads. 4. Incubation of erythrocytes at 37°C abolished the differences between acclimation temperatures, due to more pronounced increase of osmotic sensitivity by effect of high temperature in the cold-acclimated toads. Abstract-l.
INTRODUCTION
The metabolism of amphibians changes during thermal acclimation as in many other poikilotherm~c animals. In anura, some show the reasonable compensation in respiratory rate [B. horeas (Bishop & Gordon, 1967; Carey, 1979), R. temporuria (Jankowsky, 1960; Harri, 197311, while others show the inverse compensation CR. remporaria in summer (Harri, 1973), R. catesheiana (Weathers, 1976), Pseuducris triseriata (Packard, 1972; Dunlap, 1980), Acris crepitans (Dunlap, 1973. 198O)J Thermoregulation of amphibia is mainly behavioral, including selection of microclimates (Brattstrom, 1970). In aquatic anura, however, thermal acclimation seems to be essentially physiological, since they are exposed directly to the environmental temperatures from which they cannot escape. Therefore, thermal acclimation of such forms can be analyzed more easily than those of terrestrial or semi-terrestrial forms. However, despite relatively large number of studies in the latter forms, only a few ,have been done in the former (Tsugawa, 1976, 1980; De Costa et al., 1981). In the previous experiments, isozyme patterns and apparent K, of pyruvate for lactate dehydrogenase of liver tissue were found to change with cold acclimation of Xenopus laevis laevis, a typical aquatic anura (Tsugawa, 1976, 1980). The effect of cold acclimation on the tissue lactate dehydrogenase activities and isozyme patterns were also reported in another aquatic anura, Discoglossus pictus pictus (De Costa et al., 1981). But changes in metabolic rates of the intact animals during thermal acclimation were left unconfirmed. So, firstly, the effect of thermal acclimation on the rate of CO2 production of intact animals was determined. 431
Acclimation temperature affects membrane-associated phenomena and constituents of lipid fractions in many poikilothermic animals (e.g. Martner & Almon, 1981 in R. pipiens). In nurse shark erythrocytes a reduction of the acclimation temperature was reported to increase resistance to hemolysis (Abramowitz et al., 1977). So, secondly, the osmotic fragility of X. Iaecis erythrocytes was determined to demonstrate an example of the effect of thermal acclimation on membrane-associated phenomena in this toad. The data demonstrate that the African clawed toad, X. laeois, can partially compensate the temperatureinduced alteration in respiratory rate by increasing its capacity, and that acclimation temperature also affects a characteristic of erythrocytes in this animal. MATERIALS
AND METHODS
Animals Adult X. IaeGis, hatched from eggs in the laboratory, were routinely stored at 1%2o’C, then divided randomly into two groups and kept at cold (14 i 1’C) and warm (24-25°C) temperatures. Fluorescent lieht orovided a 13L: IlD photoperiod (083G-2130 light) 111 a ‘light-sealed room. They were fed on cattle liver twice a week at 1%25°C or once a week at 14’C.
