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Oxygen consumption by three species of lamellibranch mollusc in declining ambient oxygen tension
Comp. Biochem. Physiol., 1971. Vol. #A,
pp. 955 to 970. Pergamon Press. Printed in Great Britain
OXYGEN CONSUMPTION BY THREE SPECIES OF LAMELLIBRANCH MOLLUSC IN DECLINING AMBIENT OXYGEN TENSION B. L. BAYNE School of Biological Sciences, Department of Zoology, The University, Leicester LE17RH (I&?&&
18 Narch 1971)
Abstract-l. Oxygen consumption by Arctica iskzndica was dependent on oxygen tension. Oxygen uptake by Laevicardium crassurn and Mytilus edulis was independent of oxygen tension above a certain critical tension. 2. An index is derived to indicate the degree of dependence of oxygen consumption on oxygen tension. 3, In ~5. crQssutlland M. edulis larger individuals were less dependent on oxygen tension than smaller individuals. 4. In M. edulks dependence of oxygen consumption on oxygen tension increased during periods of laboratory stress. INTRODUCTION
THERE is evidence that some lamellibranch species can regulate their oxygen consumption in conditions of reduced oxygen tension; for example, Mya arena~iu (Van Dam, 1938), Ostrea edulis (Gaarder & Eliassen, 1954), Pecten grandis (Van Dam, 1954), Myths edulis (Rotthauwe, 1958), Mytilus pema (Bayne, 1967) and Mytih cuZzjmiams (Moon & Pritchard, 1970). The details of this relationship between oxygen consumption and oxygen tension are of interest both for understanding the ecology of a species, and also in elucidat~g possible mechanisms for control of gas exchange in the lame~branch gill. In thii paper the results of experiments to determine the relationship between size, oxygen tension and oxygen consumption in three lamellibranch species are described. MATERIALS
AND METHODS
L. were collected in the Oresund and experiments carried out in the Marinbiologisk Laboratorium at Helsingor, Denmark. Luevicardium crassurn (Gmelin) were collected from shell gravel off-shore from Plymouth, England and held at the Plymouth Marine Laboratory. Myths edulis L. were collected from low water at Heacham Beach in the Wash and experiments carried out in an aquarium of re-circulating sea water in Leicester. All individuals were used ir, experiments within 1 week of collection, although some additional experiments were carried out with M. edulis that had been maintained in the laboratory for periods of up to 86 days. The apparatus (Fig. 1) consisted of a water/gas exchange column (3) through which water was passed down (A) and oxygen-free nitrogen bubbled up (C). The oxygen-depleted water was passed via a glass coil for temperature equilibrium (D), through a flow meter (E)
Arctica ishdica
955 32
956
B. L. BAYNE
and thence to the experimental chamber (H). This chamber was provided with three ports; water entered and exited (L) via two three-way taps (F’ and F”) whilst the third port received a Beckman macro oxygen electrode (G) which was operated with a Beckman Model 160 Physiological Gas Analyser (M) and chart recorder (N). By manipulation of the taps
FIG. 1. The apparatus used in measuring the oxygen consumption of bivalves in declining oxygen tension. For details see text. water could be passed either through the chamber or through the by-pass (0). The water in the experimental chamber was stirred with an immersible magnetic stirrer (K) and stirring bar (J). The glass coil and the experimental chamber were immersed under water in a tem~ra~e-controlled water bath. The experimental procedure was as follows: The animal (I) was placed in the chamber on a perforated glass plate over the stirring bar and left overnight in flowing water (SO to 120 mljmin) of 95 to 100 per cent saturation with oxygen and chlorinity lS*Oy&,. At the start of the experiment the chamber was isolated from the water flow by means of the three-way taps and the by-pass, and the depletion of oxygen in the chamber, due to oxygen withdrawal by the animal, was recorded for 30 or 60 min, depending on the size of the animal. During this period flow of nitrogen was started with resultant reduction of oxygen tension in the water. After the first period of measurement the taps were turned to allow water of reduced oxygen tension to flow through the chamber. The resulting decline in oxygen tension was followed and the chamber isolated again when the tension had reached the required vslue. There followed a further period of measurement of the oxygen consumption by the animal, and the process was then repeated. Oxygen uptake values were obtained in this way for four or six oxygen tensions from 1 SO-160 mm Hg to 25-30 mm Hg PO,. In experiments with Laevkardium and Myths the flow of nitrogen was then stopped and two further determinations of oxygen consumption made at ca. 80 and 1 SOmm Hg ~0s as the oxygen tension of the water returned to normal. Values for oxygen consumption were computed from the amount of oxygen available in
OXYGEN CONSUM~ION
BY 7WtES
SPECIES OF ~~LIB~CH
MOLLUSC
957
the chamber and the measured rate of decline of oxygen. The animal flesh was removed from the shell, dried overnight at 90°C and weighed as flesh dry weight. Occasional measurements of the pH of the water indicated no change due either to the passage of nitrogen or to the metabolic activities of the animals. Rach experiment lasted 1 day. Average vahzes for the rate of change of oxygen tension during reduction with nitrogen and subsequent “recovery” to normal were as follows :
n=-nHg
ml 0, per min
SD.
155-95 95-48 48-24 2480 SO-145
0.250 0.145 0.043 0.240 0.093
0,057 0.035 0.010 0.036 0.026
This method is superior to the “closed-chamber” technique because the periods of isolation of the chamber are kept to a miniium. Following each period of isolation water is flushed through the chamber and there is a complete renewal of water available to the allimal, Experiments with Arctica were carried out at 11 & 0.5%. Experiments with Laeericaydim and Myrtles were held at 15 f 0.2”C,
RESULTS
1. Oxygen constsmption in decliningoxygen tensiott A. ihndica. A total of eleven experiments, with animals over a size range 301000 mg dry weight, were carried out with this species in July and August 1969. The relations~p between oxygen uptake at full oxygen tension, expressed as $0, per anima1 per hr, and the flesh dry weight in mg, may be expressed as : Oxygen consumption = 5.89 x dry wt”**Or (Correlation coefficient = O-977 for 10 degrees of freedom.) The results oxygen tension tensions tested, mental oxygen oxygen.
of four experiments in which oxygen consumption was related to are plotted in Fig. 2. This relationship was linear over the range of i-e. the rate of oxygen consumption was dependent on the environtension, indicating that the species is a metabolic conformer to
In Fig. 3 the slopes of the ;QOs/pO, curves from eleven experiments are plotted against the dry flesh weight in mg. This relationship was fmear on logarithmic co-ordinates and may be expressed as: (Correlation
Slope = 1.936 x dry wt.-*-4r7 coefficient = O-958 for 10 degrees of freedom.)
