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Ventilation, the heart beat and oxygen uptake by Mytilus edulis L. in declining oxygen tension
Con@. Biocha. Physiol.,1971,Vol. 4QA, pp. 1065to 1085.Pergamon Press. Printed in Great Britain
VENTILATION, THE HEART BEAT AND OXYGEN UPTAKE BY MYT1TLUS l%lUL1S L. IN DECLINING OXYGEN TENSION B. L. BAYNE Department of Zoology, University of Leicester, Leicester LEl
7RH
(Received 18 March 1971)
Abstract-l. During the decline of oxygen tension Mytilus edulis either regulated (regulators) or failed to regulate (conformers) oxygen consumption. 2. Ventilation rate, and the frequency and amplitude of heat beat, increased at slightly reduced oxygen tension. 3. At low ~0, ventilation rate and heart frequency declined, with increased amplitude of heart beat. 4. The ventilation : relative perfusion ratio decreased with decliiing ~0, in regulators but remained constant in conformers. 5. It is suggested that regulation of oxygen consumption at reduced oxygen tension is based on control of the ventilation : perfusion ratio.
INTRODUCTION
TBE RJ%PIRATORY function of the ventilation current in lamellibranch molluscs has sometimes been considered incidental to the feeding function (Krogh, 1941; Ghiretti, 1966). However, this view has recently been questioned by Hamwi & Haskin (1969) who recorded a linear relationship between ventilation and respiratory rates in ~~c~~~~ wttrcenaria and concluded that “water transport for this species may be at least partially regulated by o~gen requirements” (p. 824). Further, Brand (1968) reported a direct relationship between the flow of water through the mantle cavity of Anodmta anatina and the rate of heart beat. It is likely that lamellibranchs can control gas exchange to some extent, especially in view of the .fact that some species regulate oxygen uptake at reduced oxygen tension (Bayne, 1971). Such control probably is a function of both the ventilation current and the heart output, and an understanding of this control will be possible only if parameters of both ventilation and perfusion are monitored simultaneously on individual animals (Rahn, 1966 ; Hanson & Johansen, 1970 ; Johansen et al., 1970). Whereas the direct measurement of heart output in Mytilus edulis has not proved possible to date, estimates of heart rate and amplitude, together with simultaneous measurement of ventilation and respiration rates provide some understanding of gas exchange in this species. The animals that were used in the experiments to be recorded here were able to regulate oxygen uptake in declining 1065
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oxygen tension for up to 30 days in the laboratory, after which they became metabofic conformers to oxygen (Bayne, 1971). This provided a means to study regulation of oxygen consumption in this species, by comparing indi~fiduaIs capable of regulation with others showing conformity to oxygen tension.
Specimens of the cammon mussel, My~i~r~~e&&s, were coIlected at Wcacham Beach, in the Wash Some individuals were used in experiments within 5 days of collection, others were used after some weeks of laboratory imposed nutritive stress. The conditions of laboratory stress were similar to thase described by Rayne Pr.Thampson (1970).
b
i
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FIG. I. Examples of the recorded heart trace of N. edpllis in one experiment, with the animal regulating oxygen uptake. a, 150 mm Wg PO%, heart frequency and amplitude normal; b, 60 mm Hg PO,, amplitude increased; c, 30 mm Hg pi& frequency and amplitude decreasing; d, 20 mm Hg pOB, incipient cardiac arrest; e, 18 mm Hg PO%, cardiac arrest; f, Recovery of oxygen tension to 40 mm Hg @a, frequency and amplitude increased.
OXYGENUPTAKEBY
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O~YGBNTRNSION
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Heart-beat, frequency and amplitude were monitored from platinum electrodes implanted alongside the pericardium, recording through an impedence pneumograph which was linked by d.c. coupling to a multi-pen recorder. Heart frequency was measured directly (Fig. 1; see also Helm & Trueman, 1967). Amplitude was estimated from the height of the heart-beat trace following internal calibration of the pneumog~ph and recorded as relative amplitude in arbitrary units. Ventilation rate was estimated indirectly by following the disappearance of cells of Phaeodactylum tricornutum as the water passed through the experimental chamber containing the animal. Cell concentrations were determined with a Coulter Counter. Control of the rate of flow of water through the chamber and of the concentration of Phaeodactylum cells are of some importance in this experimental design. Rate of flow must be constant and must exceed the anticipated rn~~urn ventilation rate by the animal in order to avoid more than one passage of water through the animal’s mantle cavity. In these experiments water flow was controlled at 45 + 2-O ml mini. Davids (1964) showed that variations in the concentration of algal cells presented to Mytilus resulted in variations in filtration rate. In these experiments cell concentrations were held constant at 1000 rt: 50 cells ml-l, and the animals were given 12-15 hr undisturbed in the experimental chamber, at constant flow rate and cell concentration, before measurements were started. In estimating ventilation rate from the rate of removal of particles from the ventilation current, accurate values will be obtained only if particle retention is 100 per cent efficient. Many experiments with Mytilus in this laboratory have shown the retention for Phaeodactylum to vary between 92 and 100 per cent efficiency in healthy animals. Further, under constant conditions of open flow, ventilation rates estimated from changes in cell concentration were stable over long periods (Table 1). TARLE I-THE VENTILATION RATES OF TWO M. edulis UNDER CONSTANT CONDITIONS OF TEMPERATURE, OXYGEN TENSION,FLOW RATE OF WATER AND CELL CONCENTRATION OF Phaeo-
dactylurn Hr from
Weight-specific ventilation rate as l/hr per g dry wt.
startof
experiment
Experiment 1
Experiment 2
1
0.978 0.986 1.098 1.090 1.017 1.045 0,968 1 a070 1.031 zk0,052
2.29 2.25 2.07 2.38 2.26 2.26 2.21 2.13 2.27 zk0.096
2 3 4 5 6 7 8 Mean JoS.D.