Stundard metabolic rate (SMR) Resting metabolism of intact female toads, 2-4yr old, 17-52 g body wt, was determined by measuring the rate of CO, production using an open-circuit system. Toads were fasted 2-3 days before and during each measurement. Each animal was placed in a 1.5 1 respiratory chamber (aluminium box with plastic ceiling) with 0.3 I of tap water in which the toad was almost completely submerged. Respiratory chamber(s) and spiral ducts for supplying fresh air were kept dark and immersed into a water bath, the temperature of which was changed from the temperature of acclimation to that of measurement at a rate of about
432
KAWIJI TWGAWA
1L2”C every 5 min and controlled within &O.S’C. The Co, concentrations of water CO2 and air phases were equilibrated continuously by blowing bubbles of fresh air from the air bag to the water, with an air flow rate of 0.5-0.6, 0.7 and 0.7-0.8 jimin at 15. 20 and 25”C, respectively, The air leaving the respiratory chamber was channelled to an air dessicating chamber and COz analyzer (Hitachi-Horiba EIA-IA. full scale 600 ppm; Horiba-seisakusho, Kyoto) through a flowmeter and an automatic change-over valve, and the concentration of COZ was recorded at 1 or 3-min intervals repeatedly for 1 hr and 45 min following monitoring of the concentration of the supplied air for IS min. Preliminary experiments indicated that the rate of CO, production occasionally declined, even if the recording chart showed a relatively inactive state (low frequency of expiration), up to 8-10 hr after an animal was placed in the respiratory chamber. Therefore, after a 10 hr or longer eqL]i~ib~tion period. the rate of CO2 production was measured for i@20hr at a temperature of measurement. Often, measurements at other temperatures were followed. After measurements were carried out at an acclimation temperature, toads were transferred to the other acclimation temperature. The rates of CO, production of an animal were calculated for each 1.75-hr period. and the results were sorted using the following criteria: (I) Not more than the two highest values among the 35 observed at 3-min intervals exceeded twice the average for the 7 lowest ones among them and (2) not more than the 7 highest values exceeded 1.5~times the average for the lowest 7. An average of the lowest 1:3 among the values corresponding to the two items was regarded as the standard metabolic rate (SMR) of the measurement. SMR of toads correlated with 3i4-powers of their body wt rather than the weight itself. so that values were expressed as ,J CO, producedimin per 3/4-powers of g body wt. Body wt of each toad fluctuated during the experimental period but the changes were irregular. Over 4-10 months of experiments the curves of body wt of each toad did not exhibit net increase or decrease in weight. Therefore. the mean values for the period of experiment were used as representations of their body wt. Mean SMRs of toads acclimated to either cold or warm temperature were compared with those of the same animals after acclimation to the other temperature using the Student t-tests of paired and unpaired observations.
Male and female toads. 15-44 g body wt. were sacrificed by decapitation at 0900- 1200 in December-February. Blood obtained from the carotid artery or heart was immediately mixed with phosphate buffered saline (PBS; 6.1 g NaCI. 0.15 g KCI. 0.876 g Na,HPO, and 0.153 g KH,PO, in I I of water. pH 7.4) containing 20 units/ml heparin. After the blood suspension was diluted with 3 vol. of heparin-free PBS, a few small aggregates were removed. Then, diluted blood was centrifuged at 700 rev,‘min for 3 min and washed twice with heparin-free PBS at room temperature. Experiments were carried out on blood samples from individual toads without pooling of sample. Twenty 10 50 ~1 of fresh erythrocyte suspension in heparinfree PBS was added to a test tube containing 3 ml of prewarmed or pre-cooled NaCi solution with 2 mM Na-PO,. pH 6.2 or 7.4. After itlcubation at 0, 25 or 37°C for 10 or 30 min. the test tubes were immediately centrifuged at 800 rev;min for 3 min and the resulting supernatant was quickly removed. The absorbance of the supernatant was determined at 577 nm and plotted as a function of the concentration of
NaCl solution. Mean corpuscular fragility (MCF), the concentration of NaCl in which 505; of the erythrocytes are hemolyzed (Catlett & Millich, 1976). was determined the hemolysis curves by interpolation.
from
RESCILTS
Typical records of respiratory activity of a toad at 2S”C are shown in Fig, 1. Rapid increase in CO, concentration followed by rapid decline indicates expiratory movement. The frequency of the expiratory movement was high during active movement of the toad. and therefore used as a criterion to distinguish the resting state from the active state. Figure 2 shows the “physiological” and the “unphysiological” SMRs at different temperatures of measurement in toads acclimated to cold or warm temperature for 3-8 weeks. The “physiological” SMR means the rates measured at the acclimation temperature prior to measurement at other temperatures or those after stepwisc upward or downward change in the measurement temperature from acclimation temperature. The ‘~unphysiolo~jc~I1” SMR meitns the rates measured firstly at the temperature opposed to that of their acclimation or those at other temperatures following such measurement. SMR at 2O”C, irrespective of whether physiological or unphysiological. was higher in cold-acclimation than warm-acclimation phase. Physiological SMR of cold-acclimated toads was higher bv 9”:. than that of
PPM
600
Fig. I. Carbon dioxide production of a toad in active (A) and standard (3) states at 25°C. Air flow rate, 0.80 I/mini body wt of the toad. 43 g. Carbon dioxide concentration above 600 ppm is for reference because it was recorded in a range over full scale of analy7er used.