Expressed in another form, the mean oxygen consumption at 80 mm Hg $0, is 49.3 per cent (standard deviation 4.6 per cent) of the mean consumption at 160 mm Hg PO,, over the entire size range tested.
B. L. BAYNE
OXYGEN CONSUM~ION
BY THREE SPECIES OF L~IB~~
959
MOLLUSC
L. CYUSSURLA total of ten experiments with animals over a size range 160-3140 mg dry weight were carried out with this species in July 1970. The relationship between oxygen consumption (as ~10, per animal per hr) at 159 mm Hg pOa, and dry weight in mg may be expressed as :
Oxygen consumption = 2.57 x dry WL*.~~ (correlation coefficient = O-969 for 9 degrees of freedom.) The results of four experiments relating oxygen consumption to oxygen tension are plotted in Fig. 4. In each case there was a range of oxygen tension over which the rate of oxygen uptake was more or less independent of the ambient pOz. The
I
/--’
0
*/ I 0
N
0”
/ 0
f \
1
I
I
120
I
160
PO, FIG.
4. Oxygen cons~ption (as ml oxygen per g dry weight per hr) of cd&ma in decfining oxygen tension; the results of four experiments.
Z.5&-
degree of this independence may be assessed by estimating the “critical tension” or PC (Prosser & Brown, 1961) from these curves. However, a more reliable estimate is obtained by plotting pO,[QO, against PO,, where ~0, is the oxygen tension and QO, is the weight-specific oxygen consumption (Tang, 1933). The resulting linear
B. L. BAYNE
960
plot (Fig. 5) indicates that oxygen consumption and oxygen tension may be related according to the hyperbolic equation : QO,
=
po2
(1)
+ K, 40,
when K, and K, are constants estimated as the intercept and slope respectively the regression equation relating pO,/QO, to ~0,.
of
Izoo-
IOOO-
soo-
0” 0 ‘N 6000?
400 -
200 I
i
I
40
8’0
1
120
J
I60
% FIG. 5. The relationship between oxygen tension and the quotient of the oxygen tension and the weight-specific oxygen consumption, for Laevicardium.
From equation 1 it may be seen that as K, increases in value in relation to K, . PO,, so the oxygen consumption will be more directly proportional to oxygen tension. Conversely, as K, is reduced in value in proportion to K, . PO,, so QO, approaches a constant, i.e. becomes increasingly independent of the oxygen tension. A value for K,/K, therefore provides an index of dependence of oxygen consumption on oxygen tension. Values for this index were calculated for each experiment from regression equations fitted, by the method of least squares, to the curves of pO,/QO, against
OXYGEN CONSUMPTION
BY THREB SPECIES OF LAMBLLIBRANCH
90,. These values are plotted against the weight-specific Fig. 6, and the relationship may be expressed as: (Correlation
961
MOLLUSC
oxygen consumption
in
Qrir, = 19-Ox $jo*r**= coefficient = 0447 for 9 degrees of freedom.)
There exists, therefore, an exponential relationship between the “oxygendependence index” and oxygen consumption. Small individuals, with high
FIG. 6. The
“oxygen-dependence index” (see text) for Laevicardium, against the weight-specific oxygen consumption.
plotted
metabolic rate, are less independent of the ambient oxygen tension than larger individuals with relatively low metabolic rate. M. edulis. Many experiments have been carried out with this species. The observation by Kruger (1960) that the value of the power b in the allometric equation relating oxygen consumption to dry weight varies seasonally has been confirmed (unpub~shed data). The experiments recorded in Figs. 7-9 were carried out in the summer of 1970 and for these animals the oxygen consumption (in ~10, per hr) per animal may be related to dry weight in mg (over a size range 3401270 mg) by the expression: Oxygen consumption = O-316 x dry wt.l*o*7 (Correlation coefficient = 0,906 for 44 degrees of freedom.) The results of four experiments to relate oxygen uptake to oxygen tension in animals within 7 days of being brought into the laboratory are plotted in Fig. 7.
962
B. L. BAYNE
OXYGEN CONSUMPTION
BY THFW.3 SPECIES OF LAMELLIBRANCH
MOLLUSC
963
They indicate a regulation of oxygen consumption down to oxygen tensions of SO-70 mm Hg, and a conformity at less than 50 mm Hg ~0,. In Fig. 8 the data from these experiments are expressed aspO,/QO, againstp0, and, as in the case of
. . . .** /
. .
. .
IO 01
I
I
,,,!I
0.5
IO
Qo,
FIG. 9. The “oxygen-dependence index” (see text) for Myth, weight-specific oxygen consumption.
plotted against the
Laevkardium, the linear relationship affords an estimate of the “oxygen-dependence index”. In Fig. 9 this index (K,/K,) is related to the weight-specific oxygen consumption (QO,) by the expression : ICI/K, = 15.5 x QO,osls. (Correlation coefficient = 0648 for 9 degrees of freedom.) The degree of correlation between the oxygen-dependence index and QO, for Mytilus in these experiments was not as strong as for Laevicardium. This is due, at least in part, to the fact that Mytih began to lose the capacity to regulate oxygen consumption very soon after they were brought into the laboratory. This is illustrated in Fig. 10, where estimates of the critical tension (P,) for animals from different laboratory cultures have been plotted against a time-scale of days for which the animals were maintained in the laboratory. Each point in Fig. 10 represents an experiment with a single animal, and the different symbols represent different cultures which were initiated at different times of the year. There appeared to be a gradual loss of the capacity to regulate oxygen uptake in declining oxygen tension until, after about 30 days, most animals had become metabolic conformers to oxygen below 160 mm Hg ~0,. These cultures of Mytih were maintained under
B. I_..IjAYNE
964
conditions described by Bayne & Thompson (1970) as representing some degree of nutritive and temperature stress. Two experiments were carried out to confirm the effects of time spent in culture on the capacity to regulate oxygen consumption. Cultures of Myt~~us were set up x
I
I
15
I
35
4,
t
X
.
