Temperature dependence of “standard metabolic rate” in a poikilotherm

Corap. Biochem. PhysioL, 1968, VoL 25, pp. 427 to 436. Pergamon Press. Printed in Great Britain

TEMPERATURE DEPENDENCE OF " S T A N D A R D METABOLIC RATE" IN A POIKILOTHERM M. A. T R I B E and K. B O W L E R School of Biological Sciences, University of Sussex, Department of Zoology, University of Durham (Recdved 2 November 1967)

Abstract--1. Experiments with variously acclimatized male blowflies (Ca/h= phora erythrocephala) show that "standard metabolic rate", as measured by oxygen consumption of the whole animal and of isolated flight muscle tissue, is temperature dependent over the range 10-30°C. 2. The results contrast with the recent findings of Newell (1966, 1967) and Newell & Northcroft (1967); discussion is therefore given to the respective differences between results and their interpretation. INTRODUCTION RECENT reports on the respiratory rates of a variety of intertidal animals by Newel] (1966) and Newell & Northcroft (1967) have made the interesting observation that oxygen consumption is not linear with time but can be divided into two phases. In one phase, when the animals display active respiratory movements, the rate of oxygen consumption is temperature dependent (with Q10 values of about 2). In the other phase, when the animals are "quiescent", oxygen consumption is low and the rate is broadly speaking independent of temperature (Q10 values of about 1). The conclusiondrawn from thislatterobservationisthat basal metabolic processes of intertidalinvertebratesmay be relativelyindependent of short-termfluctuations in temperature, in the normal range. They suggest this may be of particular significance in intertidal animals as the normal regulatory mechanisms (i.e. temperature acclimatization)involved in a response to a new temperature rdgime would not have time to bc effectiveconsidering the short-term nature of the fluctuations experienced. The studies on whole animals were extended and supported by work on mitochondria isolated from a variety of poikilothermic animals, includingtwo terrestrialinvertebrates,chosen to represent a wide variety of habitats(Newell, 1966, 1967). The classicalwork on the effectof temperature on poikilothermshas led to the widely accepted view thattheirmetabolic ratewould approximately double for each 10°C rise in temperature (Qt0 value of about 2) within the normal temperature range, although it should be remembered that Qxo is also temperature dependent; see reviews by BuUock (1955), Prosser (1955) and Precht et al. (1955). However, as Newell (1966) points out, owing to the methods used to determine respiratory rates in much of the earlier work, the values obtained would not represent "basal" or "resting" metabolic rates. For example, the literature concerning the effect of 427

