Seasonal changes in the effect of temperature on the oxygen consumption of the winkle Littorina littorea (L.) and the mussel Mytilus edulis L.

Comp. Biochem.Physiol., 1970, Vol. 34, pp. 367 to 383. PergamonPress. Printedin Great Britain

SEASONAL CHANGES IN THE EFFECT OF TEMPERATURE ON THE OXYGEN C O N S U M P T I O N OF THE WINKLE L I T T O R I N A LITTOREA (L.) A N D THE M U S S E L M Y T I L U S EDULIS L. R. C. N E W E L L and V. I. PYE Department of Zoology, Queen Mary College, London E,1. (Received 3 October 1969) A b s t r a c t - - 1 . The active rates of oxygen consumption of Littorina littorea and

Mytilus edulis are markedly temperature-dependent at all seasons of the year. 2. The standard rates are only slightly affected by temperature fluctuation over much of the environmental temperature range. 3. The range of such metabolic homeostasis varies with season. 4. Such changes are reflected in the oxygen consumption of cell-free preparations of winkles and mussels. 5. This suggests that compensation for temperature fluctuation in the metabolism of the intact animals may be controlled at a subcellular level. INTRODUCTION MANY OFthe organisms living in the intertidal zone are subjected to rapid fluctua-

tions in temperature with the ebb and flow of the tide. Under extreme conditions, for example, the tissue temperature of some barnacles at Plymouth was found to be 29.9°C in the summer and that of limpets was 24.9°C (Southward, 1958). The sea temperature was meanwhile only 13.7°C so that the above organisms were subjected to a thermal flux of 16.2°C in the case of barnacles and 11.2°C in the case of limpets. It has been shown that the metabolism of many intertidal animals may be relatively unaffected by such short-term fluctuations and show almost perfect compensation over the normal environmental temperature range. Among such organisms may be included the anemone Actinia equina, the polychaete Nephtys hombergi, the winkle Littorina littorea, the cockle Cardium edule and the barnacle Balanus balanoides (Newell, 1966, 1967, 1969; Newell & Northcroft, 1965, 1967). The amphipod Gammarus oceanicus (Halcrow & Boyd, 1967), some limpets (Davies, 1966, 1967), the trochid Calliostoma zizyphinum (Micallef, 1966), fresh-water Trichoptera and gammarids belonging to the pulex group (Collardeau, 1961 ; Collardeau-Roux, 1966; Roux & Roux, 1967), fresh-water copepods belonging to the genus Diaptomus (Siefken & Armitage, 1968) and the crayfish Orconectes sp. (Wiens & Armitage, 1961) also show compensation for acute temperature fluctuation in their oxygen consumption over at least part of the range of temperatures which they experience in their natural environment. It is not clear, however, 367

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R . C . NEWELL AND V. I. P ~