A
I
I
,50
-70
.
I 95
Doys in ioborotory culture
FIG. 10. Estimates of the “critical tension” (PC) for Mytilus, plotted against the days spent in laboratory culture. The different symbols represent individual estimates for animals in different cultures.
at 15°C at a time when the ambient field temperature was 14-17°C. Cells of Tetraselmis wcica were continuously dosed into the holding tanks as food at a final concentration of between O-3 and O-5 mg organic matter per animal per day. These conditions represented a nutritive, but no temperature, stress. At indefinite periods animals were removed from the holding tanks in order to determine the oxygen consumption~o~gen tension relationship. The results of these experiments are plotted in Figs. 11 and 12. In both euitures there was a gradual apparent loss of the capacity to regulate oxygen consumption in declining oxygen tension. This loss took the form of increasing values for K, (the intercept) and decreasing values for K2 with increased time spent in culture. Values for the oxygen-dependence index were calculated for these experiments and are listed in Table 1. This index increased with time spent in culture. 2. Oxygen consumption in increasing oxygen tension On average, the animals in these experiments spent between 70 and 80 min at tensions less than 80 mm Hg PO,. In experiments with Laevicardium and ~yti~~ the oxygen tension of the water was raised from the lowest value tested
.
. .
.
FIG. 11. The quotient of oxygen tension and weight-specific oxygen consumption, related to oxygen tension, for Mytilus after different periods of time spent in laboratory culture: (o), 2 days in culture; (x), 6 days; (A), 36 days; (A), 72 days in culture. Experiment 1.
*
I
40
I
PO=
I
120
I
80
J
160
FIG. 12. The quotient of oxygen tension and weight-specific oxygen consumption, related to oxygen tension, for Myths after different periods of time spent in laboratory culture : (o), 5 days in culture; (x), 20 days; (A), 42 days; (A), 89 days in culture. Experiment 2.
iO-
,o-
B. L. BAYNE
966 TABLE I--CkxuLhTIoN
OF
PERIODS
AN “OXYGEN-DEPENDENCE INDEX"
OF TIMESPENT
IN CULTURE
INTHE
FOR M.
edulis
AFTERDIFFEREXT
LABORATORY
Time spent in culture (days)
Kl
Experiment 1
2 6 36
106 49.5 72.9
0.499 0.432 0.263
21.2 114.6 277.2
Experiment 2
5 20 42
30.7 51.8 146.1
0.214 0.085 0.025
143.5 609.4 5844.0
&
k’,lK,
The index is derived from the constants of the equation relating oxygen consumption to oxygen tension. Increase in the index indicates increasing metabolic dependence on the oxygen supply.
(20-25 mm Hg) to 80 mm Hg in approximately 8 min. It was of interest to determine the oxygen consumption during recovery from these short periods of hypoxia. The results of two experiments with each species are recorded in Figs. 13 and 14. In some experiments the oxygen uptake in increasing oxygen tension was 0,35-
OE-
OO”
O-15 -
005-
I
40
1
fl0
120
I
160
P02 FIG. 13. The oxygen consumption (as ml oxygen per g dry weight per hr) of during the decline (A and 0) and recovery (a and 0) of oxygen tension.
Laevicardium
OXYGEN CONSUMPTION
BY THREE SPECIBS OF LAMBLLIBRANCH
MOLLUSC
967
depressed slightly below the values observed during the decline of tension; in other experiments oxygen consumption during decline and increase of tension were similar. In no case was the oxygen uptake on recovery of ~0, significantly higher than at the start of the experiment. 07
r
FIG. 14. The oxygen consumption (as ml oxygen per g dry weight per hr) of during the decline (0 and A) and recovery (0 and A) of oxygen tension.
Mytilus
DISCUSSION
The values for the weight exponents (b) in the equations relating oxygen consumption by Arctica and Lumicardium to tissue dry weights (0.601 and 0.627 respectively) are similar to values recorded for other bivalve molluscs (Prosser & Brown, 1961) and they imply a surface-area dependence for metabolic rate. The weight exponent for MytiZus in the summer (l-024) is not significantly greater than 1.0, but is higher than other recorded values for this species (Rothauwe, 1958; Read, 1962). However, Kruger (1960) reported values for b as high as 0.93 in March and October and 0.88 in July. These data suggest a strict weight-dependence for metabolic rate in the summer months. In the winter months, however, the exponent b is 0,766 (unpublished data) which is in close agreement with the value for b of O-76 recorded by Zeuthen (1953). The implications of these seasonal differences in the weight-exponent will be discussed elsewhere. The degree of independence between the metabolic rate of an animal and the environmental oxygen tension is partly dependent on the animal’s level of activity (Fry, 1947; Winberg, 1956). As activity increases so does the critical tension (or incipient limiting tension) below which oxygen consumption becomes linear with change in oxygen tension. As Fry (l.c.) states “. . . respiratory dependence is generally well expressed . . . over a wide range of levels of oxygen supply only when the animals are respiring at their maximum rate” (p. 43).