25
20
15
Temp. CC) F1g ? Standard metabolic rate (SMR) and SEM of female toads accltmated to I? I4.C (open symbols) and to 24 25 C (closed symbols) for 3 8 weeks. “Phystoloplcal” (P: 0) and “unphysiological” (U; Cl) SMRs related to the order of exposure 10 the temperature of measurements. DiR’erences m “physiological” SMRs between the two acclimation temperatures were significant by paired r-test. For details. see text.
Table
1 ShiR
warm-acclimated toads. but comparisons of each toad between the cold and warm-acclimation phases showed a significant difference (P < 0.02) of about 15”;,. Difference in unphysiological SMRs was also significant at this temperature (P < 0.05). SMR at 25‘C was also higher in cold-acclimation phase. Comparisons of physiological SMR of each toad exhibited statistically significant difference (P < 0.05). At 15 C. physiological SMR was increased by 20”;, during cold acclimation as compared with warm-acclimation phase (P < 0.01 by paired t-test). Table 1 shows SMRs after different prrlods of acchmation. Physiological SMR at 20cC cxhibttcd a tendcncy to increase 1.5 2.5 weeks after acclimation to cold. though this was not statistically significant. presumably due to the small number of measurements. Three to 6 weeks after acclimation. SMR of coldacclimated toads was higher by IO”,, than that of warm-acclimated toads (P < 0.05 by palred r-test). SMR at 2o‘C of cold-acclimated toads. and therefore the difference in SMR between the two acclimation temperatures. tended to decrease with duration of acclimation. At 15 and 25’C’. however. differences in physiological SMR between the two acclimation tzmpcratures increased with extending period of acchmation. The order of exposure to the temperature of measurement affected the SMR in some cases. When toads acclimated to 25°C were exposed to 15 C immediately from their acclimation temperature, SMR showed a higher rate compared IO the value when toads were exposed via 20 C. in spite of rhe long
of toads acchmatcd to Lold or uarm temperature for ditferent periods. Vaiucs per g ” of body WI) 2 SEM wtth the number of toads measured in parentheses
arc’ mean SMR (~1 CO,;min
(‘C)
Duration of accl. (weeks)
I5
1.5 2.5
Temp. of measurement
.Acclimation
(14-C) p:
u 36 .:- 6.5
20
I.5 2.5 36 ;I b.5
‘> 6.5
temperature (25’ c‘)
c::w*
c:wt .--
1.71 x 0.07 (8)
P U P I:
1.X0 1.96 1.79 I .x4 1.82
+ + + * f
0.16(4) 0.14(12) 0.23 (6) 0.09 (101 0.15 (5)
P L! P I: P II
2.96 3.43 2.88 2 6X 2.66 3.36
2 & f * + k
0.36 (41 0.08 (3) O.l7(L?I 0.29(2) 0.16(X) 0.14 (6)
P U P t; P U
3.45 4.17 4.25 3.8? 3.69 4.79
+ * + I + ?
0.74(3) 0.25(31 0.33 (12) 0.68 (5) 0.22 (7) 0.94 (6)
1.69+ 0.I2 (1 I I I .9J * 0.34 (4) I.46 + 0.07 (8) 1.76 _c 0.17(41
I.16 0.92 1.26, 1.04
1.17”(Y)) 0.93(l) I .‘9‘ (6) 1.69 (2)
2.60 5 0.1317)
II4
l.OP(Z) .