w h e t h e r s u c h c o m p e n s a t i o n for t e m p e r a t u r e fluctuation, w h i c h results in a low v a l u e for t h e t e m p e r a t u r e coefficient (Q10), is a r e s u l t of t h e r m a l a c c l i m a t i o n o r w h e t h e r it is a fixed v a l u e c h a r a c t e r i s t i c o f t h e p a r t i c u l a r o r g a n i s m s in w h i c h it has b e e n d e s c r i b e d . W e have e a r l i e r s h o w n t h a t in c e r t a i n i n t e r t i d a l algae t h e t e m p e r a t u r e r a n g e o v e r w h i c h r e s p i r a t i o n is r e l a t i v e l y i n d e p e n d e n t of t e m p e r a t u r e is i n d e e d m o d i f i a b l e a c c o r d i n g to seasonal c h a n g e s in t h e e n v i r o n m e n t a l t e m p e r a t u r e ( N e w e l l & Pye, 1968). T h u s in t h e algae Porphyra, Griffithsia, Fucus, Ulva a n d Enteromorpha t h e curves r e l a t i n g t h e rate o f o x y g e n c o n s u m p t i o n to t e m p e r a t u r e ( R . T . curves) w e r e f l a t t e n e d b e t w e e n 0 a n d 10°C in D e c e m b e r b u t b e t w e e n 15 a n d 30°C in July. I t was t h e r e f o r e of i n t e r e s t to d e t e r m i n e w h e t h e r s u c h seasonal c o m p e n s a t i o n for t e m p e r a t u r e f l u c t u a t i o n o c c u r r e d in t w o c o m m o n i n t e r t i d a l m o l l u s c s , t h e w i n k l e Littorina littorea a n d t h e m u s s e l Mytilus edulis. T h e r e s u l t s are d i v i d e d i n t o t w o sections, t h e first o f w h i c h s h o w s t h e seasonal c h a n g e s w h i c h o c c u r in t h e o x y g e n c o n s u m p t i o n o f i n t a c t animals. T h e s e c o n d s e c t i o n d e s c r i b e s t h e r e s u l t s of a s i m i l a r series o f m e a s u r e m e n t s w h i c h w e r e c a r r i e d o u t on cell-free h o m o g e n a t e s o f w i n k l e s a n d m u s s e l s c o l l e c t e d f r o m t h e s h o r e at W h i t s t a b l e , K e n t , b e t w e e n J a n u a r y a n d M a y 1968. MATERIALS AND METHODS The animals were collected from the shore from approximately mid-tide level at Whistable, Kent, and transported to the laboratory in large vacuum flasks. T h e air temperature at the time of collection of the specimens was noted. T h e rate of oxygen consumption of 18 individual specimens of L. littorea and M. edulis was measured at a variety of temperatures in a Gilson Differential Respirometer (model G.R. 20) within 24 hr of collection of the animals. Most of the animals showed several rates of oxygen consumption which, in L. littorea, were observed to correspond with different levels of activity. Each of the rates for the animals was plotted as a function of dried tissue weight on logarithmic paper and regression lines fitted by the method of least squares to the maximal and minimal rates of oxygen consumption. T h e correlation coefficient (r) for each of the regression lines so obtained was statistically significant at the 0"001 level. Variations in the level of the maximal or "active rate" of oxygen consumption and in the minimal or "standard rate" with temperature and season could then be read off for an arbitrary-sized animal in the middle of the weight range used. This procedure is identical with that used on other invertebrates (Courtney & Newell, 1965; Newell & Northcroft, 1965, 1967; Newell, 1969). Cell-free homogenates of L. littorea and M. edulis were also prepared from animals which had been collected from the shore at various seasons and transported to the laboratory in vacuum flasks. In each case the shells were removed and the tissues placed in a chilled vessel in 0"88 M sucrose in phosphate buffer p H 7"4. T h e tissues were homogenized for 10 rain in a Virtis 23 high-speed homogenizer and were then ground lightly with a little clean sand by means of a chilled pestle and mortar. The homogenate was then centrifuged for 10 rain at approximately 600 g at 1°C to remove cellular debris and sand. T h e supernatant was finally spun for a further 10 min at approximately 1000 g at I°C and the supernatant stored in a chilled vessel at approximately 1 °C. Of the cell-free homogenate so obtained 0"75 ml was then placed in Warburg flasks together with 0"75 ml of a mixture of reagents such that the final reaction mixture in each flask contained KC1 16 m M ; MgSO4 1.6 raM; A T P 0.2 raM; Cyt c 0"42 x 10 -2 m M and succinate 50 raM. T h e final osmolarity was approximately 0.44 M and the p H 7"4. T h e centre well contained 0"3 ml of 15% K O H and air was the gas phase throughout. An equilibration period of 10 rain was allowed before the taps of the respirometer were closed and readings were made at intervals of from

EFFECT OF TEMPERATURE ON OXYGEN C O N S U M P T I O N OF W I N K L E AND MUSSEL

369

3 to 5 min, depending upon the activity of the preparation, over a period of up to 30 rain. The pH remained constant at 7.4 throughout the experiment. Then a new series of measurements was made at another temperature on another sample of homogenate which had meanwhile been stored at approximately 1°C. All measurements were complete within 8 hr of the initial preparation of the homogenate whose nitrogen content was finally estimated by a semi-micro-Kjeldahl method using a catalyst consisting of 5 parts by weight CuSO4, 5HzO (MAR), 15 parts by weight K~SO4 (MAR), 5 parts by weight HgO and 1 part by weight of selenium (MAR) (Jacobs, 1959). The ammonia was absorbed into 5 ml of 2% boric acid and was estimated by titration against N/100 HCI using an indicator of 2 vol. 0-05% ethanolic methyl red and 1 vol 0-05% ethanolic methylene blue. All values for the mean rate of oxygen consumption are expressed as/~10Jmg N per hr and the number of vessels from which the mean was calculated is indicated in the legends to the figures. The standard deviation was in general small, but where it exceeded + 1-0/zl 02/mg N per hr its magnitude is indicated in the figure. RESULTS