968
B. L. BAYNE
One aspect of the effect of activity on the QO,/pO, relationship is illustrated by experiments using closed containers, in which the animal’s own metabolic activity is relied upon to reduce the oxygen tension. As a result of handling the animal at the start of the experiment, the level of activity or of “excitement” rises, with the result that the oxygen consumption shows dependence on oxygen tension during the early stages of the experiment as the tension is lowered from saturation. There follows a period of respiratory independence as the activity falls, and a final stage of dependence as the tension is reduced below the incipient limiting tension. The result of such an experiment is often a tri-phasic QO,/pO, curve, the proper interpretation of which requires an assessment of the animal’s activity as well as its metabolism (cf. Moon & Pritchard, 1970). By similar argument, the relationship between body size and the degree of independence of metabolic rate from oxygen tension is conditioned by the higher weight-specific oxygen consumption rate of smaller animals. This results in higher values for the incipient limiting tension for smaller than for larger animals. To recognize these effects of activity on the QO,/pO, relationship is not to suggest that a true dependence of routine oxygen consumption on environmental oxygen tension does not occur. In the case of both Arctica and “stressed” Mytilus animals were undisturbed and given 12-15 hr to reach a metabolic equilibrium within the experimental chamber before measurements were begun. And in each case oxygen consumption was reduced linearly with declining oxygen tension. The ecological significance of an animal’s ability (or lack of it) to regulate oxygen uptake at reduced ~0, is not clear. However, the apparent loss of the ability to regulate by Mytihs under a nutritive stress may be linked to changes in the animal’s energy metabolism, which are also induced by stress. This possibility is being examined at present. It is important to distinguish between “routine” and “standard” metabolism in experiments such as those recorded here. At the standard rate of oxygen consumption, with no “scope for activity” (Fry, 1947), oxygen consumption may remain independent of oxygen tension down to the “incipient lethal tension” below which there is insufficient oxygen for the animal to meet its basic metabolic requirements. Whereas an increase in the “incipient limiting tension” imposes a limit only on the animal’s scope for activity, an increase in incipient lethal tension imposes a more severe ecological limit, by increasing the oxygen tension at which energy metabolism must become anaerobic. Some of these relationships are illustrated for Myths in Fig. 15. Generalized curves have been drawn for routine oxygen consumption in declining oxygen tension for two animals of different size in conditions of no stress, and for a single animal under stress. The latter shows both the decline in oxygen consumption that results from prolonged stress in the laboratory as well as the loss of the capacity to regulate oxygen uptake at reduced ~0,. Also indicated in Fig. 15 are generalized values for standard oxygen consumption (derived from experiments on starved animals before and during stress) and the relevant incipient lethal tensions. With increase in size, and the consequent increase in the capacity to regulate oxygen
OXYGEN CONSUMPTION
BY THREE SPECIES OF LAMELLIBRANCH
MOLLUSC
969
consumption, there is a reduction of the incipient lethal tension from 42 to 35 mm HgpO,. This predicts an increase in resistance to anoxia accompanying size 0.6-
FIG. 15. Generalized curves for the weight-specific oxygen consumption (as ml oxygen per g dry weight per hr) of Myth in declining oxygen tension, to show the effects of sire and stress on the incipient lethal tension. The continuous lines represent the QO, for two unstressed animals of different size; the dotted lines represent QO, for a single animal at two levels of stress. (A), estimated basal metabolism of a small unstressed individual; (A), estimated basal metabolism of a larger unstressed individual; (0), estimated basal metabolism of a small slightly stressed animal; (o), estimated basal metabolism of a small severely stressed animal; 1, 2, 3 and 4 indicate values for the incipient lethal tensions of the four hypothetical individuals. increase.
With stress, however, there is an increase in the incipient lethal tension, from 42 to 70 mm HgpO,, with predicted decrease in resistance to anoxia.
Acknowledgements-This research formed part of a programme supported by the Natural Environment Research Council under Grant No. GR/3/516. My thanks are due also to Dr. J. E. Smith and Professor G. Thorson for the use of facilities at the Plymouth and Helsingor Laboratories, respectively.
970
B. L. BAYNE REFERENCES
BAYNEB. L. (1967) The respiratory response of Mytilusperna L. (Mollusca: Lamellibranchia) to reduced environmental oxygen. Physiol. 2001. 40, 307-3 13. BAYNEB. L. & THOMPSONR. J. (1970) Some physiological consequences of keeping Mytilus edulis in the laboratory. Helgolander wiss. Meeresunters. 20, 526-552. FRY F. E. J. (1947) Effects of the environment on animal activity. Univ. Toronto Stud. Biol. 55 (Publ. Ontario Fish. Res. Lab.) 68, l-62. GAARDERT. & ELIA~~.ENE. (1954) The energy metabolism of Ostrea edulis. Univ. Bergen Arbok. Naturv. 3, 1-7. KRUGERF. (1960) Zur Frage der Grossenabhlngigkeit des Sauerstoffverbrauchs von Mytilus edulis L. Helgolander wiss. Meeresuntevs. 7, 125-148. MOON T. W. & PRITCHARDA. W. (1970) Metabolic adaptations in vertically-separated populations of Mytilus californianus Conrad. J. exp. mar. Biol. Ecol. 5, 35-46. PROSSERC. L. & BROWN F. A. (1961) Comparative Animal Physiology, p. 688. Saunders, Philadelphia. READ K. R. H. (1962) Respiration of the bivalved molluscs Mytilus edulis L. and Brachidontes demissus plicatulus Lamark as a function of size and temperature. Comp. Biochem. Physiol. 7, 89-101. ROTTHAUWEH. W. (1958) Untersuchungen zur Atmungsphysiologie und Osmoregulation bei Mytilus edulis mit einen kurzen Anhang iiber die Blutkonzentration von Dreissensia polymorpha in Abtingigkeit von Elektrolytgehalt des Aussenmediums. Veroff. Inst. Meeresforsch. Bremerhaven 5, 143-159. TANG P.S. (1933) Oxygen consumption as a function of oxygen pressure. Q. Rev. Biol. 8, 260-274. VANDAM K. (1938) On the utilisation of oxygen and regulation of breathing in some aquatic animals. Dissertation: Drukkerij “Volharding”, Groningen. VANDAM K. (1954) On the respiration of scallops. Biol. Bull. mar. biol. Lab., Woods Hole 107,192-202. WINBERGG. G. (1956) Rate of metabolism and food requirements of fishes, Nauch. Trudy Beloruss. gos. Univ. Lenin, p. 253 (Fish. Res. Bd Can. Transl. Ser. No. 194). ZEUTHENE. (1953) Oxygen uptake as related to body size in organisms. Q. Rev. Biol. 28, l-12. Key Word Index-Oxygen consumption of lamellibranchs; Arctica islandica; Laevicardium crassurn; Mytilus edulis; oxygen tension; regulation and conformity of metabolic rate; stress-induced changes in metabolic rate.
