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Effects of Environmental Stress on Marine Bivalve Molluscs

He Be Akberali

Departments of Zoology and Botany, University of Manchester, England

and

E. R. Trueman

Department of Zoology, University of Manchester, England

I. Introduction . . . . . . . . . . . . . . . . . . A. Definition of stress . . . . . . . . . . . . . . B. Occurrence of natural and man-made stresses . . . . . . C. Threshold levels of pollutant stress . . . . . . . . . . D. Development of experimental techniques . . . . . . . .

11. Behavioural Responses to Stress . . . . . . . . . . . . A. Valve closure as a protective mechanism . . . . . . . . B. Relationship between heart rate, valve movement, and pumping

activity . . . , . . . . . . . . . . . . .. C. Behavioural response to some pollutant stressors . . . . D. Relevance of valve closure in epifaunal and infaunal species . . E. Effect of subthreshold levels on behaviour . . .. .. F. Effect of temperature on heart rate . . . . . . . . . .

111. Detection of Stress . . . . . . . . . . . . . . . . A. The significance of registering changes in the environment . . B. Sites of reception . . . . . . . . . . . . . . C. Detection and response to environmental changes . . . .

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ADVANCES IN MARINE BIOLOGY, VOL. 22 Copyright Q 1985. by Academic Press Inc. (London1 Ltd. All righlr o f reproduction in any form reserved.

ISBN 0- 12-026122-7

102 H . B . AKBERALI AND E. R . TRUEMAN

IV. Respiratory Physiology during Stress . . . . . . . . . . Valve closure and cessation of aerobic processes. . . . . . Relationship between heart rate, valve movements, PO>. and pC02

A. B. C. Anaerobic respiration during valve closure.. . . . . . . D. Valve activity and pH changes . . . . . . . . . .

V. The Role of the Shell . . . . . . . . . . . . . . A. Physical protection and isolation from environmental stress . . B. Shell closure and calcium reabsorption . . . . . . . . C. Effect of prolonged stress on shell strength . . . . . .

VI. Action of Heavy Metal Stressors . . . . . . . . . . . . A. Accumulation of heavy metals . . . . . . . . .. B. Effects of heavy metals on tissues . . . . . . . . . .

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. . 163 C . Effects of heavy metals on released gametes and embryonic and juvenile

stages . . . . . . . . . . . . . . . . . . . . 168 D. Effects of heavy metals on cellular organelles . . . . . . . . 175

VII. Conclusions . . . . . . . . . . . . . . . . . . . . 182 References . . . . . . . . . . . . . . . . . . . . 183

I. Introduction

A. Definition of Stress

Concern about changes in the environment and their effect on animals has recently evoked extensive research on Bivalvia based on behavioural, physiological, and biochemical responses at organismal, tissue, or cellular levels. Bayne (1975) offered the following working definition of stress as applied to marine bivalve molluscs: “Stress is a measurable alteration of a physiological (behavioural, biochemical, or cytological) steady-state which is induced by an environmental change, and which renders the individuals (or the population) more vulnerable to further environmental change.”

Most adult bivalves are relatively immobile, and any change in the environment of transient, recurrent, or permanent occurrence has to be accommodated by the animal. In the aquatic environment, many low levels of natural or man-made changes are within their normal adaptive range and are tolerated without serious consequences. More extreme changes may be temporarily resisted but can ultimately result in the death of organisms. Between these limits of tolerance and resistance to environmental stressors, a region of sublethal response occurs whereby the capa- bility for survival may be reduced as a consequence of the stressor.

The species used in this article are listed in Table I with their authorities.

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS 103

TABLE I. THE LIST OF SPECIES WITH THEIR AUTHORITIES USED IN THIS ARTICLE

Species Authority

Anadara s e n i h Anodonta anatina Anodonta cygnea Arctica islandica Cardium (Cerastoderma) edule Cardium (Cerastoderma) gluucum Chlamys opercularis Choromytilus meridionalis Crassostrea commercialis Crassostrea gigas Crassostrea margaritacea Crassostrea uirginica Donax denticulatus Donax juliane = Donax trunculus Donax serra Donax trunculus Helix uspersa Isognomon alatus Ligumia subrostrata Lima scabra Loligo pealii Lolliquneula breuis Macoma batthica Mercenaria (Venus) mercenaria Modiolus demissus Modiolus modiolus Mya arenaria Mytilus californianus Mytilus edulis Mytilus edulis planulatus Mytilus galloprovincialis Mytilus uiridis Neotrigonia margaritacea Ostrea edulis Pecten irradians Pecten maximus Perna perna Pimpehales promelas Pleurobema coccineum Scrobicularia plana Spisula solidissima Tellina fabula Unio tumidus Venerupis decussuta Venus striatula