A. Variations in the rate of oxygen consumption of intact L. littorea and M. edulis 1. Seasonal changes in the active rate of oxygen consumption. Specimens of L. littorea were collected in January, February, March, April and May from a position at approximately mid-tide level at Whitstable, Kent. T h e rate of oxygen consumption of 18 specimens of widely different sizes was then measured at a variety of temperatures and the results plotted on logarithm paper as a function of dried tissue weight. T h e results for an arbitrary animal of 30 mg dry tissue weight are shown in Fig. 1 from which it is evident that in general the maximal, or "active rate" of oxygen consumption varied markedly according to temperature as has been previously described by Newell & Northcroft (1967). Seasonal changes are to some extent obscured by the variability of the response to temperature, nevertheless it is apparent that the temperature beyond which a decline occurs is progressively increased from January to May. T h u s in January and February a decline occurs at approximately 20°C, in March decline occurs at temperatures above 23°C whilst in April and May decline in the rate of oxygen consumption with temperature does not occur until temperatures above 27.5°C are reached. In much the same way, small specimens of the mussel M. edulis were collected from the shore at approximately mid-tide level during February and April. Again the rates of oxygen consumption of a wide variety of sizes of animal were recorded over a range of temperatures and plotted on logarithmic paper as a function of dried tissue weight. T h e regression line fitting the maximal rate of oxygen consumption was regarded as the "active rate" and probably corresponded with ciliary feeding. At other times a m u c h slower rate of oxygen consumption was recorded even though the valves were open, and it seems likely that such rates approach the quiescent or "standard rate" of oxygen consumption of the animal. Variations in the level of the active rate of an arbitrary animal of 30 mg dry tissue weight with temperature are shown in Fig. 2. As in that of L. littorea, the active rate of oxygen consumption of M. edulis increases markedly with temperature and, moreover, shows a similar seasonal alteration in the temperature at which a decline in oxygen consumption occurs. A decline in the active rate of oxygen

370

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consumption occurs at temperatures above 20°C in animals collected during February whereas no such decline is evident below 27.5°C in mussels collected during April. A modification of the upper limit of thermal tolerance of the gill

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FIG. 1. Graphs showing seasonal variations in the effect of acute temperature change on the active rate of oxygen consumption of L. littorea. Data expressed in terms of an animal of dried tissue weight 30 mg. The air temperature on the shore at the time of collection of the winkles is shown by an arrow, a, January; b, February; c, March; d, April, e; May.

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EFFECT OF TEMPERATURE ON OXYGEN CONSUMPTION OF WINKLE AND MUSSEL

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cilia of the intertidal bivalves Modiolus demissus and Crassostrea virginica in response to acclimation temperature has been noted by Vernberg et al. (1963) and there seems little doubt that the seasonal variations in the point of thermal decline of the active rate of oxygen consumption in both L. littorea and M. edulis reflect a similar process of thermal acclimation. 2. Seasonal changes in the standard rate of oxygen consumption. T h e effect of temperature on the minimal or "standard rate" of oxygen consumption of L. littorea is quite different from that on the active rate. Instead of a general increase with temperature, the standard rate of oxygen consumption is only slightly affected by temperature variation over much of the range of thermal tolerance of the animal (see also Newell & Northcroft, 1967). In order to compare seasonal changes in the standard rate with those noted above for the active rate, measurements were made on the standard rate of oxygen consumption of winkles collected during January, February, March, April and May at the same time as those made on the active rate. T h e effect of temperature on the standard rate of an animal of 30 mg dry tissue weight is shown in Fig. 3. It is evident that a similar extension in the

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372

R. C. NEWELL AND V. I. PYE

range of thermal tolerance occurs as in the active rate. A decline in the standard rate occurs at temperatures above 20°C in animals collected during January and this value increases to as high as 27.5°C in animals collected during April and May. A second feature of some importance is that the level of the standard rate of oxygen consumption is in general suppressed at high temperatures in animals collected during April and May compared with those collected earlier in the year. Thus the line relating the rate of oxygen consumption to temperature (R.T. curve) has a shallower slope during the spring and early summer than during the winter. Finally, it will be noticed that, as in certain intertidal algae (Newell & Pye, 1968), the temperature range over which the standard rate of oxygen consumption is virtually independent of temperature is appropriate to the seasonal temperatures prevailing in the habitat at the time of collection of the winkles. This demonstrates that the metabolic homeostasis described earlier for specimens of L. littorea collected at Whitstable during the summer (Newell & Northcroft, 1967) is not a fixed feature of the species but is part of a sequence of compensatory changes, or acclimation to the higher temperatures occurring on the shore at that time. In M. edulis collected during February, the standard rate of oxygen consumption declined at temperatures above 17.5°C but this value was increased to 22.5°C in the mussels collected during April (Fig. 4). As in L. littorea the temperature range over which the standard rate was virtually independent of temperature increased with the onset of warmer conditions from - 1 to 15°C in February to < I°C to 20°C in April. On the other hand, the upper limit of thermal tolerance in the oxygen consumption of quiescent mussels was not the same as that for the active rate (Fig. 2) neither was there any suppression of the standard rate at high temperatures in the animals collected during April.