pp. 955 to 970. Pergamon Press. Printed in Great Britain
OXYGEN CONSUMPTION BY THREE SPECIES OF LAMELLIBRANCH MOLLUSC IN DECLINING AMBIENT OXYGEN TENSION B. L. BAYNE School of Biological Sciences, Department of Zoology, The University, Leicester LE17RH (I&?&&
18 Narch 1971)
Abstract-l. Oxygen consumption by Arctica iskzndica was dependent on oxygen tension. Oxygen uptake by Laevicardium crassurn and Mytilus edulis was independent of oxygen tension above a certain critical tension. 2. An index is derived to indicate the degree of dependence of oxygen consumption on oxygen tension. 3, In ~5. crQssutlland M. edulis larger individuals were less dependent on oxygen tension than smaller individuals. 4. In M. edulks dependence of oxygen consumption on oxygen tension increased during periods of laboratory stress. INTRODUCTION
THERE is evidence that some lamellibranch species can regulate their oxygen consumption in conditions of reduced oxygen tension; for example, Mya arena~iu (Van Dam, 1938), Ostrea edulis (Gaarder & Eliassen, 1954), Pecten grandis (Van Dam, 1954), Myths edulis (Rotthauwe, 1958), Mytilus pema (Bayne, 1967) and Mytih cuZzjmiams (Moon & Pritchard, 1970). The details of this relationship between oxygen consumption and oxygen tension are of interest both for understanding the ecology of a species, and also in elucidat~g possible mechanisms for control of gas exchange in the lame~branch gill. In thii paper the results of experiments to determine the relationship between size, oxygen tension and oxygen consumption in three lamellibranch species are described. MATERIALS
AND METHODS
L. were collected in the Oresund and experiments carried out in the Marinbiologisk Laboratorium at Helsingor, Denmark. Luevicardium crassurn (Gmelin) were collected from shell gravel off-shore from Plymouth, England and held at the Plymouth Marine Laboratory. Myths edulis L. were collected from low water at Heacham Beach in the Wash and experiments carried out in an aquarium of re-circulating sea water in Leicester. All individuals were used ir, experiments within 1 week of collection, although some additional experiments were carried out with M. edulis that had been maintained in the laboratory for periods of up to 86 days. The apparatus (Fig. 1) consisted of a water/gas exchange column (3) through which water was passed down (A) and oxygen-free nitrogen bubbled up (C). The oxygen-depleted water was passed via a glass coil for temperature equilibrium (D), through a flow meter (E)
Arctica ishdica
955 32
956
B. L. BAYNE
and thence to the experimental chamber (H). This chamber was provided with three ports; water entered and exited (L) via two three-way taps (F’ and F”) whilst the third port received a Beckman macro oxygen electrode (G) which was operated with a Beckman Model 160 Physiological Gas Analyser (M) and chart recorder (N). By manipulation of the taps
FIG. 1. The apparatus used in measuring the oxygen consumption of bivalves in declining oxygen tension. For details see text. water could be passed either through the chamber or through the by-pass (0). The water in the experimental chamber was stirred with an immersible magnetic stirrer (K) and stirring bar (J). The glass coil and the experimental chamber were immersed under water in a tem~ra~e-controlled water bath. The experimental procedure was as follows: The animal (I) was placed in the chamber on a perforated glass plate over the stirring bar and left overnight in flowing water (SO to 120 mljmin) of 95 to 100 per cent saturation with oxygen and chlorinity lS*Oy&,. At the start of the experiment the chamber was isolated from the water flow by means of the three-way taps and the by-pass, and the depletion of oxygen in the chamber, due to oxygen withdrawal by the animal, was recorded for 30 or 60 min, depending on the size of the animal. During this period flow of nitrogen was started with resultant reduction of oxygen tension in the water. After the first period of measurement the taps were turned to allow water of reduced oxygen tension to flow through the chamber. The resulting decline in oxygen tension was followed and the chamber isolated again when the tension had reached the required vslue. There followed a further period of measurement of the oxygen consumption by the animal, and the process was then repeated. Oxygen uptake values were obtained in this way for four or six oxygen tensions from 1 SO-160 mm Hg to 25-30 mm Hg PO,. In experiments with Laevkardium and Myths the flow of nitrogen was then stopped and two further determinations of oxygen consumption made at ca. 80 and 1 SOmm Hg ~0s as the oxygen tension of the water returned to normal. Values for oxygen consumption were computed from the amount of oxygen available in
OXYGEN CONSUM~ION
BY 7WtES
SPECIES OF ~~LIB~CH
MOLLUSC
957
the chamber and the measured rate of decline of oxygen. The animal flesh was removed from the shell, dried overnight at 90°C and weighed as flesh dry weight. Occasional measurements of the pH of the water indicated no change due either to the passage of nitrogen or to the metabolic activities of the animals. Rach experiment lasted 1 day. Average vahzes for the rate of change of oxygen tension during reduction with nitrogen and subsequent “recovery” to normal were as follows :
n=-nHg
ml 0, per min
SD.
155-95 95-48 48-24 2480 SO-145
0.250 0.145 0.043 0.240 0.093
0,057 0.035 0.010 0.036 0.026
This method is superior to the “closed-chamber” technique because the periods of isolation of the chamber are kept to a miniium. Following each period of isolation water is flushed through the chamber and there is a complete renewal of water available to the allimal, Experiments with Arctica were carried out at 11 & 0.5%. Experiments with Laeericaydim and Myrtles were held at 15 f 0.2”C,
RESULTS
1. Oxygen constsmption in decliningoxygen tensiott A. ihndica. A total of eleven experiments, with animals over a size range 301000 mg dry weight, were carried out with this species in July and August 1969. The relations~p between oxygen uptake at full oxygen tension, expressed as $0, per anima1 per hr, and the flesh dry weight in mg, may be expressed as : Oxygen consumption = 5.89 x dry wt”**Or (Correlation coefficient = O-977 for 10 degrees of freedom.) The results oxygen tension tensions tested, mental oxygen oxygen.
of four experiments in which oxygen consumption was related to are plotted in Fig. 2. This relationship was linear over the range of i-e. the rate of oxygen consumption was dependent on the environtension, indicating that the species is a metabolic conformer to
In Fig. 3 the slopes of the ;QOs/pO, curves from eleven experiments are plotted against the dry flesh weight in mg. This relationship was fmear on logarithmic co-ordinates and may be expressed as: (Correlation
Slope = 1.936 x dry wt.-*-4r7 coefficient = O-958 for 10 degrees of freedom.)