Animal 1 weighed l-20 g dry wt., animal 2 weighed O-31 g dry wt. The oxygen tension of water flowing into and out of the experimental chamber was continuously monitored with Beckman “macro” oxygen electrodes using a Model 160 Physiological Gas Analyser, and the animal’s oxygen consumption was computed from these values. The complete apparatus was as follows (Fig. 2): Water (A) was passed down a gasliquid exchange column (B), with nitrogen (C) bubbled through to reduce oxygen tension. The water was passed via a ~mpera~re-equilibration coil (0) and flow meter (E) to a small
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flask (F) into which was dosed a suspension of Phaedoctylum cells at constant rate from a peristaltic pump (G). This flask (and others) was stirred by an immersible magnetic stirrer (H). The water then flowed to the first measuring chamber (I) in which the “inflow” oxygen tension was measured by the oxygen electrode (I’). A T-piece and tap (K’) allowed samples
I
I
I
FIG. 2. The apparatus used in the measurement of oxygen consumption, heart beat and ventilation rate of M. edulis in an “open flow” system. For details of lettering see text. to be taken for the “inflow” cell concentration count. The water then passed into the experimental chamber (L) containing the animal with the electrodes implanted. The water in the experimental chamber was also stirred by an immersible magnetic stirrer (H). The “outflow” water passed via a T-piece and tap (K”; for sampling for “outflow” cell concentration) to the second measuring chamber (M) and oxygen probe J” for monitoring “outflow” oxygen tension. Signals from the oxygen electrodes and the heart preparation were fed via the oxygen analyser (N) and impedence pneumograph (P) respectively to the multichannel pen recorder (Q). The experimental design was as follows: The animal, with electrodes implanted on the pericardium, was placed in the experimental vessel and left undisturbed for 12-15 hr at 15°C. Samples were then taken for cell counts, and oxygen tension and heart-beat recordings begun. The oxygen tension of the water was then reduced in four to six steps to 20 mm Hg ~0~ or less; at each step in this reduction the preparation was allowed 1 hr for equilibration followed by sampling for cell counts. Heart beat and oxygen tension recordings were continuous. After 2 hr at the lowest oxygen tension to be tested, the pOa was increased to full saturation in three to four steps, with samples for cell counts taken at each step after allowing 1 hr for equilibration. The animal was left at full oxygen saturation and standard
OXYGEN UPTAKE BY MYZ’ILUS
EDULAS IN DECLINING
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conditions of flow and cell concentration overnight, and the experiment repeated the following day. A total of ten such experiments were carried out in the summer and autumn of 1970. In addition, many supporting experiments, not of the complete design as described, were carried out. The carrying capacity of the blood for oxygen was estimated by bleeding a number of individuals directly from the ventricle, pooling the blood and centrifuging to remove the blood cells. The blood was then equilibrated to different partial pressures of oxygen by evacuating a small tonometer containing the blood sample, and the oxygen content of the blood estimated by the micro gasometric method of Roughton & Scholander (1943) as modified by Scholander & Van Ram (1956). In addition, some samples were analysed by the Van Slyke method. The blood of M. e&&s contains no blood pigment. RESULTS Oxygen consumption
The results of five experiments in which the animals regulated oxygen consumption in declining oxygen tension are plotted in Fig. 3A. The degree of regulation by these animals varied, depending partly on size and partly on the length of time for which the animal had been maintained in the laboratory, as discussed by 2.4r
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FIG. 3A. The oxygen consumption (as ml oxygen per g dry weight per hr) by five animals showing regulation of uptake in declining oxygen tension. 3. Th e oxygen consumption (as ml oxygen per g dry weight per hr) by four animals showing conformity of uptake in declining oxygen tension. C. Th e oxygen consumption (as ml oxygen per g dry weight per hr) during the decline (a> and recovery (a> of oxygen tension ; two experiments.
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OXYGEN UPTAKE BY MYXZLUS
EDULIS
IN DECLINING OXYGEN TENSION
1071
Bayne (1971). In Fig. 3B the results of four experiments in which the animals did not regulate their oxygen consumption have been plotted. These animals had been maintained in the laboratory under nutritive stress for more than 30 days. In these experiments animals were held at oxygen tensions of less than 50 mm Hg for 3-4 hr before recovery to normal saturation values. In Fig. 3C values for oxygen uptake during decline and recovery of oxygen tension are plotted for two experiments ; in both cases, and in all similar experiments, oxygen consumption during increase in ~0, was higher than during decline of oxygen tension. This suggested the incurment of an “oxygen debt” at low oxygen tensions which was ‘%epaid” on more oxygen becoming available. This was in contrast to other experiments (Bayne, 1971) in which no oxygen debt was apparent after shorter periods of hypoxia.
Vmtilativn rate During the decline of oxygen tension the ventilation rate by animals showing regulation of oxygen uptake (called “regulators”) increased slightly down to 80-100 mm HgpO,, followed by decline at lower tensions (Fig. 4A). VentiIation rates for animals not regulating oxygen uptake (called “conformers”) either increased slightly or were maintained down to about 40 mm Hg PO,, below which they declined (Fig. 4B). On recovery from periods of hypoxia ventilation rates did not differ from rates recorded during the decline of oxygen tension (Fig. 4C). 4,0-
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FIG. 5A. The v01ume of oxygen made available to the animal (as ml oxygen per hr) during the decline in oxygen tension: animals regulating oxygen uptake. The bar represents the 95 per cent confidence limit; the different symbols are results from different experiments. B. The volume of oxygen made available to the animal (as ml oxygen per hr) during the decline in oxygen tension: animals conforming in oxygen uptake. The bar represents the 95 per cent confidence limit; the different symbols represent results from different experiments.
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From the measured values for ventilation rate and the oxygen tension of the “inflow” water, it is possible to compute values for the amount of oxygen made available to the animal in unit time by the ventilation current. Some of these values (as ml oxygen made available per hr) have been plotted in Fig. 5A for regulators and Fig. 5B for conformers (data from four experiments in each case). The amount of oxygen made available per hr declined linearly with reduced oxygen tension. The percentage extraction of oxygen (or extraction efficiency) from water passing through the mantle cavity may be calculated from values for the amount of oxygen made available and the oxygen consumption. In water of full saturation with oxygen the extraction efficiency was low (5-10 per cent; see Fig. 6A) as recorded for other bivalves (Hazelhoff, 1938; Bayne, 1967). However, in animals regulating their oxygen uptake, there was an increase in extraction efficiency with declining oxygen tension, reaching 3040 per cent at less than 30 mm Hg ~0, (Fig, 6A). In contrast, in animals conforming in their oxygen uptake, the extraction efficiency did not alter with decline in oxygen tension, but remained between 4 and 10 per cent (Fig. 6A). On return from periods of hypoxia to higher oxygen tensions both conformers and regulators showed increased extraction efficiency for oxygen (Fig. 6B). Comparisons between animals, especially when they differ in physiological condition, are made easier if absolute ventilation (and perfusion) rates are converted to rates per unit of oxygen consumed (Dejours et al., 1970). In Fig. 7A data for ventilation rates (as ml water pumped per g dry weight per hr) are plotted per unit of oxygen consumed (as ml oxygen consumed per g dry weight per hr) for three experiments, in two of which the animals regulated their oxygen uptake, and in one of which the animal was a conformer. There was an increase in the ratio in all cases, with decline in oxygen tension. In Fig. 7B the same ratio VW/vo, (VW/V,,) is plotted for two experiments during both decline and recovery of PO,. Less water was pumped by the animals per unit oxygen consumption during recovery than during the decline of oxygen tension. Heart frequency
and amplitude
Results for the frequency and amplitude of heart beat in declining oxygen tension are plotted in Fig. 8A for an animal regulating oxygen uptake and in Fig. 8B for a conformer. These are taken as broadly typical of the experimental results as a whole. In both cases there was a slight increase in frequency as the oxygen tension was lowered to 4060 mm Hg, followed by a decline that eventually resulted in cardiac arrest at less than 20 mm Hg PO,. The pattern of heart beat at the point of arrest varied in different preparations but was generally one of two types. In the first, there was a marked decline from the normal frequency to a slower one, followed by gradual bradycardia to total arrest (Fig. 1). In the second there was more sudden cessation of beat followed by occasional “bursts” of activity of slightly reduced frequency (similar to Fig. 4 in Helm & Trueman, 1967). The overall pattern of eventual arrest at low oxygen tension was common to all preparations.