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Despite such differences between the winkle and mussel, it is evident that certain general inferences may be made on the seasonal changes noted above. In both the active and standard rate of such molluscs, for example, seasonal changes

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involve an increase in the upper limit of thermal tolerance or point at which a decline ill oxygen consumption occurs. The second important similarity is that in both Littorina and Mytilus, the rate of oxygen consumption of the quiescent animals (the standard rate) is virtually independent of temperature over the thermal range occurring on the shore. This effect has been noted earlier in a number of intertidal organisms (Newell & Northcroft, 1965, 1967; Newell, 1966, 1969) but the possibility of a seasonal adjustment in the range of thermal independence has been described before only in certain intertidal algae (Newell & Pye, 1968). The rate of oxygen consumption of crude mitochondrial preparations of a variety of intertidal organisms also shows a low temperature coefficient (Newell, 1966, 1967). It is therefore clearly of interest to determine whether seasonal changes occur also in sub-cellular fractions of L. littorea and M. edulis.

B. Variations in the rate of oxygen consumption of cell-free homogenates of L. littorea and M. edulis 1. The effect of osmotic pressure of the suspending medium on the rate of oxygen consumption of cell-free homogenates. It has been pointed out that the rate of oxygen consumption of quiescent Littorina and Mytilus was found to be relatively unaffected by temperature change. In order to determine whether the same general effect could be detected in a cell-free homogenate, a series of preparations was made up so that the sucrose molarity in the final reaction mixture was 0.11 M, 0.22 M, 0.33 M, 0-44 M and 0.66 M. The pH was 7.4 and added succinate 50 mM ; other reagents are listed on p. 368. The results for a cell-free homogenate of L. littorea are shown in Fig. 5 from which it is apparent that in 0.11 M sucrose the rate of oxygen consumption was markedly temperature-dependent. At higher osmotic pressures, however, a region with a low temperature coefficient occurred. The slope of this part of the curve is reflected in the value for the temperature coefficient over the range 1 to 11°C in each of the suspending media. Thus in 0.11 M sucrose the Q10 over this temperature range was 2"083, in 0.22 M was 1.92, in 0.33 M was 1.8, in 0.44 M sucrose was 1.416 and in 0.66 M sucrose was 1.362. There was also a general suppression in the level of oxygen consumption at high temperatures in 0.44 M and 0.66 M sucrose. The air temperature at the time of collection of the animals was 9°C and it is evident that the form of the curve for a homogenate suspended in 0.44 M or 0.66 M sucrose most closely approached that for the standard rate of intact animals. This is perhaps not surprising, since the equivalent molarity of the body fluids of marine invertebrates is likely to be of the order of 0.85 M. In squid nerve, for example, the total ionic constituents amount to a glucose equivalent of 0.980 molal (Baker et al., 1962), so that the mitochondria would be expected to function at relatively high osmotic pressures in the intact animal. The results for a similar experiment on a homogenate of Mytilus edulis are shown in Fig. 6. The animals were collected at the same time as the winkles used in the experiment described above and the cell-free homogenate was suspended in

374

R. C. NEWELL AND V. I. PYE

an identical sequence of reaction mixtures. I n this animal the Q10 over the temperature range 1 to l l ° C was low in all the suspending media, but a similar suppression of the level of oxygen consumption occurred at high temperatures in the homogenates suspended in 0.44 M and 0.66 M sucrose as that noted above in homogenates of L. littorea.

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FIG 5. Graphs showing the effect of the osmotic pressure of the reaction medium on the relation between oxygen consumption and temperature in cell-free homogenates of L. littorea. Of the final reaction mixture 1"5 ml at pH 7"4 contained KC1 16mM; MgSO~ l ' 6 m M ; ATP 0.2mM; Cyt c 0.42×10-SmM and succinate 50 mM. The final sucrose concentration was a, 0'11 M; b, 0"22 M; c, 0"33 M; d, 0"44M; e, 0"66 M. Each point represents the mean of three vessels and where the standard deviation exceeds + 1"0/4 Oz/mg N per hr it is indicated by a line. The air temperature at the time of collection of the animals is indicated by an arrow.