428

M. A. T a m s AND K. BowLm

t e m p e r a t u r e on respiratory rates of insects is very large and clearly shows that oxygen consumption is t e m p e r a t u r e dependent in a wide variety of insect species; for a recent review see Keister & Buck (1964). However, f r o m m u c h of the work it is not clear whether the respiratory rates reported truly represent a basal rate. Frequently, too, the previous thermal history of the animals is not considered. I n view of Newell's suggestion that intertidal animals and probably other terrestrial poikilotherms m a y combat rapid fluctuations in environmental t e m p e r a ture by maintaining metabolic activities at a constant low level, it seems important to re-examine the effect of t e m p e r a t u r e on the metabolic rate of another terrestrial poikilotherm. W e have therefore studied the effect of t e m p e r a t u r e on the respiratory rate of variously acclimatized adult male blowflies, Calliphora erythrocephala, animals which m i g h t also b e expected to experience wide and rapid fluctuations in their environmental temperature. MATERIALS AND M E T H O D S The flies used in this study were wild type male imagoes (Caltiphora erythrocephala). These flies were reared from the same generation and were obtained from the same stock (wild type "S") as had been used in earher studies (Tribe, 1966a). Immediately after eclosion the flies were placed in 9-in. diameter crystallizing dishes covered with sleeve muslin (thirty animals per dish). The bottoms of the dishes were lined with filter-paper and flies were provided with sugar, water and yeast ad//b. The dishes were placed in the dark at either |0, 20 or 30°C (±0"5°C) for 8 days to accIimatize. All respiratory measurements were made by standard Warburg respirometry in 15-mi capacity flasks. The centre well contained 0.3 ml 10% K O H with a filter paper wick (Whatman No. 40). As far as possible, ten determinations of single flies were obtained at each experimental temperature (10, 20 and 30°C±0"1°C) for each acclimatized group. Each experiment continued for 1 hr and readings were taken every 10 rain. The method used to estimate standard metabolic rate in terms of oxygen consumption of whole animals was the same as that described earlier (Tribe, 1966b), the results being expressed both in terms of wet and dry weights. This method is considered to be a good estimate of "standard metabolic rate" because although flies were physically restricted in their movements, any struggling which did take place was readily observable from the non-linearity of the oxygen uptake with time, and such records (less than 5 per cent) were excluded. Indeed in many instances, a linear uptake has been obtained for periods of 2 hr or more using this technique (Tribe, 1966b). Half-thorax preparations of flight muscle tissue were made by immobilizing the flies at 0°C, and then adopting the method of Ctegg & Evans (1961). This procedure was used successfully in an earlier study (Tribe, 1967a). The reaction medium used was Bodenstein's (1946) saline at pH 7.2 with 30 mM glucose as added substrate. As far as possible, ten respiratory rate determinatiaus were made for both 10 and 30°C-adapted animals at 10 and 30°C, and the results are shown in Table 3. In addition, values are also shown for tissue from flies acclimatized at 20°C, and measured at 20°C, as a check against the other two sets of readings. Oxygen uptake in all cases was linear with time during the 1-hr observation period of each experiment. RESULTS T h e results of the oxygen consumption of whole animals acclimatized at 10, 20 and 30°C measured at 10, 20 and 30°C are shown in T a b l e 1 and in Fig. 1.

TEMPERATURE DEPENDENCE OF "STANDARD METABOLIC RATE"

429

A n analysis of variance showed a significant increase in the respiratory rate of all acclimatized groups of animals with a rise in experimental t e m p e r a t u r e ( P < 0-01), as can be seen f r o m T a b l e 1 and Fig. 1. T h e Qxo values for these data TABLE I~ExPERIM~TS

W I T H WHOLE ANIMALS

Acclimatized temperature

(oc)

n

pl Os/mg dry wt. per hr

pl Os/mg wet wt. per hr

A. Oxygen uptake at 10°C 10 10 20 10 30 10

0"60 ± 0.08 0.59 ± 0.04 0-69 _+0.07

1.53 ± 0.24 1.96 ± 0.17 1.94 ± 0.07

B. Oxygen uptake at 20°C 10 10 20 10 30 9

1.75 ± 0.13 1.51 _+0"12 1.36 ± 0.10

5.96 ± 0.43 4.73 _+0.39 3.58_+0.32

C. Oxygen uptake at 30°C 10 10 20 9 30 10

2"93 ± 0"13 2'70 ± 0"19 1"82+0"17

10.17±0.69 8.44 ± 0.56 5.11 _+0.70

12 11 I0 L: 9J=

>, 7"? 6-

~'5_~_ 3-

lb o

2'o °

3'o °

Temperature °C

Fxo. 1. Oxygen uptake of variously acclimatized male blowflies at three experimental temperatures. Mean values and standard errors are shown. A, IO°Cacclimatized flies; 0 , 20°C-acclimatized flies; x, 30°C-acclimatized flies. are shown in T a b l e 3, and as is usually the case, larger values were obtained in the lower t e m p e r a t u r e ranges (1.96-2.90) t h a n in the u p p e r t e m p e r a t u r e range (1.341.78), on a wet weight basis. Q10 values on a dry weight basis are also given in T a b l e 3.