OXYGEN UPTAKE BY MYTZLUS
El?ULIS
IN DECLINING
OXYGEN TENSION
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B FIG. 7A. Ventilation rate (VW) calculated per ml of oxygen consumed (Vo,) in three experiments: ( x and O), animals regulating oxygen uptake; (A), animal conforming in oxygen uptake. B. Ventilation rate (VW) calculated per ml oxygen consumed (Voz) during decline (0 and A) and during recovery (0 and A) of oxygen tension, in two experiments.
The pattern for amplitude of the heart beat differed between regulators and conformers. During regulation of oxygen uptake the amplitude gradually increased with decline in oxygen tension, with a rapid increase just prior to cardiac arrest. During conformity of oxygen uptake heart amplitude altered less during decline of $0, with only a slight increase prior to cardiac arrest (Figs, 8A, 3).
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FKG. 8C. FIG. 8A. Heart frequency (0 ; beats per min) and amplitude (0 ; arbitrary units) during decline of oxygen tension: animal regulating oxygen uptake. B. Heart frequency (’ ; beats per min) and amplitude (0 ; arbitrary units) during decline of oxygen tension: animal conforming in oxygen uptake. C. Heart frequency (beats per min) and amplitude (arbitrary units) during decline (0) and recovery (A) of oxygen tension. On recovery from hypoxia values for heart frequency increased from zero over a course similar to that recorded during decline of oxygen tension (Fig. 8C),However, the amplitude of heart beat increased from cardiac arrest to exceed values recorded during decline inpt),, and then remained high for some minutes following recovery to full oxygen saturation. A relative index of heart rate multiplied by amplitude was calculated from these experiments and three examples (taken from the same experiments as illustrated in Fig. 7) are plotted in Fig. 9A on a basis of heart output per unit oxygen consumption As with VW/V,, this relative perfusion index (Q’JVo,) increased in all experiments during decline of oxygen tension. Differences in the relative perfusion index during recovery of oxygen tension and during decline (Fig. 9B) were slight,
OXYGZW UPTAKE BY MYTLLUS EDULIS IN DECLINING
OXYGEN TENSION
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OXYGEN UPTAKE BY MYTILUS
EDULIS IN DECLINING
OXYGBN TBNSION
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Ventilation : relative perfusion ratio The patterns of change for ventilation rate and relative perfusion rate for regulating and conforming animals do not immediately suggest an explanation of differences in the apparent capacity to control oxygen uptake in declining oxygen tension, However, combination of these two parameters in a ventilation : relative perfusion ratio, when plotted against ~0, (Fig. 1OA) does suggest a basic diEerence. In regulators there was a linear decline in this ratio with reduced oxygen tension; in conformers there was little change over a wide range of PO,. This ratio should be used with caution. Neither was the heart output measured directly, nor is there evidence that increased output from the heart resulted in increased perfusion of the gills. Nevertheless, changes in the ventilation : relative perfusion ratio are suggestive. When plotted against percentage extraction of oxygen (Fig. 11) the relationship is seen to be one of decreasing efficiency with increasing values for the ratio. In Fig. 10B values for the ventilation : relative perfusion ratio are plotted for animals recovering from anoxia. In all experiments the ratios were smaller during recovery than during decline of PO,. This correlated with the increased extraction efficiency achieved during recovery of oxygen tension. Blood oxygen capacity The oxygen content of blood was measured at six partial pressures of oxygen, and the results plotted in Fig. 12 together with values for sea water. There was no significant difference between the two. DISCUSSION
The low extraction efliciency recorded here for Mytih at full oxygen saturation agrees with other recorded values for bivalves (Hazelhoff, 1938; Bayne, 1967). There are likely to be many reasons for this low efficiency but two possibilities can be stated, viz. lack of an oxygen-transporting pigment in the blood which results in a low carrying capacity for oxygen, and a high ventilation : perfusion ratio. The low oxygen-carrying capacity of the blood may partly be offset by the large blood volume, estimated as 50 per cent of the wet weight of the tissues (Martin et al., 1958). This would provide an oxygen “store” that is large in proportion to the mass of respiring tissue. The ventilation : relative perfusion ratios recorded in this paper are not absolute vahres due to a lack of direct measurements of either stroke volume or gill perfusion rate. A crude estimate of stroke volume was determined by observing the heart through a window cut in the shell and measuring the dimensions of the ventricle at systole and diastole. This yielded estimates of heart minute volume of between O-3 and 0.7 ml. Assuming that these also represent perfusion rates, and taking an average ventilation rate of 1 l/hr, ventilation : perfusion ratios may be between 22 and 55. These values are high on the scale of ventilation : perfusion ratios for aquatic animals (Rahn, 1956; Johansen et al., 1970; Holeton, 1970) and, if confirmed, would help to explain the low extraction efficiencies for oxygen.
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These low extraction efficiencies for oxygen may be increased by accelerating the perfusion of the gills. Aiello & Guideri (1965) reported occasional “accordion-like shortening of the inter-lamellar blood vessel” (p. 436) in M$%.u which was caused by contraction of muscles in the inter-lamellar connexions. The authors point out that this would increase the efficiency of the branchial circulation. Such a mechanism might operate in conjunction with increased stroke volume of the heart to reduce the ventilation : perfusion ratio. The possibilities for regulation of oxygen uptake in aquatic animals by variation of the ventilation : perfusion ratio was implied by Rahn (1966) and by Hanson & Johansen (1970) for dogfish and Holeton (1970) for the antarctic ice-fish.
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FIG. 11. The relationship between the percentage extraction of oxygen and the ventifation : relative perfusion ratio: (A, A, 0, a), data from experiments in which the animal regulated oxygen uptake; ( x , @), data from two experiments in which the animals conformed in oxygen uptake.
During the decline of oxygen tension “unstressed” Myths were abIe to regulate oxygen uptake, whereas animals under a nutritive stress were not. However, the “stressed” animals had not lost the capacity to increase the effectiveness of oxygen extraction from the water, as evidenced during recovery of the animals from anoxia. That they fail to do so during the decline of p0, may be linked with wider aspects of energy metabolism as related to stress, and this possibility is being studied at present. However, the results of experiments during recovery from anoxia do confirm the suggestion that some control of oxygen uptake is exercised through variation in the ventilation : perfusion ratio affecting the physiological diffusion barrier to oxygen.