2. The effect of concentration of added succinate on the rate of oxygen consumption of cell-free homogenates. T h e cell-free homogenates prepared and suspended in the medium described in p. 368 probably contained variable amounts of metabolic substrate. In Balanus balanoides, both qualitative and quantitative changes in such

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materials occur seasonally (Barnes et al., 1963) and such variations may profoundly affect the level of metabolism of the organism concerned. We have therefore investigated the effect of substrate concentration on the rate of oxygen consumption of cell-free homogenates of L. littorea and M . edulis.

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Fro. 8. Graphs showing seasonal variations during 1968 in the effect of acute temperature change on the rate of oxygen consumption of cell-free homogenates of L. littorea. Of the final reaction mixture 1"5 ml at pH 7"4 contained KC1 16 raM; MgSO, 1-6 mM; ATP 0"2 mM; Cyt c 0"42 x 10 -3 ml and succinate 50 raM. The final sucrose concentration was 0.44 M. Each point is the mean of four vessels and where the standard deviation exceeded + 1"0/zl O~/mg N per hr it is indicated by a line. The air temperature at the time of collection of the winkles is indicated by an arrow, a, 22 February; b, 7 March; c, 18 March; d, 21 April; e, 22 May. T h e rate of oxygen consumption of a cell-free homogenate of L. littorea suspended in 0-44 M sucrose in phosphate buffer p H 7"4 in the presence of KC1 16 raM, M g S O 4 1.6 raM, A T P 0-2 raM, Cyt c 0.42 × 10 -2 m M plus succinate at a final concentration in 1.5 ml of reaction m e d i u m of 500 mM, 50 m M or zero is shown in Fig. 7A. As would be expected in a cell-free homogenate of a whole winkle, the endogenous substrate level was evidently so high that added succinate had little effect on the rate of oxygen consumption. M u c h the same situation was found to apply to cell-free homogenates of the mussel M . edulis (Fig. 7B). Nevertheless, to prevent possible seasonal fluctuations in endogenous substrate from

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becoming limiting, we have used 50 mM succinate in all of the following experiments on cell-free homogenates of both L. littorea and M. edulis the p H being 7.4 and the sucrose molarity 0.44 M. 3. Seasonal variations in the rate of oxygen consumption of cell-free homogenates of L. littorea and M. edulis. In order to determine whether a seasonal variation in the rate of oxygen consumption of cell-free homogenates of L. littorea and M. edulis occurs, specimens were collected from mid-tide level at Whitstable during February, March, April and May 1968. Cell-free homogenates were prepared as described on p. 368, and suspended in reaction media consisting of final concentrations in 1-5 ml of KC1 16 mM, MgSO4 1-6 mM, A T P 0-2 mM, Cyt c 0-42 x 4O 60 30 g

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FIG. 9. Graphs showing seasonal variations during 1968 in the effect of acute temperature change on the rate of oxygen consumption of cell-free homogenates of m . edulis. Reaction mixture as in Fig 8. Each point represents the mean of five vessels and where the standard deviation exceeded + 1-0/~10z]mg N per hr, it is indicated by a line. T h e air temperature at the time of collection of the mussel is indicated by an arrow, a, February; b, March; c, April; d, NIay.

10 .2 mM, succinate 50 mM in 0.44 M sucrose. The pH was maintained at 7.4 with phosphate buffer to which had been added the appropriate volume of K O H or HC1. Figure 8 shows the seasonal variations which occurred in the rate of oxygen consumption of cell-free homogenates of L. littorea, the air temperature at the time of collection of the specimens being shown by an arrow. The main feature which is apparent from the graphs is that during the earlier months of the year the level of oxygen consumption was more temperature-dependent than during April or May. That is, a shallow slope or "plateau" occurs which extends over the