430

M. A. TRIBEANDK. BowLm~

When the respiratory rates of the variously acclimatized animals are compared at the same experimental temperature, it is usual to find that the animals adapted to the lower temperature have a higher metabolic rate (oxygen consumption) than the animals adapted to a higher temperature, i.e. they show a "type 3" adaptation (see Precht, 1958). From Table 2 it can be seen that there are no significant differences on either a wet or dry weight basis between the variously acclimatized animals run at 10°C. TABLE2--LsvsLS oF SIGNIFICANCE(STUDENT'S t-DISTRIBUTION) Acclimatized groups

(°C)

Wet wt. basis

Dry wt. basis

(1) Whole animal experiments at 10°C 20

Not significant

Not significant

10 30 20 30

Not significant

Not significant

Not significant

Not significant

(2) Whole animal experiments at 20°C 20

Not significant

10 P
1

10 30 20 30

Not significant P < 0"05

Not significant

Not significant

Not significant

P<0.001

P<0.001

P < 0.01

P<0.01

Significance at the 5 per cent level. However, there is a significant difference between the 10 and 30°C-acclimatized animals at 20°C (P < 0.05), and in the experiments run at 30°C significant differences on both a wet and dry weight basis are seen between all the variously acclimatized groups, with the exception of the difference between the 10 and 20°C-acclimatized groups. In general therefore a "type 3" partial compensation pattern is seen with a rise in temperature. It can be seen from Fig. 1 that the 10°C-adapted group have the highest, and the 30°C-adapted group have the lowest, respiratory rates, the oxygen consumption of the 20°C-adapted animals being intermediate between the other groups. The results of the isolated thorax experiments are shown in Table 4 and Fig. 2. It is evident that the respiratory rates of the isolated thoraces are very dependent on

TEMPERATURE DEPENDENCE OF "STANDARD METABOLIC RATE"

431

the experimental t e m p e r a t u r e ; the rate of oxygen consumption at 30°C w a s ' f o u n d to be about three times as large as the rate at 10°C, for b o t h acclimatized groups of animals ( P < 0.001 in b o t h cases); again the values at 20°C are found to be intermediate between the other two groups. O n a Q10 basis, a value of 1.46 is obtained TABLE 3--Qt0 VALUES (°C)

Wet wt.

Dry wt.

(1) 10°C acclimatized animals 10~Qlo = 2"90 30

Qto

3"89 1 "70

1"67

(2) 20°C acclimatized animals 10 "~ )-Qx o =

2"56

2"24

1"79

1"79

/

30

Qto

(3) 30°12 acclimatized animals

TABLE 4

10"~ Qto =

1"97

1"85

30

1"34

1"43

Qxo

HALF-THORAX PREPARATIONS OF FLIGHT MUSCI.,B TISSUB

(i) QOI values as pl Oi/mg dry wt. muscle per hr Acclimatized temperature (°C) n pl Oi/mg dry wt. muscle per hr A. Oxygen uptake at 10°C 10 6 30 6 B. Oxygen uptake at 20°C 20 6 C. Oxygen uptake at 30°C 10 10 30 11

6.74 +_0"30 5"03 ± 0"28 11"08 ± 0"39 19"67 ± 0"54 15.83 ± 0"61

(ii) P values between 10 and 30°C adapted tissues At 10°C At 30°C

P<0.01 P < 0-001 t-distribution; significant at the 5% level

432

M. A. TRIBEANDK. BOWLm~

for the 10°C-adapted group, and a value of 1"58 for the 30°C-adapted animals. As in the experiments on the whole animal, there is a tendency for the half-thorax preparations from animals acclimatized at the lower temperature to have a higher 20. L2