OXYGEN UPTAKE BY MYTILUS
EDVLIS
IN
DECLINING
OXYGEN TENSION
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Trueman (1967) and Helm & Trueman (1967) used a similar recording technique to that reported in this paper to record heart beat in Mytilus and other bivalves in relation to air exposure. They observed a reduction of heart frequency (bradycardia) d urm * g ex p osure, followed by rapid increase and “overshoot” of frequency and amplitude on re-immersion in sea water. They interpreted these results in terms of a response to anoxia followed by increased cardiac output during repayment of an oxygen debt. These conclusions are supported by the results recorded here, a reduction of the $0, in the mantle cavity eventually causing reduction of the heart beat. Moon & Pritchard (1970) recorded the decline inp0, in the mantle cavity fluid of Mytilus californianus both in the laboratory and in the
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FIG. 12. The oxygen-carrying capacity of the blood of M. edulis at different oxygen tensions. The means and the ranges for five or seven determinations are represented; the dotted line shows values determined for sea water at 20°C and 18x,, chlorinity.
field. In the laboratory (at 10°C; humidity not stated) the $0, reached < 20 mm Hg in less than 30 min although in the field population the ~0, did not decline below 40 mm Hg during exposure. At these latter oxygen tensions M. edulis would be expected to show bradycardia without complete cardiac arrest. There would appear to be a direct causal link between the $0, of the mantle cavity water and the heart beat, presumably acting through changes in the oxygen tension of the blood. Similarly, the oxygen tension of the water has a direct effect on the activity of the gill cilia (Theede et al., 1969) and on the ventilation rate. In water of reduced oxygen tension, changes in heart beat and in ventilation can work in unison to reduce the ventilation : perfusion ratio and so control the effectiveness of oxygen extraction. During air exposure ventilation ceases and heart beat slows in response to decliningpo, in the mantle cavity. On re-immersion, and with 36
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rapid change in the oxygen tension of the water in the mantle cavity, the ventilation and perfusion rates are increased in such a way as to maintain low ventilation : perfusion ratios during the period of repayment of an oxygen debt acquired during exposure to air. Acknowledgements-This research formed part of a programme supported by the Natural Environment Research Council under Grant No. GR/3/516. REFERENCES AIELLO E. & GUIDERIG. (1965) Distribution and function of the branchial nerve in the mussel. Biol. Bull. mar. biol. Lab., Woods Hole 129, 431-438. BAYNE B. L. (1967) The respiratory response of Mytilus perna L. (Mollusca: Lamelhbran&a) to reduced environmental oxygen. Physiol. Zoiil. 40, 307-313. BAYNE B. L. (1971) Oxygen consumption by three species of lamellibranch mollusc in declining ambient oxygen tension. Comp. Biochem. Physiol. 40, 955-970. BAYNEB. L. & THOMPSONR. J. (1970) Some physiological consequences of keeping Mytilus edulis in the laboratory. Helgolander wiss. Meeresunters. 20, 526-552. BRANDA. R. (1968) Some adaptations to the burrowing habit in the class Bivalvia. Ph.D. thesis, University of Hull. DAVIDS C. (1964) The influence of suspensions of micro-organisms of different concentrations on the pumping and retention of food by the mussel (Mytilus edulis L.). Neth.J. Sea Res. 2, 233-249. DEJOURSP., GAREY W. F. & RAHN H. (1970) Comparison of ventilatory and circulatory flow rates between animals in various physiological conditions. Resp. Physiol. 9, 108-l 17. GHIRETTIF. (1966) Respiration. In Physiology of Mollusca (Edited by WILBUR K. M. & YONCE C. M.), Vol. 2, pp. 175-208. Academic Press, New York. HAMWI A. & HASKINH. H. (1968) Oxygen consumption and pumping rates in the hard clam Mercenaria mercenaria: a direct method. Science, N. Y. 163, 823-824. HANSOND. & JOHANSEN K. (1970) Relationship of gill ventilation and perfusion in Pacific dogfish, Squalus suckleyi. J. Fish. Res. Bd Can. 27, 551-564. HAZELHOFFE. H. (1938) Uber die Ausnutzung des Sauerstoffs bei verschiedenen Wassertieren. 2. vergl. Physiol. 26, 306-327. HELM M. M. & TRUEMANE. R. (1967) The effect of exposure on the heart rate of the mussel, Mytilus edulis L. Comp. Biochem. Physiol. 21, 171-177. HOLETONG. F. (1970) Oxygen uptake and circulation by a haemoglobinless antarctic fish (Chaenocephalus aceratus Lonnberg) compared with three red-blooded antarctic fish. Comp. Biochem. Physiol. 34,457+71. JOHANSEN K., LENFANTC. & MECKLENBURC T. A. (1970) Respiration in the crab, Cancer magister. Z. vergl. Physiol. 70, 1-19. KROGH A. (1941) The Comparative Physiology of Respiratory Mechanisms. University of Pennsylvania Press, Philadelphia. MARTINA. W., HARRISONF. M., HUSTONM. J. & STEWARTD. M. (1958) The blood volumes of some representative molluscs. J. exp. Biol. 35, 260-279. MOON T. W. & PRITCHARDA. W. (1970) Metabolic adaptations in vertically-separated populations of Mytilus californianus Conrad. J. exp. mar. Biol. Ecol. 5, 35-46. RAHN H. (1966) Aquatic gas exchange: theory. Resp. Physiol. 1,1-12. ROUGHTONF. J. W. & SCHOLANDER P. F. (1943) Micro gasometric estimation of the blood gases-I. Oxygen. J. biol. Chem. 148, 541-550. SCHOLANDER P. F. & VAN DAM L. (1956) Micro gasometric determination of oxygen in fish blood. J. cell. camp. physiol. 48, 529-532.
OXYGEN
UPTAKE
BY
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THEIXDEH., PONAT A., HIROKI K. & SCHLIEPERC. (1969) Studies on the resistance of marine
bottom invertebrates to oxygen-deficiency and hydrogen sulphide. Marine Biol. 2, 325-337. TRUEMAN E. R. (1967) Activity and heart rate of bivalve molluscs in their natural habitat. Nature, Lond. 214, 832-833. Key Word Index-Myths edulis; oxygen consumption; ventilation rate; heart beat; oxygen tension; blood oxygen capacity; oxygen debt; control of oxygen uptake; ventilation: perfusion ratio.
VENTILATION, THE HEART BEAT AND OXYGEN UPTAKE BY MYT1TLUS l%lUL1S L. IN DECLINING OXYGEN TENSION B. L. BAYNE Department of Zoology, University of Leicester, Leicester LEl
7RH
(Received 18 March 1971)
Abstract-l. During the decline of oxygen tension Mytilus edulis either regulated (regulators) or failed to regulate (conformers) oxygen consumption. 2. Ventilation rate, and the frequency and amplitude of heat beat, increased at slightly reduced oxygen tension. 3. At low ~0, ventilation rate and heart frequency declined, with increased amplitude of heart beat. 4. The ventilation : relative perfusion ratio decreased with decliiing ~0, in regulators but remained constant in conformers. 5. It is suggested that regulation of oxygen consumption at reduced oxygen tension is based on control of the ventilation : perfusion ratio.