Expressed in another form, the mean oxygen consumption at 80 mm Hg $0, is 49.3 per cent (standard deviation 4.6 per cent) of the mean consumption at 160 mm Hg PO,, over the entire size range tested.
B. L. BAYNE
OXYGEN CONSUM~ION
BY THREE SPECIES OF L~IB~~
959
MOLLUSC
L. CYUSSURLA total of ten experiments with animals over a size range 160-3140 mg dry weight were carried out with this species in July 1970. The relationship between oxygen consumption (as ~10, per animal per hr) at 159 mm Hg pOa, and dry weight in mg may be expressed as :
Oxygen consumption = 2.57 x dry WL*.~~ (correlation coefficient = O-969 for 9 degrees of freedom.) The results of four experiments relating oxygen consumption to oxygen tension are plotted in Fig. 4. In each case there was a range of oxygen tension over which the rate of oxygen uptake was more or less independent of the ambient pOz. The
I
/--’
0
*/ I 0
N
0”
/ 0
f \
1
I
I
120
I
160
PO, FIG.
4. Oxygen cons~ption (as ml oxygen per g dry weight per hr) of cd&ma in decfining oxygen tension; the results of four experiments.
Z.5&-
degree of this independence may be assessed by estimating the “critical tension” or PC (Prosser & Brown, 1961) from these curves. However, a more reliable estimate is obtained by plotting pO,[QO, against PO,, where ~0, is the oxygen tension and QO, is the weight-specific oxygen consumption (Tang, 1933). The resulting linear
B. L. BAYNE
960
plot (Fig. 5) indicates that oxygen consumption and oxygen tension may be related according to the hyperbolic equation : QO,
=
po2
(1)
+ K, 40,
when K, and K, are constants estimated as the intercept and slope respectively the regression equation relating pO,/QO, to ~0,.
of
Izoo-
IOOO-
soo-
0” 0 ‘N 6000?
400 -
200 I
i
I
40
8’0
1
120
J
I60
% FIG. 5. The relationship between oxygen tension and the quotient of the oxygen tension and the weight-specific oxygen consumption, for Laevicardium.
From equation 1 it may be seen that as K, increases in value in relation to K, . PO,, so the oxygen consumption will be more directly proportional to oxygen tension. Conversely, as K, is reduced in value in proportion to K, . PO,, so QO, approaches a constant, i.e. becomes increasingly independent of the oxygen tension. A value for K,/K, therefore provides an index of dependence of oxygen consumption on oxygen tension. Values for this index were calculated for each experiment from regression equations fitted, by the method of least squares, to the curves of pO,/QO, against
OXYGEN CONSUMPTION
BY THREB SPECIES OF LAMBLLIBRANCH
90,. These values are plotted against the weight-specific Fig. 6, and the relationship may be expressed as: (Correlation
961
MOLLUSC
oxygen consumption
in
Qrir, = 19-Ox $jo*r**= coefficient = 0447 for 9 degrees of freedom.)
There exists, therefore, an exponential relationship between the “oxygendependence index” and oxygen consumption. Small individuals, with high
FIG. 6. The
“oxygen-dependence index” (see text) for Laevicardium, against the weight-specific oxygen consumption.
plotted
metabolic rate, are less independent of the ambient oxygen tension than larger individuals with relatively low metabolic rate. M. edulis. Many experiments have been carried out with this species. The observation by Kruger (1960) that the value of the power b in the allometric equation relating oxygen consumption to dry weight varies seasonally has been confirmed (unpub~shed data). The experiments recorded in Figs. 7-9 were carried out in the summer of 1970 and for these animals the oxygen consumption (in ~10, per hr) per animal may be related to dry weight in mg (over a size range 3401270 mg) by the expression: Oxygen consumption = O-316 x dry wt.l*o*7 (Correlation coefficient = 0,906 for 44 degrees of freedom.) The results of four experiments to relate oxygen uptake to oxygen tension in animals within 7 days of being brought into the laboratory are plotted in Fig. 7.
962
B. L. BAYNE
OXYGEN CONSUMPTION
BY THFW.3 SPECIES OF LAMELLIBRANCH
MOLLUSC
963
They indicate a regulation of oxygen consumption down to oxygen tensions of SO-70 mm Hg, and a conformity at less than 50 mm Hg ~0,. In Fig. 8 the data from these experiments are expressed aspO,/QO, againstp0, and, as in the case of
. . . .** /
. .
. .
IO 01
I
I
,,,!I
0.5
IO
Qo,
FIG. 9. The “oxygen-dependence index” (see text) for Myth, weight-specific oxygen consumption.
plotted against the
Laevkardium, the linear relationship affords an estimate of the “oxygen-dependence index”. In Fig. 9 this index (K,/K,) is related to the weight-specific oxygen consumption (QO,) by the expression : ICI/K, = 15.5 x QO,osls. (Correlation coefficient = 0648 for 9 degrees of freedom.) The degree of correlation between the oxygen-dependence index and QO, for Mytilus in these experiments was not as strong as for Laevicardium. This is due, at least in part, to the fact that Mytih began to lose the capacity to regulate oxygen consumption very soon after they were brought into the laboratory. This is illustrated in Fig. 10, where estimates of the critical tension (P,) for animals from different laboratory cultures have been plotted against a time-scale of days for which the animals were maintained in the laboratory. Each point in Fig. 10 represents an experiment with a single animal, and the different symbols represent different cultures which were initiated at different times of the year. There appeared to be a gradual loss of the capacity to regulate oxygen uptake in declining oxygen tension until, after about 30 days, most animals had become metabolic conformers to oxygen below 160 mm Hg ~0,. These cultures of Mytih were maintained under
B. I_..IjAYNE
964
conditions described by Bayne & Thompson (1970) as representing some degree of nutritive and temperature stress. Two experiments were carried out to confirm the effects of time spent in culture on the capacity to regulate oxygen consumption. Cultures of Myt~~us were set up x
I
I
15
I
35
4,
t
X
.