-. 2.62 2.31 2.51 2.61 3.x.3 3.89 3.76 4.08 3.11
+ & * I
0.15(12) 0 IY (3) O.lO(7l 0.20(5)
f 0.26(7) f 0.13(2) f 0.23 (9) 1 0.330) rf- 0.1 I(6l 3.832 1.06(5)
1.10 1.15~(llll I 16 106 1.03(5, 1.19” I.lX’(ZI 0.90
I .07 1.13 l.lh(7l 1.05 1.06(lJ 1.19h 1.19(4) I.25 1.36(4)
* Ratio of mean SMR during cold acclimation to that during warm acchmaiton a. h and c Indicate statistically significant or doubtful drfferences by unpaired r-test (P < 0.01, 0.05 and 0.1. respectively). + Mean of ratios of SMR during cold to rhat durmg warm m the same animals with the level of significance by paired t-rest (symbols as in ‘). : P, physlological SMR; U. unphyslologcal SMR (for detaila see rest)
434
KATSU~
Tsuc.4~~ l?lSCLSSiON
The body wt of 13 toads used for SMR determination ranged from 16.6 to 52,Og with the mean of 33.3 and standard deviation of 9.8. In most cases. SMR has been expressed per g body wt in the study of thermal acclimation. However, metabolic rate is not simply proportional to body wt. but proportional to body mass to the power 0.75 for varieties of vcrtebrates. In this experiment, the regression line for SMR (pi CO,/min) of ZYC-acclimated toads at 2O’C was in SMR = 1.22 + 0.67 In (body wt) r = 0.72. II = I?
Fig. 3. Osmotic
fragility of X. becis erythrocytes. Twenty ~1 of erythrocyte suspensions in PBS were incubated in 3 ml of various concentrations of NaCl solutions containing 2 mM Na-PO,, pH 6.2 (closed symbols) or pH 7.4 (open symbols) at 0°C (A). 25°C (0) or 37’C (a) for 30 min. 0 indicates combined symbols of A. o and D.
equilibrium period (Fig. 2). When SMR of toads acciimated to 14°C for 6.5 weeks or longer was measured at 20°C the unphysiological SMR was significantly higher (P < 0.01) than the physiological SMR (Table 1).
A typical hemolysis curve of a toad acclimated to 25’C is shown in Fig. 3. As complete hemoiysis was achieved with 20-4OmM NaCi solution, the maximum absorbances of su~ernatants obtained from these solutions were considered as 100%. Erythrocytes hemoiyzed in saline-free solution occasionally contained amounts of hemoglobin, presumably due to drastic hemolysis and re-sealing of fragments. Figure 4 shows osmotic fragility measured at three different temperatures. Percent hemolvsis at pH 7.4 increased with increasing duration of-incubation at every temperature examined (statistically significant except for the difference between the values of warmacclimated toads at 37C; Table 2). Erythrocytes of X. laevis were less stable at 37 than at 25’C, irrespective of the acclimation temperature, while there was no difference in stability between the extremely- cold and moderate temperatures except the values of warmacclimated toads for 30min. Incubation at low pH accelerated hemolysis irrespective of acclimation or incubation temperature, but the stimulating effect of low pH was more pronounced with increasing temperature. Erythrocytes from cold-acclimated toads exhibited conditions higher stability under physiological (pH 7.4, 25°C) as well as at extremely cold temperature compared with erythrocytes from warm-acclimated toads (Fig. 4 and Table 2). At 37C, however, the difference in MCFs between the two acclimation temperatures disappeared after incubation for 30 min, due to more pronounced increase in MCF in erythrocytes from cold-acclimated than from warm-acclimated toads (but the difference in increase of MCFs due to elevated temperature of elongated period of incubation between the two acclimation temperatures was not statistically significant).
and coefficients of variation (CV; $13 of SMR per g body wt were higher than those per g3’4 or g2 3 (e.g. for SMRs at 20°C; 25, 20 and 20%. respectively), indicating that it is better to express the SMR as ~1 CO, producedjmin per 0.75 power of the body wt. For a hypothetical individual whose body wt corresponded with the mean body wt of 33.3g, SMRs expressed classically as ~1 C02/min per g body wt are given by multiplying the values shown in this paper by 0.416. Carey (1979) reported that the rates of oxygen consumption of B. boreus and R. pip&s were lower between 0300 and 0600 than at any other time. X. inevis examined in the present experiment. however, were occasionally active before dawn and least active at mid-day, even after one day in the dark respiratory chamber: so that the time of measurement was neglected.