INTRODUCTION
TBE RJ%PIRATORY function of the ventilation current in lamellibranch molluscs has sometimes been considered incidental to the feeding function (Krogh, 1941; Ghiretti, 1966). However, this view has recently been questioned by Hamwi & Haskin (1969) who recorded a linear relationship between ventilation and respiratory rates in ~~c~~~~ wttrcenaria and concluded that “water transport for this species may be at least partially regulated by o~gen requirements” (p. 824). Further, Brand (1968) reported a direct relationship between the flow of water through the mantle cavity of Anodmta anatina and the rate of heart beat. It is likely that lamellibranchs can control gas exchange to some extent, especially in view of the .fact that some species regulate oxygen uptake at reduced oxygen tension (Bayne, 1971). Such control probably is a function of both the ventilation current and the heart output, and an understanding of this control will be possible only if parameters of both ventilation and perfusion are monitored simultaneously on individual animals (Rahn, 1966 ; Hanson & Johansen, 1970 ; Johansen et al., 1970). Whereas the direct measurement of heart output in Mytilus edulis has not proved possible to date, estimates of heart rate and amplitude, together with simultaneous measurement of ventilation and respiration rates provide some understanding of gas exchange in this species. The animals that were used in the experiments to be recorded here were able to regulate oxygen uptake in declining 1065
8 L. BAYNiX
1066
oxygen tension for up to 30 days in the laboratory, after which they became metabofic conformers to oxygen (Bayne, 1971). This provided a means to study regulation of oxygen consumption in this species, by comparing indi~fiduaIs capable of regulation with others showing conformity to oxygen tension.
Specimens of the cammon mussel, My~i~r~~e&&s, were coIlected at Wcacham Beach, in the Wash Some individuals were used in experiments within 5 days of collection, others were used after some weeks of laboratory imposed nutritive stress. The conditions of laboratory stress were similar to thase described by Rayne Pr.Thampson (1970).
b
i
60 set I
i
FIG. I. Examples of the recorded heart trace of N. edpllis in one experiment, with the animal regulating oxygen uptake. a, 150 mm Wg PO%, heart frequency and amplitude normal; b, 60 mm Hg PO,, amplitude increased; c, 30 mm Hg pi& frequency and amplitude decreasing; d, 20 mm Hg pOB, incipient cardiac arrest; e, 18 mm Hg PO%, cardiac arrest; f, Recovery of oxygen tension to 40 mm Hg @a, frequency and amplitude increased.
OXYGENUPTAKEBY
MYTXLUSEDULISIN
DECLINING
O~YGBNTRNSION
1067
Heart-beat, frequency and amplitude were monitored from platinum electrodes implanted alongside the pericardium, recording through an impedence pneumograph which was linked by d.c. coupling to a multi-pen recorder. Heart frequency was measured directly (Fig. 1; see also Helm & Trueman, 1967). Amplitude was estimated from the height of the heart-beat trace following internal calibration of the pneumog~ph and recorded as relative amplitude in arbitrary units. Ventilation rate was estimated indirectly by following the disappearance of cells of Phaeodactylum tricornutum as the water passed through the experimental chamber containing the animal. Cell concentrations were determined with a Coulter Counter. Control of the rate of flow of water through the chamber and of the concentration of Phaeodactylum cells are of some importance in this experimental design. Rate of flow must be constant and must exceed the anticipated rn~~urn ventilation rate by the animal in order to avoid more than one passage of water through the animal’s mantle cavity. In these experiments water flow was controlled at 45 + 2-O ml mini. Davids (1964) showed that variations in the concentration of algal cells presented to Mytilus resulted in variations in filtration rate. In these experiments cell concentrations were held constant at 1000 rt: 50 cells ml-l, and the animals were given 12-15 hr undisturbed in the experimental chamber, at constant flow rate and cell concentration, before measurements were started. In estimating ventilation rate from the rate of removal of particles from the ventilation current, accurate values will be obtained only if particle retention is 100 per cent efficient. Many experiments with Mytilus in this laboratory have shown the retention for Phaeodactylum to vary between 92 and 100 per cent efficiency in healthy animals. Further, under constant conditions of open flow, ventilation rates estimated from changes in cell concentration were stable over long periods (Table 1). TARLE I-THE VENTILATION RATES OF TWO M. edulis UNDER CONSTANT CONDITIONS OF TEMPERATURE, OXYGEN TENSION,FLOW RATE OF WATER AND CELL CONCENTRATION OF Phaeo-
dactylurn Hr from
Weight-specific ventilation rate as l/hr per g dry wt.
startof
experiment
Experiment 1
Experiment 2
1
0.978 0.986 1.098 1.090 1.017 1.045 0,968 1 a070 1.031 zk0,052
2.29 2.25 2.07 2.38 2.26 2.26 2.21 2.13 2.27 zk0.096
2 3 4 5 6 7 8 Mean JoS.D.
Animal 1 weighed l-20 g dry wt., animal 2 weighed O-31 g dry wt. The oxygen tension of water flowing into and out of the experimental chamber was continuously monitored with Beckman “macro” oxygen electrodes using a Model 160 Physiological Gas Analyser, and the animal’s oxygen consumption was computed from these values. The complete apparatus was as follows (Fig. 2): Water (A) was passed down a gasliquid exchange column (B), with nitrogen (C) bubbled through to reduce oxygen tension. The water was passed via a ~mpera~re-equilibration coil (0) and flow meter (E) to a small
1068
B. L. BAYNE
flask (F) into which was dosed a suspension of Phaedoctylum cells at constant rate from a peristaltic pump (G). This flask (and others) was stirred by an immersible magnetic stirrer (H). The water then flowed to the first measuring chamber (I) in which the “inflow” oxygen tension was measured by the oxygen electrode (I’). A T-piece and tap (K’) allowed samples
I
I
I
FIG. 2. The apparatus used in the measurement of oxygen consumption, heart beat and ventilation rate of M. edulis in an “open flow” system. For details of lettering see text. to be taken for the “inflow” cell concentration count. The water then passed into the experimental chamber (L) containing the animal with the electrodes implanted. The water in the experimental chamber was also stirred by an immersible magnetic stirrer (H). The “outflow” water passed via a T-piece and tap (K”; for sampling for “outflow” cell concentration) to the second measuring chamber (M) and oxygen probe J” for monitoring “outflow” oxygen tension. Signals from the oxygen electrodes and the heart preparation were fed via the oxygen analyser (N) and impedence pneumograph (P) respectively to the multichannel pen recorder (Q). The experimental design was as follows: The animal, with electrodes implanted on the pericardium, was placed in the experimental vessel and left undisturbed for 12-15 hr at 15°C. Samples were then taken for cell counts, and oxygen tension and heart-beat recordings begun. The oxygen tension of the water was then reduced in four to six steps to 20 mm Hg ~0~ or less; at each step in this reduction the preparation was allowed 1 hr for equilibration followed by sampling for cell counts. Heart beat and oxygen tension recordings were continuous. After 2 hr at the lowest oxygen tension to be tested, the pOa was increased to full saturation in three to four steps, with samples for cell counts taken at each step after allowing 1 hr for equilibration. The animal was left at full oxygen saturation and standard
OXYGEN UPTAKE BY MYZ’ILUS
EDULAS IN DECLINING
OXYGEN TENSION
1069
conditions of flow and cell concentration overnight, and the experiment repeated the following day. A total of ten such experiments were carried out in the summer and autumn of 1970. In addition, many supporting experiments, not of the complete design as described, were carried out. The carrying capacity of the blood for oxygen was estimated by bleeding a number of individuals directly from the ventricle, pooling the blood and centrifuging to remove the blood cells. The blood was then equilibrated to different partial pressures of oxygen by evacuating a small tonometer containing the blood sample, and the oxygen content of the blood estimated by the micro gasometric method of Roughton & Scholander (1943) as modified by Scholander & Van Ram (1956). In addition, some samples were analysed by the Van Slyke method. The blood of M. e&&s contains no blood pigment. RESULTS Oxygen consumption
The results of five experiments in which the animals regulated oxygen consumption in declining oxygen tension are plotted in Fig. 3A. The degree of regulation by these animals varied, depending partly on size and partly on the length of time for which the animal had been maintained in the laboratory, as discussed by 2.4r
2.0-
16-
N a” IZ-
0.8 -
0,4-
L
I
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t 80 %
FIG. 3A.