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environmental temperature range at the time of collection of the animals. The temperature at which the maximal rate of oxygen consumption occurs also shows a marked seasonal change. Thus in February the maximal rate occurred at 8.5°C, in early March at 12.5°C, in late March at 15°C, in April at 18.5°C and in May at 18-5°C. In both of these respects the influence of temperature on the rate of oxygen consumption by cell-free homogenates of L. littorea is similar to that on the standard rate of oxygen consumption of the intact winkle. In M. edulis too, a marked seasonal variation in the influence of temperature on the rate of oxygen consumption by cell-free homogenates occurs. Figure 9 shows that the R.T. curve for a cell-free homogenate of specimens collected during February was markedly temperature-dependent with a peak value occurring at 10°C. In March the air temperature was 15°C at the time of collection of the animals and by this time an extensive shallow slope between 5 ° and 15°C had developed. The maximum rate of oxygen consumption occurred at 16.5°C at this season. In April the cell-free homogenate was apparently more active, perhaps due to a decrease in the proportion of non-respiratory proteins in the preparation, but nevertheless the temperature coefficient was 1.50 between 6 and 16°C. Maximum activity of the homogenate was at 22"5°C compared with 16.5°C in April. Finally, in May a considerable suppression had occurred and the Qlo was 1.0 between 0.75 and 12.5°C. CONCLUSION The results presented above demonstrate that the effects of temperature on the active and standard rates of oxygen consumption of L. littorea and M. edulis are modifiable according to season. In the active rate, such modification primarily involves an increase in the thermal tolerance of the system. For example, in L. littorea a decline in the active rate occurs at 20°C in animals collected from the shore in February, in March a decline occurred at higher temperatures than 23°C whilst in April and May the peak in the active rate of oxygen consumption occurred at 27.5°C after which a decline ensued. Similarly, in M. edulis a decline in the active rate of oxygen consumption occurred at temperatures above 20°C in animals collected in February whereas no such decline occurred below 27.5°C in specimens collected during April. The active rate of oxygen consumption was, however, markedly temperaturedependent throughout the temperature range at which measurements were made. This result is not surprising since direct measurements on activity itself in many organisms have shown it to be profoundly influenced by temperature fluctuation. An increase in the active rate of oxygen consumption with temperature approximates to the observed effects of temperature on ciliary activity (Gray, 1923; Schlieper et al., 1958) on cirral activity in barnacles (for review, see Southward, 1964; Ritz & Foster, 1968) and on activity in a large number of other invertebrates (Fox, 1936, 1938b, 1939; Fox & Wingfield, 1937). A similar influence of temperature on the active rate of oxygen consumption has been noted in a number of other invertebrates including Balanus balanoides (Newell & Northcroft, 1965),

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Actinia equina, Nephtys hombergi, Cardium edule and Littorina littorea (Newell & Northcroft, 1967) and the amphipod Gammarus oceanicus (Halcrow & Boyd, 1967). On the other hand, in some other animals the active rate of oxygen consumption is scarcely affected by temperature fluctuation. In the shrimp Palaemonetes vulgaris for example, the Q10 values for both active and standard rates are less than 2.0 (McFarland & Pickens, 1965) whilst in the fresh-water copepod Diaptomus the Qlo for the rate of oxygen consumption of active specimens was also low over much of the range of thermal tolerance (Siefken & Armitage, 1968). In many invertebrates no distinction has been made between active and standard rates of oxygen consumption, but nevertheless accounts of low temperature coefficients for oxygen consumption are widespread. In Orconectes immunis and O. nais the mean Q10 for oxygen consumption between 16 and 30°C was only 1.42 (Wiens & Armitage, 1961). Oxygen consumption of certain Trichoptera and gammarids is also almost independent of temperature over the range in which the animals live (Collardeau, 1961; Collardeau-Roux, 1966; Roux & Roux, 1967), and the same is true of the trochid Calliostoma zizyphinum (Mieallef, 1966) and of the limpet Patella vulgata (Davies, 1966, 1967). Again, in the echinoid Eucidaris tribuloides the Q10 between < 15 and 20°C for the mean rate of oxygen consumption by well-fed sea urchins collected during the summer was 1.0 compared with a value of 3.4 over this range in winter animals (McPherson, 1968).* No definite generalization can thus be made on the effect of temperature on the active rate of oxygen consumption of such animals but the environmental temperature appears to be implicated in the range over which temperature-independent oxygen consumption occurs. A modification in the upper limit of thermal tolerance in response to acclimation temperature has been noted in gill cilia of the mussel Modiolus demissus by Vernberg et al. (1963). It seems likely, therefore, that the seasonal changes noted in the maximal or active rate of oxygen consumption of L. littorea and M. edulis are directly comparable with observations on the activity of such organisms, although the factors inducing such changes have not been investigated here. The minimal rate of oxygen consumption (the standard rate) of L. littorea and M. edulis is much less affected by temperature fluctuation than the active rate. This feature has already been noted in L. littorea and in a number of other intertidal invertebrates collected during the summer months (Newell, 1966, 1969; Newell & Northcroft, 1965, 1967) and is probably also reflected in the low value for the Q10 noted in invertebrates at undefined activity levels. On the basis of the present results, however, it is possible to add that in L. littorea and M. edulis a seasonal adjustment occurs in the temperature range of such metabolic homeostasis. For example, in L. littorea collected during January, when the air temperature was 3°C, the slope of the line relating the standard rate of oxygen consumption to temperature was very shallow between this temperature and 8.5°C. In February, * L a s k e r et al. (1970) have similarly s h o w n that t h e respiration rate of the c o p e p o d Asellopsis is relatively unaffected by t e m p e r a t u r e b e t w e e n 6 a n d 15°C; the Qlo over this