~15

E -~10 0

~.5

/f

1'0 ° 2'0 ° 3'0' Temperature 'C

FIG. 2. Oxygen uptake of isolated flight muscle tissue from variously acclimatized flies at three experimental temperatures. Mean values and standard errors are shown. A, 10°C-acclimatized flies; 0, 20°C-acclimatized flies; x, 30°Cacclimatized flies. oxygen consumption at both of the experimental temperatures than those from animals adapted at 30°C (P < 0.01, 10°C-adapted; P < 0.001, 30°C-adapted). Thus the half-thorax preparations show a typical "type 3" adaptation to temperature (Precht, 1958). DISCUSSION The resuks presented here clearly show that standard oxygen consumption of blowfly imagoes and their tissues is temperature dependent and add support to similar studies by other workers on a variety of insects (for a review see Keister & Buck, 1964). The values for the oxygen consumption of adult Calliphora (between 0.6 and 3-0/zl Os/mg wet wt. per hr, depending upon temperature) obtained in this study are considered to be a good estimate of "standard metabolic rate", firstly for the reasons given above (see Materials and Methods) and, secondly, the values obtained are very comparable with the results obtained with aUectomized adults of this species (Thomsen, 1949). Thirdly, the pattern of respiration shown by the half-thoraces is essentially similar to those of the whole animals, both in the response to temperature during measurement and to adaptation. These data then, together with the evidence for temperature dependence of "basal" metabolic rate from a wide variety of poikilotherms (for example, Uta stansburiana and Scleoporus occidentalis, Dawson & Bartholomew, 1956; Astacus pallipes eggs and Artemia salina, Grainger, 1956; Carassius auratus, Kanugo & Prosser, 1959; Salmo trutta, Beamish, 1964; Porcellio laevis and Armadillidium vulgate, Edney, 1964; Palaeomonetes vulgaris, McFarland & Pickens 1965; Patella

TEMPERATURE DEPENDENCE OF "STANDARD METABOLIC RATE"

433

aspersa and Patella vulgata, Davies, 1966), contrast with the work and suggestions of Newell and co-workers (Newell, 1966; 1967; NeweU & Northcroft, 1967). It may well be, as NeweU suggests, that the intertidal animals he studied respond differently from other poikilotherms, a difference which is dictated by the peculiar rigors of intertidal life. However, it appears from the literature that intertidal animals do not necessarily maintain a low-temperature independent metabolic rate during low tide, as Newell suggests, but rather that they can, and normally do, sustain varying periods of anaerobic respiration (Dugal, 1935; yon Brand, 1946; Martin, 1961). It appears therefore that in the majority of the animals studied, the oxygen debt incurred is paid back when the animal is submerged (yon Brand, 1946). Consequently, it may be that during the "quiescent" phase (Newell, 1966), the animals are essentially respiring anaerobically, particularly as it is possible that the low level of oxygen consumption registered during this phase (when the animals show no respiratory movements) is insufficient to support aerobic metabolism of the whole animal: Thus it is questionable whether one can break up a process such as respiration into phases in this context, for these phases would be from their origin related, and thus interdependent (van Dam, 1935). Furthermore, several intertidal molluscs have been shown to exhibit "normal" patterns of temperature acclimatization; for example, in their heart beat (Segal, 1961, 1962) and in their oxygen consumption (Davies, 1967). Kenny (1958) has also shown a "resistance" adaptation (Precht, 1958) to occur to a temperature in the amphineuran Clavarizona hirtosa. Thus the usual temperature compensatory pathways seem to exist in many intertidal animals. Newell (1967) also offers additional evidence in support of his theory. He has measured the respiratory rates of mitochondria isolated from a variety of poikilothermic animals, i.e. from aquatic, intertidal and terrestrial environments, and has found that oxygen uptake is essentially temperature independent over the "normal" temperature range in which the particular animal lives. Newell's work therefore conflicts with the results with isolated tissues presented here (see Table 4), and there appear to be a number of reasons for this. First, the rate of oxygen uptake by isolated mitochondria need not, and often does not, follow the rate of uptake in the whole animal, nor indeed the rate in isolated tissues (see Tribe 1966b, 1967a, b). Secondly, as many authors have pointed out previously (e.g. Lewis & Slater, 1954; van den Bergh & Slater, 1962; Balboni, 1965; Carney, 1966), slight variations in the procedure for isolating mitochondria, including variations in the isolation media used, can bring about marked changes in their intactness and hence their oxidative behaviour. Newell's results are remarkable in two ways. First, the quantities and oxidation rates of both substrates used are low and, secondly, pyruvate is not easily oxidized by mitochondria without a "primer" Krebs cycle acid (see Beevers, 1960; Lehninger, 1965). Although Newell does not give the amount of mitochondrial protein used at each determination, his results contrast with similar results obtained for the locust with succinate as substrate, and pyruvate plus malate as substrate obtained by Rees (1954) and Klingenberg & Slenczka (1959). It is likely therefore that the low values obtained by Newell