I20
1
I
160
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0”
i
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I- 6-
FIG. 3A. The oxygen consumption (as ml oxygen per g dry weight per hr) by five animals showing regulation of uptake in declining oxygen tension. 3. Th e oxygen consumption (as ml oxygen per g dry weight per hr) by four animals showing conformity of uptake in declining oxygen tension. C. Th e oxygen consumption (as ml oxygen per g dry weight per hr) during the decline (a> and recovery (a> of oxygen tension ; two experiments.
0.2-
@6-
r
l-8
OXYGEN UPTAKE BY MYXZLUS
EDULIS
IN DECLINING OXYGEN TENSION
1071
Bayne (1971). In Fig. 3B the results of four experiments in which the animals did not regulate their oxygen consumption have been plotted. These animals had been maintained in the laboratory under nutritive stress for more than 30 days. In these experiments animals were held at oxygen tensions of less than 50 mm Hg for 3-4 hr before recovery to normal saturation values. In Fig. 3C values for oxygen uptake during decline and recovery of oxygen tension are plotted for two experiments ; in both cases, and in all similar experiments, oxygen consumption during increase in ~0, was higher than during decline of oxygen tension. This suggested the incurment of an “oxygen debt” at low oxygen tensions which was ‘%epaid” on more oxygen becoming available. This was in contrast to other experiments (Bayne, 1971) in which no oxygen debt was apparent after shorter periods of hypoxia.
Vmtilativn rate During the decline of oxygen tension the ventilation rate by animals showing regulation of oxygen uptake (called “regulators”) increased slightly down to 80-100 mm HgpO,, followed by decline at lower tensions (Fig. 4A). VentiIation rates for animals not regulating oxygen uptake (called “conformers”) either increased slightly or were maintained down to about 40 mm Hg PO,, below which they declined (Fig. 4B). On recovery from periods of hypoxia ventilation rates did not differ from rates recorded during the decline of oxygen tension (Fig. 4C). 4,0-
3.0 -
P) 5 6 f 2-o= E 3
I.0
FIG. 4A.
1072
B. Id. BAYNE
0
I-
f
!5-
0 ,/, x
.
40
A
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.
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%
60
,
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t
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FIG. 5A. The v01ume of oxygen made available to the animal (as ml oxygen per hr) during the decline in oxygen tension: animals regulating oxygen uptake. The bar represents the 95 per cent confidence limit; the different symbols are results from different experiments. B. The volume of oxygen made available to the animal (as ml oxygen per hr) during the decline in oxygen tension: animals conforming in oxygen uptake. The bar represents the 95 per cent confidence limit; the different symbols represent results from different experiments.
Ti
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9 b
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5 P
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B. L. BAYNE
1074
From the measured values for ventilation rate and the oxygen tension of the “inflow” water, it is possible to compute values for the amount of oxygen made available to the animal in unit time by the ventilation current. Some of these values (as ml oxygen made available per hr) have been plotted in Fig. 5A for regulators and Fig. 5B for conformers (data from four experiments in each case). The amount of oxygen made available per hr declined linearly with reduced oxygen tension. The percentage extraction of oxygen (or extraction efficiency) from water passing through the mantle cavity may be calculated from values for the amount of oxygen made available and the oxygen consumption. In water of full saturation with oxygen the extraction efficiency was low (5-10 per cent; see Fig. 6A) as recorded for other bivalves (Hazelhoff, 1938; Bayne, 1967). However, in animals regulating their oxygen uptake, there was an increase in extraction efficiency with declining oxygen tension, reaching 3040 per cent at less than 30 mm Hg ~0, (Fig, 6A). In contrast, in animals conforming in their oxygen uptake, the extraction efficiency did not alter with decline in oxygen tension, but remained between 4 and 10 per cent (Fig. 6A). On return from periods of hypoxia to higher oxygen tensions both conformers and regulators showed increased extraction efficiency for oxygen (Fig. 6B). Comparisons between animals, especially when they differ in physiological condition, are made easier if absolute ventilation (and perfusion) rates are converted to rates per unit of oxygen consumed (Dejours et al., 1970). In Fig. 7A data for ventilation rates (as ml water pumped per g dry weight per hr) are plotted per unit of oxygen consumed (as ml oxygen consumed per g dry weight per hr) for three experiments, in two of which the animals regulated their oxygen uptake, and in one of which the animal was a conformer. There was an increase in the ratio in all cases, with decline in oxygen tension. In Fig. 7B the same ratio VW/vo, (VW/V,,) is plotted for two experiments during both decline and recovery of PO,. Less water was pumped by the animals per unit oxygen consumption during recovery than during the decline of oxygen tension. Heart frequency
and amplitude
Results for the frequency and amplitude of heart beat in declining oxygen tension are plotted in Fig. 8A for an animal regulating oxygen uptake and in Fig. 8B for a conformer. These are taken as broadly typical of the experimental results as a whole. In both cases there was a slight increase in frequency as the oxygen tension was lowered to 4060 mm Hg, followed by a decline that eventually resulted in cardiac arrest at less than 20 mm Hg PO,. The pattern of heart beat at the point of arrest varied in different preparations but was generally one of two types. In the first, there was a marked decline from the normal frequency to a slower one, followed by gradual bradycardia to total arrest (Fig. 1). In the second there was more sudden cessation of beat followed by occasional “bursts” of activity of slightly reduced frequency (similar to Fig. 4 in Helm & Trueman, 1967). The overall pattern of eventual arrest at low oxygen tension was common to all preparations.