loor
Fig. 4. Mean corpuscular fragility {MCF) wrth SEM of erythrocytes from toads acclimated to 14°C (open symbols) and to 2s”C (closed ones) for 411 weeks. MCF, the concentration of NaCl where 5046 of erythrocytes are hemoivzed, was determined at pH 6.2 (Cl) or pH 7.4 (A and 01. I&ubation time; at pH 6.2 for 30 min, at pH 7.4 for 10 (G) or 30min 40). lvtCF at 37’C, nH 6.2 were higher than f 10 mM irrespective of the &chmation te&erature. Number of animals examined: ZYC-acclimated, 10: 14’C-acclimated, 8 (pH 7.4 at OT for 30min and at 37°C). 9 (pH 7.4 at 0°C for IOmin and at 25°C) or 10 (pH 6.2).
435
Thermal acclimation of Xenopus laeuis Table 2. Statistical analysis of MCF shown in Fig. 4 Experimental conditions pH 7.4 10 min 30min pH 6.2 30 min
Between incubation temperatures* 14”Cacclimated ZSC-acclimated 0 vs 25 25 vs 37 0 vs 31 0 vs 25 25 vs 37 0 vs 31 c(NS) NS(NS) a(a)
b(a) a(a) _(a)$
b(a) b(b) _(a)’
NS(NS) b(b) a(a)
b(b) a(a) (b)’
c(NS) NS(NS) +a)’
Between incubation conditions* 14”C-acclimated 25”Cacclimated (0°C) (25°C) (37°C) (0°C) (25°C) (37°C) 10’ vs 30’ (pH 7.4) pH 6.2 vs 7.4 (30’)
a(a) a(a)
a(a) a(a)
b(b) ga)’
a(a) a(b)
Between acclimation temperaturest (25°C) (0°C) pH 7.4 10 min 30 min pH 6.2 30 min
b(b) b(b) b(c)
c(c) c(b) NS(NS)
a(b) a(a)
C(NS) 4aY
(37°C) NS(NS) NS(NS) -(-)
* Paired r-test and Wilcoxon’s signed rank test or sign test where indicated by s (in parentheses). t Unpaired r-test and Mann-Whitney’s U-test (in parentheses). a = significant at P < 0.01. b = significant at P < 0.05. c = not sienificant but doubtful (P < 0.1) for t- and U-tests. NS = not-significant.
In this experiment, an adaptive metabolic adjustment in response to thermal acclimation has been documented at the level of intact animal (Fig. 2 and Table 1). The same type of acclimation was reported in R. temporaria (Jankowsky, 1960; Harri & Hedenstam, 1972; Harri, 1973) and B. boreas (Bishop & Gordon, 1967; Carey, 1979). On the other hand, the inverse compensation was reported in R. catesbeiana (Weathers, 1976) summer R. temporaria (Harri, 1973), P. triseriata (Packard, 1972; Dunlap, 1980) and A. crepitans (Dunlap, 1973, 1980). Unlike most common anuran amphibians (Brattstrom, 1970) thermal acclimation of X. laeuis is expected to be physiological rather than behavioral, because their aquatic environment imposes a relatively narrow range of temperatures on them. Therefore, it is reasonable that the type of thermal acclimation of this aquatic toad is compensative (Precht’s type 3; see Precht et al., 1973). A slight degree of thermal compensation observed may be due to the stenothermal nature of the toad. In nature, the species is found throughout the temperate regions of South and West Africa (Deuchar, 1975). Callaghan (1953) reported that ion transport of isolated skin of X. laeuis was reduced at temperatures above 25°C and autolysis and death of the skin were caused at even higher temperatures. Deuchar (1975) noted that 25°C was the maximum temperature that intact animals will tolerate, although according to our exprience adult intact toads can tolerate higher temperatures (about 30°C). Cullen & Webster (1977) found that exposure of swimming tadpoles of this toad to 10°C induced abnormalities in optic nerves. In this experiment, SMR was measured during April 1979-February 1980. In amphibia, interactions of season and temperature acclimation were observed (Harri, 1973; Lagerspetz, 1977; Pasanen, 1977). But