A
I
I
,50
-70
.
I 95
Doys in ioborotory culture
FIG. 10. Estimates of the “critical tension” (PC) for Mytilus, plotted against the days spent in laboratory culture. The different symbols represent individual estimates for animals in different cultures.
at 15°C at a time when the ambient field temperature was 14-17°C. Cells of Tetraselmis wcica were continuously dosed into the holding tanks as food at a final concentration of between O-3 and O-5 mg organic matter per animal per day. These conditions represented a nutritive, but no temperature, stress. At indefinite periods animals were removed from the holding tanks in order to determine the oxygen consumption~o~gen tension relationship. The results of these experiments are plotted in Figs. 11 and 12. In both euitures there was a gradual apparent loss of the capacity to regulate oxygen consumption in declining oxygen tension. This loss took the form of increasing values for K, (the intercept) and decreasing values for K2 with increased time spent in culture. Values for the oxygen-dependence index were calculated for these experiments and are listed in Table 1. This index increased with time spent in culture. 2. Oxygen consumption in increasing oxygen tension On average, the animals in these experiments spent between 70 and 80 min at tensions less than 80 mm Hg PO,. In experiments with Laevicardium and ~yti~~ the oxygen tension of the water was raised from the lowest value tested
.
. .
.
FIG. 11. The quotient of oxygen tension and weight-specific oxygen consumption, related to oxygen tension, for Mytilus after different periods of time spent in laboratory culture: (o), 2 days in culture; (x), 6 days; (A), 36 days; (A), 72 days in culture. Experiment 1.
*
I
40
I
PO=
I
120
I
80
J
160
FIG. 12. The quotient of oxygen tension and weight-specific oxygen consumption, related to oxygen tension, for Myths after different periods of time spent in laboratory culture : (o), 5 days in culture; (x), 20 days; (A), 42 days; (A), 89 days in culture. Experiment 2.
iO-
,o-
B. L. BAYNE
966 TABLE I--CkxuLhTIoN
OF
PERIODS
AN “OXYGEN-DEPENDENCE INDEX"
OF TIMESPENT
IN CULTURE
INTHE
FOR M.
edulis
AFTERDIFFEREXT
LABORATORY
Time spent in culture (days)
Kl
Experiment 1
2 6 36
106 49.5 72.9
0.499 0.432 0.263
21.2 114.6 277.2
Experiment 2
5 20 42
30.7 51.8 146.1
0.214 0.085 0.025
143.5 609.4 5844.0
&
k’,lK,
The index is derived from the constants of the equation relating oxygen consumption to oxygen tension. Increase in the index indicates increasing metabolic dependence on the oxygen supply.
(20-25 mm Hg) to 80 mm Hg in approximately 8 min. It was of interest to determine the oxygen consumption during recovery from these short periods of hypoxia. The results of two experiments with each species are recorded in Figs. 13 and 14. In some experiments the oxygen uptake in increasing oxygen tension was 0,35-
OE-
OO”
O-15 -
005-
I
40
1
fl0
120
I
160
P02 FIG. 13. The oxygen consumption (as ml oxygen per g dry weight per hr) of during the decline (A and 0) and recovery (a and 0) of oxygen tension.
Laevicardium
OXYGEN CONSUMPTION
BY THREE SPECIBS OF LAMBLLIBRANCH
MOLLUSC
967
depressed slightly below the values observed during the decline of tension; in other experiments oxygen consumption during decline and increase of tension were similar. In no case was the oxygen uptake on recovery of ~0, significantly higher than at the start of the experiment. 07
r
FIG. 14. The oxygen consumption (as ml oxygen per g dry weight per hr) of during the decline (0 and A) and recovery (0 and A) of oxygen tension.
Mytilus
DISCUSSION
The values for the weight exponents (b) in the equations relating oxygen consumption by Arctica and Lumicardium to tissue dry weights (0.601 and 0.627 respectively) are similar to values recorded for other bivalve molluscs (Prosser & Brown, 1961) and they imply a surface-area dependence for metabolic rate. The weight exponent for MytiZus in the summer (l-024) is not significantly greater than 1.0, but is higher than other recorded values for this species (Rothauwe, 1958; Read, 1962). However, Kruger (1960) reported values for b as high as 0.93 in March and October and 0.88 in July. These data suggest a strict weight-dependence for metabolic rate in the summer months. In the winter months, however, the exponent b is 0,766 (unpublished data) which is in close agreement with the value for b of O-76 recorded by Zeuthen (1953). The implications of these seasonal differences in the weight-exponent will be discussed elsewhere. The degree of independence between the metabolic rate of an animal and the environmental oxygen tension is partly dependent on the animal’s level of activity (Fry, 1947; Winberg, 1956). As activity increases so does the critical tension (or incipient limiting tension) below which oxygen consumption becomes linear with change in oxygen tension. As Fry (l.c.) states “. . . respiratory dependence is generally well expressed . . . over a wide range of levels of oxygen supply only when the animals are respiring at their maximum rate” (p. 43).