OXYGEN UPTAKE BY MYTZLUS
El?ULIS
IN DECLINING
OXYGEN TENSION
1075
1076
B. L. BAYNE
I
I
!
I
80 P 02
40
120
I I60
A
i
I
I
40
80
I
120
I 160
P02
B FIG. 7A. Ventilation rate (VW) calculated per ml of oxygen consumed (Vo,) in three experiments: ( x and O), animals regulating oxygen uptake; (A), animal conforming in oxygen uptake. B. Ventilation rate (VW) calculated per ml oxygen consumed (Voz) during decline (0 and A) and during recovery (0 and A) of oxygen tension, in two experiments.
The pattern for amplitude of the heart beat differed between regulators and conformers. During regulation of oxygen uptake the amplitude gradually increased with decline in oxygen tension, with a rapid increase just prior to cardiac arrest. During conformity of oxygen uptake heart amplitude altered less during decline of $0, with only a slight increase prior to cardiac arrest (Figs, 8A, 3).
OXYGEN UPTAKE BY MYTlLUS
EDULIS
up
s&~Ufl
L 4
r(JDJ+!qJD:
IN DECLINING
Jed KauenbeJ~
epnt!ldluD
:
”
I B
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&JD~H
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OXYGEN TENSION
1077
1078
B. L. BAYNE 35
r
-
A---------A-
----___ --)I\
‘\
2
“r
'5 25-
FKG. 8C. FIG. 8A. Heart frequency (0 ; beats per min) and amplitude (0 ; arbitrary units) during decline of oxygen tension: animal regulating oxygen uptake. B. Heart frequency (’ ; beats per min) and amplitude (0 ; arbitrary units) during decline of oxygen tension: animal conforming in oxygen uptake. C. Heart frequency (beats per min) and amplitude (arbitrary units) during decline (0) and recovery (A) of oxygen tension. On recovery from hypoxia values for heart frequency increased from zero over a course similar to that recorded during decline of oxygen tension (Fig. 8C),However, the amplitude of heart beat increased from cardiac arrest to exceed values recorded during decline inpt),, and then remained high for some minutes following recovery to full oxygen saturation. A relative index of heart rate multiplied by amplitude was calculated from these experiments and three examples (taken from the same experiments as illustrated in Fig. 7) are plotted in Fig. 9A on a basis of heart output per unit oxygen consumption As with VW/V,, this relative perfusion index (Q’JVo,) increased in all experiments during decline of oxygen tension. Differences in the relative perfusion index during recovery of oxygen tension and during decline (Fig. 9B) were slight,
OXYGZW UPTAKE BY MYTLLUS EDULIS IN DECLINING
OXYGEN TENSION
1079
1080
B. L. BAYNE
OXYGEN UPTAKE BY MYTILUS
EDULIS IN DECLINING
OXYGBN TBNSION
1081
Ventilation : relative perfusion ratio The patterns of change for ventilation rate and relative perfusion rate for regulating and conforming animals do not immediately suggest an explanation of differences in the apparent capacity to control oxygen uptake in declining oxygen tension, However, combination of these two parameters in a ventilation : relative perfusion ratio, when plotted against ~0, (Fig. 1OA) does suggest a basic diEerence. In regulators there was a linear decline in this ratio with reduced oxygen tension; in conformers there was little change over a wide range of PO,. This ratio should be used with caution. Neither was the heart output measured directly, nor is there evidence that increased output from the heart resulted in increased perfusion of the gills. Nevertheless, changes in the ventilation : relative perfusion ratio are suggestive. When plotted against percentage extraction of oxygen (Fig. 11) the relationship is seen to be one of decreasing efficiency with increasing values for the ratio. In Fig. 10B values for the ventilation : relative perfusion ratio are plotted for animals recovering from anoxia. In all experiments the ratios were smaller during recovery than during decline of PO,. This correlated with the increased extraction efficiency achieved during recovery of oxygen tension. Blood oxygen capacity The oxygen content of blood was measured at six partial pressures of oxygen, and the results plotted in Fig. 12 together with values for sea water. There was no significant difference between the two. DISCUSSION
The low extraction efliciency recorded here for Mytih at full oxygen saturation agrees with other recorded values for bivalves (Hazelhoff, 1938; Bayne, 1967). There are likely to be many reasons for this low efficiency but two possibilities can be stated, viz. lack of an oxygen-transporting pigment in the blood which results in a low carrying capacity for oxygen, and a high ventilation : perfusion ratio. The low oxygen-carrying capacity of the blood may partly be offset by the large blood volume, estimated as 50 per cent of the wet weight of the tissues (Martin et al., 1958). This would provide an oxygen “store” that is large in proportion to the mass of respiring tissue. The ventilation : relative perfusion ratios recorded in this paper are not absolute vahres due to a lack of direct measurements of either stroke volume or gill perfusion rate. A crude estimate of stroke volume was determined by observing the heart through a window cut in the shell and measuring the dimensions of the ventricle at systole and diastole. This yielded estimates of heart minute volume of between O-3 and 0.7 ml. Assuming that these also represent perfusion rates, and taking an average ventilation rate of 1 l/hr, ventilation : perfusion ratios may be between 22 and 55. These values are high on the scale of ventilation : perfusion ratios for aquatic animals (Rahn, 1956; Johansen et al., 1970; Holeton, 1970) and, if confirmed, would help to explain the low extraction efficiencies for oxygen.
1082
B. L.
BAYNE
These low extraction efficiencies for oxygen may be increased by accelerating the perfusion of the gills. Aiello & Guideri (1965) reported occasional “accordion-like shortening of the inter-lamellar blood vessel” (p. 436) in M$%.u which was caused by contraction of muscles in the inter-lamellar connexions. The authors point out that this would increase the efficiency of the branchial circulation. Such a mechanism might operate in conjunction with increased stroke volume of the heart to reduce the ventilation : perfusion ratio. The possibilities for regulation of oxygen uptake in aquatic animals by variation of the ventilation : perfusion ratio was implied by Rahn (1966) and by Hanson & Johansen (1970) for dogfish and Holeton (1970) for the antarctic ice-fish.
I
I
I
2
I
3
I
4
I
5
-
I
6
I
7
I
8
J 9
VW/vcl,
FIG. 11. The relationship between the percentage extraction of oxygen and the ventifation : relative perfusion ratio: (A, A, 0, a), data from experiments in which the animal regulated oxygen uptake; ( x , @), data from two experiments in which the animals conformed in oxygen uptake.