frogs used by Harri (1973), for example, were collected from field in each season prior to experiments and kept in laboratory for a period without food. X. laeuis used in this experiment, however, were hatched from eggs in the laboratory and reared under constant temperature and light conditions for many years. In nature, X. laeuis aestivates during summer when pools dry up and breeding usually takes place during a period of 3-5 months per year when the pools are full, while under laboratory conditions, the seasonal pattern of gonadal growth and breeding activity is usually disturbed (Deuchar, 1975). X. laeuis, reared in the same way in our laboratory, could also produce fertile eggs after administration of an adequate dose of luteinizing hormone in any season of the year. Therefore, seasonal effect on thermal acclimation can be expected to be less in this toad. The data did not exhibit a contradiction to this expectation. The order of exposure to the temperature of measurement affected the SMR (Fig. 2 and Table 1). Direct exposure to extreme temperature may induce some disturbance in metabolism and even after half a day at the temperature the “stress effect” may partially remain. Pruitt & Dimock (1979) reported the different effects of the order of exposure to experimental temperatures on metabolic rate in differently acclimated crayfish. As in nature a sudden change in the pool water temperature over 10°C never seems to happen, the “physiological” SMR is expected to be more adequate to estimate the capacity of thermal acclimation of the toad than the “unphysiological” SMR. In this experiment, high ability of Xenopus erythrocytes to tolerate low osmotic solution (Zeidler, 1979) was confirmed. Half the erythrocytes hemolyzed at around 40 mM concentration at physiological pH and
KATSUII TSUGAWA
436
temperature, although Zeidler (1979) reported that MCF of unwashed erythrocytes of X. laeris hemolyzed in 20mM NaCl solution when they were incubated at 22°C. Aging of erythrocytes in citro decreased osmotic resistance, but erythrocytes of X. laer*is were stable. MCF of erythrocytes kept in PBS for 30 hr at room temperature was 47.2 mM (at 2S”C, pH 7.4 for 30min) which was comparable to that of fresh erythrocytes (35.3 mM). Storage for 7 hr did not affect MCF. Cold
acclimation
of toads
stabilized
their
erythro-
cytes (Fig. 4 and Table 2). Abramowitz et ui. (1977) reported that the stability of erythrocytes at 15 C in isotonic urea solution was increased by cold acclimation of nurse shark, Ginglymostoma cirratum. In R. pipierzs an alteration of membrane fluidity by thermal acclimation was suggested by a change in the afinity of the muscarinic cholinergic antogonist for its receptor (Martner & Almon, 1981). Increased stabitity toads
r&u&ion
bf
plasma
osmolality.
Catlett
may
&
relate
a
Millich
(1976) found decreased osmolality in goldfish. while Bourne & Cossins (I 981) reported an increased osmolality in carp. MCF at high temperature suggested higher thermolability in erythrocytes from cold acclimated than from warm-acclimated toads (Fig. 4). These results confirmed
that
thermal
acclimation
also
phenomena in this of the difference remains
membrane-associated although the nature
affected
toad, to be
clarified. ,4clinawledgements-The author wishes to thank Miss R. Tomitani for her assistance in determining SMR. Mr T. Kimura for taking photographs and Dr fi. Tsukuda for critical reading of the manuscript. REFERENCES ABRAMOWITZ J., HONN K. V, & CHAVIN W. (1977) Effect of environmental temperature upon rates and duration of hemolysis of squaliform erythrocytes. Camp. B~uc~z~rn. Physiol.
57A,
229-230.