range being 1-16 and the environmental temperature 9°C. t4

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when the air temperature was 5°C, the shallow slope extended from - 1 to 7-5°C. In March the air temperature was 7°C and apart from an increase at 8°C, the standard rate of oxygen consumption was virtually independent of temperature from - 0 . 5 to 17"5°C. In April the air temperature was 15°C and the curve was flattened between 6 and 27.5°C whilst in May the air temperature at the time of collection of the animals was only 12°C and the curve was flattened between 6 and 22-5°C. In much the same way the curves for the standard rate of oxygen consumption of M. edulis in relation to temperature were flattened between - 1 and 15°C in February, when the air temperature was 5°C, but were flattened up to 20°C in April when the air temperature was 15°C. This seasonal variation in the extent of the shallow slope may account for the absence of similar metabolic compensation in organisms not normally subjected to temperature fluctuation. In the sublittoral Branckiostoma lanceolatum the standard rate shows no trace of a shallow slope over the temperature range at which measurements have been made (Courtney & Newell, 1965) neither does the standard rate of the tubicolous polychaete Diopatra (Mangum & Sassaman, 1969). It seems that metabolic compensation for temperature fluctuation does not occur in such organisms which largely evade thermal stress. The curves obtained with cell-free homogenates of L. l#torea and M. edulis also show similar seasonal trends to those noted in the standard rate of oxygen consumption of intact animals. Thus in cell-free homogenates of winkles collected during February, when the air temperature was 5°C, the slope of the curve relating the rate of oxygen consumption to temperature was relatively shallow between 1.5 and 7°C. In early March, when the air temperature was 6°C at the time of collection, a shallow slope extended between - 1 and 10°C whereas in late March, when the air temperature was 10.5°C, the shallow slope extended between 0.5 and 12.5°C. In April the air temperature was 15°C and the shallow slope was between 1 and 17.5°C. Finally in May the air temperature at the time of collection of the animals was 12°C and the shallow slope extended from 2 to 17.5°C. Cell-free homogenates of the mussel M. edulis also show a seasonal extension in the range over which metabolic compensation occurs. As in L. littorea, such an extension is associated with a comparable change in the temperature at which maximum oxygen consumption occurs. Both of such trends are similar to those noted in the standard rate of oxygen consumption of winkles and mussels and suggest that metabolic compensation for acute temperature fluctuation may be controlled at a sub-cellular level. The oxygen consumption of crude mitochondrial preparations of a variety of other invertebrates, including M. edulis, have a low temperature coefficient (Newell, 1966, 1967, 1969) and similar results have been reported for minced preparations of white muscle of two species of tuna (Gordon, 1968). The latter author showed that the Qx0 for oxygen consumption lies between 1.0 and 1-2 over the temperature range 5-35°C in these preparations. Possible mechanisms underlying this phenomenon have been discussed by Hochachka & Somero (1968), Hochachka (1968) and Dean (1969). ttochachka & -

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Somero (1968) showed that the substrate affinity of lactic dehydrogenase ( L D H ) isozymes of fishes from arctic, temperate and tropical habitats reached its maxim u m values at the normal environmental temperature of the fish. Above such temperatures there was a marked decline in substrate affinity (i.e., an increase in the value for the Michaelis constant Kin) which would tend to reduce the rate of reaction as temperatures increased beyond their normal values. Additional L D H isozymes with different substrate affinities could be synthesized in response to prolonged temperature change (Hochachka, 1967). Somero & Hochachka (1969) have recently shown that in leg muscle L D H of the Alaskan king crab Paralithodes and in epaxial muscle L D H of the rainbow trout Salmo gairdneri, the changes in K m which occur as the temperature is lowered serve to activate certain isozymes appropriate to the new temperature regime. At physiological substrate concentrations of 0.01 to 0.10 mM, such isozymes are inactive at high temperatures. Thus, both an increase in enzyme-substrate affinity and the activation of different isozymes may occur in response even to short-term temperature change. Another mechanism by which the rate of oxygen consumption may be rendered virtually independent of temperature is by competition for common substrate at certain branch points in glucose metabolism. For example, Hochachka (1968) has suggested that at high temperatures lipid biosynthesis may be facilitated and compete increasingly effectively for carbon with the Krebs cycle. As temperatures are increased, therefore, an increasing proportion of carbon would be channelled into lipids with carbon flow through the Krebs cycle being maintained at a constant rate. Dean (1969) working on the rainbow trout Salmo gairdneri has recently shown with labelled acetate that carbon flow through the Krebs cycle is indeed held constant between 5 and 11.5°C with an increasing proportion of 14C appearing as lipids at high temperatures. T h e seasonal modification which we have described above and in intertidal algae (Newell & Pye, 1968) suggests that the metabolic control system is modifiable although we have not demonstrated whether temperature itself or photoperiod is responsible. It is clearly of some interest to determine whether a modification in the range of metabolic homeostasis could also be induced by storage of L. littorea and M. edulis at appropriate temperatures.