During the decline of oxygen tension “unstressed” Myths were abIe to regulate oxygen uptake, whereas animals under a nutritive stress were not. However, the “stressed” animals had not lost the capacity to increase the effectiveness of oxygen extraction from the water, as evidenced during recovery of the animals from anoxia. That they fail to do so during the decline of p0, may be linked with wider aspects of energy metabolism as related to stress, and this possibility is being studied at present. However, the results of experiments during recovery from anoxia do confirm the suggestion that some control of oxygen uptake is exercised through variation in the ventilation : perfusion ratio affecting the physiological diffusion barrier to oxygen.
OXYGEN UPTAKE BY MYTILUS
EDVLIS
IN
DECLINING
OXYGEN TENSION
1083
Trueman (1967) and Helm & Trueman (1967) used a similar recording technique to that reported in this paper to record heart beat in Mytilus and other bivalves in relation to air exposure. They observed a reduction of heart frequency (bradycardia) d urm * g ex p osure, followed by rapid increase and “overshoot” of frequency and amplitude on re-immersion in sea water. They interpreted these results in terms of a response to anoxia followed by increased cardiac output during repayment of an oxygen debt. These conclusions are supported by the results recorded here, a reduction of the $0, in the mantle cavity eventually causing reduction of the heart beat. Moon & Pritchard (1970) recorded the decline inp0, in the mantle cavity fluid of Mytilus californianus both in the laboratory and in the
0.7r
FIG. 12. The oxygen-carrying capacity of the blood of M. edulis at different oxygen tensions. The means and the ranges for five or seven determinations are represented; the dotted line shows values determined for sea water at 20°C and 18x,, chlorinity.
field. In the laboratory (at 10°C; humidity not stated) the $0, reached < 20 mm Hg in less than 30 min although in the field population the ~0, did not decline below 40 mm Hg during exposure. At these latter oxygen tensions M. edulis would be expected to show bradycardia without complete cardiac arrest. There would appear to be a direct causal link between the $0, of the mantle cavity water and the heart beat, presumably acting through changes in the oxygen tension of the blood. Similarly, the oxygen tension of the water has a direct effect on the activity of the gill cilia (Theede et al., 1969) and on the ventilation rate. In water of reduced oxygen tension, changes in heart beat and in ventilation can work in unison to reduce the ventilation : perfusion ratio and so control the effectiveness of oxygen extraction. During air exposure ventilation ceases and heart beat slows in response to decliningpo, in the mantle cavity. On re-immersion, and with 36
1084
B. L. BAYNE
rapid change in the oxygen tension of the water in the mantle cavity, the ventilation and perfusion rates are increased in such a way as to maintain low ventilation : perfusion ratios during the period of repayment of an oxygen debt acquired during exposure to air. Acknowledgements-This research formed part of a programme supported by the Natural Environment Research Council under Grant No. GR/3/516. REFERENCES AIELLO E. & GUIDERIG. (1965) Distribution and function of the branchial nerve in the mussel. Biol. Bull. mar. biol. Lab., Woods Hole 129, 431-438. BAYNE B. L. (1967) The respiratory response of Mytilus perna L. (Mollusca: Lamelhbran&a) to reduced environmental oxygen. Physiol. Zoiil. 40, 307-313. BAYNE B. L. (1971) Oxygen consumption by three species of lamellibranch mollusc in declining ambient oxygen tension. Comp. Biochem. Physiol. 40, 955-970. BAYNEB. L. & THOMPSONR. J. (1970) Some physiological consequences of keeping Mytilus edulis in the laboratory. Helgolander wiss. Meeresunters. 20, 526-552. BRANDA. R. (1968) Some adaptations to the burrowing habit in the class Bivalvia. Ph.D. thesis, University of Hull. DAVIDS C. (1964) The influence of suspensions of micro-organisms of different concentrations on the pumping and retention of food by the mussel (Mytilus edulis L.). Neth.J. Sea Res. 2, 233-249. DEJOURSP., GAREY W. F. & RAHN H. (1970) Comparison of ventilatory and circulatory flow rates between animals in various physiological conditions. Resp. Physiol. 9, 108-l 17. GHIRETTIF. (1966) Respiration. In Physiology of Mollusca (Edited by WILBUR K. M. & YONCE C. M.), Vol. 2, pp. 175-208. Academic Press, New York. HAMWI A. & HASKINH. H. (1968) Oxygen consumption and pumping rates in the hard clam Mercenaria mercenaria: a direct method. Science, N. Y. 163, 823-824. HANSOND. & JOHANSEN K. (1970) Relationship of gill ventilation and perfusion in Pacific dogfish, Squalus suckleyi. J. Fish. Res. Bd Can. 27, 551-564. HAZELHOFFE. H. (1938) Uber die Ausnutzung des Sauerstoffs bei verschiedenen Wassertieren. 2. vergl. Physiol. 26, 306-327. HELM M. M. & TRUEMANE. R. (1967) The effect of exposure on the heart rate of the mussel, Mytilus edulis L. Comp. Biochem. Physiol. 21, 171-177. HOLETONG. F. (1970) Oxygen uptake and circulation by a haemoglobinless antarctic fish (Chaenocephalus aceratus Lonnberg) compared with three red-blooded antarctic fish. Comp. Biochem. Physiol. 34,457+71. JOHANSEN K., LENFANTC. & MECKLENBURC T. A. (1970) Respiration in the crab, Cancer magister. Z. vergl. Physiol. 70, 1-19. KROGH A. (1941) The Comparative Physiology of Respiratory Mechanisms. University of Pennsylvania Press, Philadelphia. MARTINA. W., HARRISONF. M., HUSTONM. J. & STEWARTD. M. (1958) The blood volumes of some representative molluscs. J. exp. Biol. 35, 260-279. MOON T. W. & PRITCHARDA. W. (1970) Metabolic adaptations in vertically-separated populations of Mytilus californianus Conrad. J. exp. mar. Biol. Ecol. 5, 35-46. RAHN H. (1966) Aquatic gas exchange: theory. Resp. Physiol. 1,1-12. ROUGHTONF. J. W. & SCHOLANDER P. F. (1943) Micro gasometric estimation of the blood gases-I. Oxygen. J. biol. Chem. 148, 541-550. SCHOLANDER P. F. & VAN DAM L. (1956) Micro gasometric determination of oxygen in fish blood. J. cell. camp. physiol. 48, 529-532.
OXYGEN
UPTAKE
BY
MYTZLJJS EDULZS IN DECLINING OXYGEN TENSION
1085
THEIXDEH., PONAT A., HIROKI K. & SCHLIEPERC. (1969) Studies on the resistance of marine
bottom invertebrates to oxygen-deficiency and hydrogen sulphide. Marine Biol. 2, 325-337. TRUEMAN E. R. (1967) Activity and heart rate of bivalve molluscs in their natural habitat. Nature, Lond. 214, 832-833. Key Word Index-Myths edulis; oxygen consumption; ventilation rate; heart beat; oxygen tension; blood oxygen capacity; oxygen debt; control of oxygen uptake; ventilation: perfusion ratio.