968
B. L. BAYNE
One aspect of the effect of activity on the QO,/pO, relationship is illustrated by experiments using closed containers, in which the animal’s own metabolic activity is relied upon to reduce the oxygen tension. As a result of handling the animal at the start of the experiment, the level of activity or of “excitement” rises, with the result that the oxygen consumption shows dependence on oxygen tension during the early stages of the experiment as the tension is lowered from saturation. There follows a period of respiratory independence as the activity falls, and a final stage of dependence as the tension is reduced below the incipient limiting tension. The result of such an experiment is often a tri-phasic QO,/pO, curve, the proper interpretation of which requires an assessment of the animal’s activity as well as its metabolism (cf. Moon & Pritchard, 1970). By similar argument, the relationship between body size and the degree of independence of metabolic rate from oxygen tension is conditioned by the higher weight-specific oxygen consumption rate of smaller animals. This results in higher values for the incipient limiting tension for smaller than for larger animals. To recognize these effects of activity on the QO,/pO, relationship is not to suggest that a true dependence of routine oxygen consumption on environmental oxygen tension does not occur. In the case of both Arctica and “stressed” Mytilus animals were undisturbed and given 12-15 hr to reach a metabolic equilibrium within the experimental chamber before measurements were begun. And in each case oxygen consumption was reduced linearly with declining oxygen tension. The ecological significance of an animal’s ability (or lack of it) to regulate oxygen uptake at reduced ~0, is not clear. However, the apparent loss of the ability to regulate by Mytihs under a nutritive stress may be linked to changes in the animal’s energy metabolism, which are also induced by stress. This possibility is being examined at present. It is important to distinguish between “routine” and “standard” metabolism in experiments such as those recorded here. At the standard rate of oxygen consumption, with no “scope for activity” (Fry, 1947), oxygen consumption may remain independent of oxygen tension down to the “incipient lethal tension” below which there is insufficient oxygen for the animal to meet its basic metabolic requirements. Whereas an increase in the “incipient limiting tension” imposes a limit only on the animal’s scope for activity, an increase in incipient lethal tension imposes a more severe ecological limit, by increasing the oxygen tension at which energy metabolism must become anaerobic. Some of these relationships are illustrated for Myths in Fig. 15. Generalized curves have been drawn for routine oxygen consumption in declining oxygen tension for two animals of different size in conditions of no stress, and for a single animal under stress. The latter shows both the decline in oxygen consumption that results from prolonged stress in the laboratory as well as the loss of the capacity to regulate oxygen uptake at reduced ~0,. Also indicated in Fig. 15 are generalized values for standard oxygen consumption (derived from experiments on starved animals before and during stress) and the relevant incipient lethal tensions. With increase in size, and the consequent increase in the capacity to regulate oxygen
OXYGEN CONSUMPTION
BY THREE SPECIES OF LAMELLIBRANCH
MOLLUSC
969
consumption, there is a reduction of the incipient lethal tension from 42 to 35 mm HgpO,. This predicts an increase in resistance to anoxia accompanying size 0.6-
FIG. 15. Generalized curves for the weight-specific oxygen consumption (as ml oxygen per g dry weight per hr) of Myth in declining oxygen tension, to show the effects of sire and stress on the incipient lethal tension. The continuous lines represent the QO, for two unstressed animals of different size; the dotted lines represent QO, for a single animal at two levels of stress. (A), estimated basal metabolism of a small unstressed individual; (A), estimated basal metabolism of a larger unstressed individual; (0), estimated basal metabolism of a small slightly stressed animal; (o), estimated basal metabolism of a small severely stressed animal; 1, 2, 3 and 4 indicate values for the incipient lethal tensions of the four hypothetical individuals. increase.
With stress, however, there is an increase in the incipient lethal tension, from 42 to 70 mm HgpO,, with predicted decrease in resistance to anoxia.
Acknowledgements-This research formed part of a programme supported by the Natural Environment Research Council under Grant No. GR/3/516. My thanks are due also to Dr. J. E. Smith and Professor G. Thorson for the use of facilities at the Plymouth and Helsingor Laboratories, respectively.
970
B. L. BAYNE REFERENCES
BAYNEB. L. (1967) The respiratory response of Mytilusperna L. (Mollusca: Lamellibranchia) to reduced environmental oxygen. Physiol. 2001. 40, 307-3 13. BAYNEB. L. & THOMPSONR. J. (1970) Some physiological consequences of keeping Mytilus edulis in the laboratory. Helgolander wiss. Meeresunters. 20, 526-552. FRY F. E. J. (1947) Effects of the environment on animal activity. Univ. Toronto Stud. Biol. 55 (Publ. Ontario Fish. Res. Lab.) 68, l-62. GAARDERT. & ELIA~~.ENE. (1954) The energy metabolism of Ostrea edulis. Univ. Bergen Arbok. Naturv. 3, 1-7. KRUGERF. (1960) Zur Frage der Grossenabhlngigkeit des Sauerstoffverbrauchs von Mytilus edulis L. Helgolander wiss. Meeresuntevs. 7, 125-148. MOON T. W. & PRITCHARDA. W. (1970) Metabolic adaptations in vertically-separated populations of Mytilus californianus Conrad. J. exp. mar. Biol. Ecol. 5, 35-46. PROSSERC. L. & BROWN F. A. (1961) Comparative Animal Physiology, p. 688. Saunders, Philadelphia. READ K. R. H. (1962) Respiration of the bivalved molluscs Mytilus edulis L. and Brachidontes demissus plicatulus Lamark as a function of size and temperature. Comp. Biochem. Physiol. 7, 89-101. ROTTHAUWEH. W. (1958) Untersuchungen zur Atmungsphysiologie und Osmoregulation bei Mytilus edulis mit einen kurzen Anhang iiber die Blutkonzentration von Dreissensia polymorpha in Abtingigkeit von Elektrolytgehalt des Aussenmediums. Veroff. Inst. Meeresforsch. Bremerhaven 5, 143-159. TANG P.S. (1933) Oxygen consumption as a function of oxygen pressure. Q. Rev. Biol. 8, 260-274. VANDAM K. (1938) On the utilisation of oxygen and regulation of breathing in some aquatic animals. Dissertation: Drukkerij “Volharding”, Groningen. VANDAM K. (1954) On the respiration of scallops. Biol. Bull. mar. biol. Lab., Woods Hole 107,192-202. WINBERGG. G. (1956) Rate of metabolism and food requirements of fishes, Nauch. Trudy Beloruss. gos. Univ. Lenin, p. 253 (Fish. Res. Bd Can. Transl. Ser. No. 194). ZEUTHENE. (1953) Oxygen uptake as related to body size in organisms. Q. Rev. Biol. 28, l-12. Key Word Index-Oxygen consumption of lamellibranchs; Arctica islandica; Laevicardium crassurn; Mytilus edulis; oxygen tension; regulation and conformity of metabolic rate; stress-induced changes in metabolic rate.