BISHOP L. G. & GORDON M. S. (1967) Thermal adaptation of metabolism in anuran amphibians. In Molecular Mechanisms of Temperature Adaptation (Edited by PROSSER C. L.), pp. 263-280. American Association for the Advancement of Science, Washington, DC. BOURNE P. K. & COSSINS A. R. (1981) The effects of thermal acclimation upon ion transport in erythrocytes. J. therm. Bioi. 6, 179-181.
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CULLEN
temperature on myelin formation in optic nerves of Xenopus tadpoles. Tiss~r Cell 9, 1~ 10. DE COSTA J., ALONSO-SEDATE M. & FRAU A. (19X1) Temperature acciimation in amphibians: Changes in lactate dehydrogenase activities and isoenzyme patterns in sevcral tissues from adult Discuylo~s.~~.s pictrrs picru< (Otth.). Corn{‘. Biochem. Phpsiol. 708, 33 l-339. DEUCHAR E, M. (1975) Xvnop~.s: Thr Soz~lk A/T~UUI CIukt& Frog. John Wiley. London. DUNLAP D. G. (1973) Latitudinal effects on metabohc rates in the frog, itcrris crupituns: Seasonal comparisons. Bioi. Bull. 145, 103-I 18. DUNLAP D. G. (1980) Comparative effects of thermal acciimation and season on metabolic compensation to temperature in the hylid frogs, Psrudtrcris triswiarcl and Awis crrpirtrns. Camp. Biochem. Physioi. 66A, 243 249. HARRI M. N. E, (1973) The rate of metabolic temperature acclimation in the frog. Raw f~,~7~p~~r~~ri~~, ?‘h~x&i. Zniii. 46, 14% 156. HAKRI M. & HEDEUSTAM R. (1972) Calorigemc etrect of adrenaline and noradrenaline in the frog, Rurw twnportrriu. Comp. Biochem.
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JANKOWSKY H.-D. (1960) iiber die hormonale Beeinflussung der Temperaturadaptation beim Grasfrosch (ROIILI f~~~izp~~~ff~i~ L.). 2. t:eryl. Phvsioi. 43, 392-410. LAGERSPETZ K. Y. H. (1977) Interactions of season and temperature acclimation in the control of metabolism in Amphihiu. J. therm. Biol. 2, 223-23 1. MARTKER J. E. & ALMON R. R. (1981) Membrane structure and muscarinic function: A temperature acclimation annroach. Gen. Pharmaco/. 12. 89-91. PA&ARD G. C. (1972) Inverse compensation for temperature in oxygen consumption of the hylid frog P~~~~dacri.s triseriara. ~h~sio~. Z&i. 45, 270-275.. PASANE~’ S. (1977) Seasonal variations in certain enzyme activities in the common frog, Runu temporariu L.. under natural and experimental conditions. Camp. Biochmm. Physio/. 568, 163- 167. PKECHT H., LAUDIEN H. & HAVSTEEN B. (1973) Poikilothermic organisms. animals II. The normal temperature range. In T~~~~p~~r(?t~r~cmd Lif; (Edited by PRE~HT CI ~1.). pp. 302--399. Springer-Verlag, Berlin. PKL‘ITT N. L. & DIMOCK R. D.. JR. (1979) The etTects 01 temperature and eyestalk extracts on oxygen consumption of the crayfish Cumhurus acuminrrrrcs (Faxon). Corn/>. Biochun.
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TSUGAWA K. (1976) Direct adaptation of cells to temperature: Similar changes of LDH isozyme patterns by in rirro and itz siru adaptations in d’utrt,ptcs luer:i.c. Comi>. Biocham. Physioi. 55B, 259-263. TSUGAWA K. (1980) Thermal dependence in kinetic properties of lactate dehydrogenase from the African clawed toad, Xenopus lowis. Comp. Bioc,hcvn. Ph~sioi. 66B. 459-466. WEATHERS W. W. (1976) Influence of temperature acclimation on oxygen ~ollsumption, haemodyila-rni~ and oxygen transport in bullfrogs. Ausr. J. Zooi. 24, 321 330. ZE~DLER R. B. (1979) Hemolysis and transport by red blood cells from the South African clawed toad, Xrnopus Iwri\. Camp. Biochum.
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