Acknowledgements--We are grateful to the Royal Society of London for an equipment grant and to the technical staff of the Zoology Department, Queen Mary College, for their assistance. We should also like to thank Professor T. I. Shaw, for his helpful comments on the results, and the staff of the Computer Centre, Queen Mary College, for their help in programming the statistical data. REFERENCES BAKERP. F., HODGKINA. L. & SHAW T. I. (1962) Replacement of the axoplasm of giant nerve fibres with artificial solutions. J. Physiol., Lond. 164, 330-354. BARNES H., BARNESM. ~; FINLAYSOND. M. (1963) The metabolism during starvation of Balanus balanaides. J. mar. biol. Ass. U.K. 43, 213-233. COLLARDEAU C. (1961) Influence de la temp6rature sur la consommation d'oxyg~ne de quelques larves de Trichopt~res. Hydrobiologia 18, 252-264.

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NEWELL R. C. & NORTHCROFT H. R. (1965) T h e relationship between cirral activity and oxygen uptake in Balanus balanoides. 07. mar. biol. Ass. U.K. 45, 387-403. NEWELL R. C. & NORTnCRO~T H. R. (1967) A re-interpretation of the effect of temperature on the metabolism of certain marine invertebrates. 07. Zool., Lond. 151, 277-298. NEWELL R. C. & PYE V. I. (1968) Seasonal variations in the effect of temperature on the respiration of certain intertidal algae. 07. mar. biol. Ass. U.K. 48, 341-348. RITZ D. A. & FOSTER B. A. (1968) Comparison of the temperature responses of barnacles from Britain, South Africa and New Zealand, with special reference to temperature acclimation in Eliminius modestus. 07. mar. biol. Ass. U.K. 48, 545-559. Roux C. & Roux A. L. (1967) Temperature et m~tabolisme respiratoire d'espbces sympatriques de gammares du groupe pulex (Crustac~s, Amphipodes). Ann. Limnol. 3, 3-16. SCHLIEPER C. R., KOWALSKI R. & ERMAN P. (1958) Beitrag zur 6kologischzellphysiologischen Characterisierung des borealen Lamellibranchiers Modiolus modiolus L. Kieler Meeresforsch 14, 3-10. SIEFKEN M. & ARMITAGE K. B. (1968) Seasonal variations in metabolism and organic nutrients in three Diaptomus (Crustacea: Copepoda). Comp. Biochem. Physiol. 24, 591-609. SOMERO G. N. & HOCHACHKA P. W. (1969) Isoenzymes and short-term temperature compensation in poikilotherms: Activation of lactate dehydrogenase isoenzymes by temperature decreases. Nature, Lond. 223, 194-195. SOUTHWARD A. J. (1958), Note on the temperature tolerance of some intertidal animals in relation to environmental temperatures and geographical distribution, o7. mar. biol. Ass. U.K. 37, 49-66. SOUTHWARDA. J. (1964) T h e relationship between temperature and rhythmic cirral activity in some Cirripedia considered in connection with their geographical distribution. Helgoldnder wiss. Meeresunters. 10, 391-403. WIENS A. W. & ARMITAGEK. B. (1961) T h e oxygen consumption of the crayfish Orconectes immunis and Orconectes nais in response to temperature and to oxygen saturation. Physiol. Zo6l. 34, 39-54. VERNBERG F. J., SCHLIEPER C. & SCHNEIDER D. E. (1963) T h e influence of temperature and salinity on ciliary activity of excised gill tissue of molluscs from North Carolina. Comp. Biochem. Physiol. 8, 271-285. K e y Word Index--Active metabolism; standard metabolism; metabolic homeostasis; temperature fluctuation; cell-free homogenate ; substrate affinity; isozymes; lipid shunt; Littorina littorea; Mytilus edulis; seasonal compensation; thermal tolerance; temperature coefficient.