434

M. A. TRIBI~AND K. BOWL~

reflect the unusual isolation and incubation media used and the time of storage of mitochondria. Consequently, mitochondria treated in this way may not have responded normally to temperature. Therefore, experiments with isolated tissues, rather than isolated mitochondria, have been preferred in this present study, because we think they provide a more reliable picture of in vivo ceRular events in response to temperature. T h u s we conclude that standard metabolic rate as measured by oxygen consumption is temperature dependent in the blowfly, Calh'phora erythrocephala, and so the suggestion implicit in Newell's work does not hold for this insect. T h e intertidal animals studied by NeweU (1966) and N e w e r & Northcroft (1967), however, may well respond differently to temperature, but anaerohiosis could be a factor which has not been considered by these authors. It seems essential therefore that a more thorough investigation should be made of the "quiescent phase" if there is to be a "re-interpretation of the effect of temperature on the metabolism of certain marine invertebrates". Preliminary investigations are therefore under way using excised giR from Carcinus maenas and Mytilus edulis, two intertidal poikilotherms, to see whether these tissues behave in a similar way to blowfly flight muscle in their response to temperature. Acknowledgements---We are grateful to Professor J. Maynard Smith (University of Sussex) and to Dr. M. Hollingsworth (St. Bartholomew's Medical School) for most valuable discussion during the preparation of this paper. REFERENCES BALBONIE. R. (I 965) Influence of preparative procedure on the ox/dative activity of honeybee flight muscle sarcosomes. ~. Insect Physiol. II, 1559-1572. BEAMISHF. W. H. (1964) Respiration of fishes with special emphasis on standard oxygen consumption--II. Influence of weight and temperature on respL,~tion of several species. Can.~. Zool. 42, 177-188. BE~v'm~sH. (1960) The TCA cycle. The utilization of pyruvate. In Respiratory Metabolism in Plants, Chap. 3. Harper & Row, New York. BOD~NsTsn~ D. (1946) Investigation on the locus of action of DDT in flies (Drosophila). Biol. Bull., Woods Hole 90, 148-157. BULLOCKT. H. (1955) Temperature adaptation in poikilothermic animals. Biol. Rev. 30, 311-342. CARNEY G. C. (1966) The effects of different isolation media on the respiration and morphology of housefly sarcosomes, je. Insect Physiol. 12, 1093-1103. CLEC,G J. S. & EVANSD. R. (1961) The physiology of blood trehalose and its function during flight in the blowfly, ft. exp. Biol. 38, 771-793. D^vr~s P. S. (1966) Physiological ecology of Patella--I. The effect of body size and temperature on metabolic rate..y, mar. Biol. 46, 647--658. DAwes P. S. (1967) Physiological ecology of Patella--II. Effect of environment acclimation on the metabolic rate. ~. mar. Biol. 47, 61-74. DAWSON W. R. & BAnTHOLOMKWG. A. (1956) Relation of oxygen consumption to body weight, and temperature acclimation in lizards Uta stansburlana and Scelophorus occidentalis. Physiol. ZooL 29, 40-51. DUOALL. P. (1935) The use of calcareous shell to buffer the product of anaerobic glycolysis in Venus mercenaria, j~. cell. comp. Physiol. 13, 235-251.

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