Linnaeus Linnaeus Linnaeus Linnaeus Linnaeus Poiret Linnaeus Krauss Iredale and Roughley Thunberg Lamarck Grnelin Linnt Linnaeus Roding Linnaeus Linnaeus Gmelin

Born Lesueur Blainville Linnaeus Linnaeus Dillwyn Linnaeus Linnaeus Conrad Linnaeus Lamarck Lamarck LinnC Lamarck Linnaeus Lamarck Linnaeus Linnaeus Rafinesque Conrad da Costa Dillwyn Gmelin Philipsson Linnaeus da Costa

Say

104 H. B. AKBERALI A N D E. R. TRUEMAN

B. Occurrence of Nuturul and Man-made Stresses

Bivalve molluscs are found in aquatic habitats ranging from oceanic waters to fresh water. The open marine environment is relatively stable with respect to its physicochemical composition, but in coastal and estuarine habitats significant fluctuations may occur in salinity, temperature, oxygen levels, and turbidity. Bivalves may also be subjected to further stresses arising from man’s activities, such as off-shore drilling, dredging, or the release of pollutants.

C. Threshold Levels of Pollutant Stress

Bivalve molluscs may be either suspension or deposit feeders, and these feeding strategies pose different problems with respect to threshold levels of environmental stress. Suspension-feeding bivalves, e.g., Mytilus edulis, Cerastoderma edule, or Anodonta cygnea, filter suspended particles from the water current entering the mantle cavity, whereas deposit-feeding bivalves, e.g., Scrobiculuria plana, feed on the sediment surface with long extensible siphons but are also capable of suspension feeding (Hughes, 1969; Earll, 1975b). The principal source of pollutant stress in suspension feeders is from pollutants in solution and associated with suspended particles, whereas in deposit-feeding animals the most important source will be the sediment. In Scrobicularia, which is both a deposit and suspension feeder, the uptake of pollutants will be related to both modes of feeding. Different feeding strategies may determine the stressors likely to affect different bivalves; for example, several heavy metals, such as zinc, manganese, cadmium, or selenium, are largely taken up with particulate matter either in suspension or from the sediment, whereas the more acutely toxic metals, such as mercury, copper, and silver, are absorbed most rapidly from solution (Bryan, 1976, 1979). Evidence of significant differential absorption of metals from solution by various tissues has been reported; for example, copper is absorbed from solution about three times more rapidly by the gills and mantle/siphons than by the digestive gland and other tissues (Bryan and Uysal, 1978). The uptake of heavy metals bound to suspended food or inorganic particulate matter is less harmful than direct availability from solution since bound heavy metals have first to be isolated by digestive processes. They may thus be dealt with by metal scavenging systems, such as metallothionein proteins, which have been shown to exist in bivalve molluscs (see Section VI,A). It is also possible that the low toxicity threshold level for mercury and copper is partly related to their direct uptake from the solution. Metals such as zinc and cadmium have a higher toxicity threshold level (Bryan, 1976, 1979)

EFFECTS OF STRESS ON MARINE BlVALVE MOLLUSCS 105

because they are less readily taken up in the free form from the solution. The sensitivity of bivalve molluscs to zinc in, for example, Mya arenaria (Eider, 1977), Mytilus viridis (D’Silva and Kureishy, 1978), Mytilus edulis planulatus and Neotrigonia margaritacea (Ahsanullah, 1976), and S. plana (Akberali et al., 1981), in terms of both the threshold for any behavioral response and acute toxicity is one order of magnitude lower than the corresponding values for copper in S . plana (Akberali and Black, 1980), M. edulis (Davenport and Manley, 1978), and other bivalve molluscs (D’Silva and Kureishy, 1978; Manley and Davenport, 1979). Differences in the threshold levels of response to salinity decline have also been reported in terms of siphonal closure and shell valve adduction, which occur at about 25 and 20%0, respectively, in M . edulis (Davenport, 1979) and S. plana (Akberali and Davenport, 1981).

D. Development of Experimental Techniques

The development of electronic and other analytical techniques has led to significant advances in our knowledge of bivalve behaviour. These techniques allow continuous monitoring of heart activity and valve movements both in the laboratory and under field conditions (Hoggarth and Trueman, 1967; Trueman, 1967; Vero and Salanki, 1969; Trueman et al., 1973; Coleman, 1974; Earl1 and Evans, 1974; Brand, 1976) and have stimulated interest in the physiological and biochemical adaptations associated with behavioural changes. Electronic techniques are a considerable advance over previous studies, which involved cutting holes in the shell to observe the heart or the use of a cumbersome system of threads and levers to record valve movements (Koch, 1917; Brown, 1954; Barnes, 1955; Schleiper, 1955; Segal, 1956; Pickens, 1965). To minimize distur- bance to the animals, attempts have been made to observe heart activity without cutting the shell, e.g., in A. cygnea (Koch, 1917) and in M. edulis (Schleiper, 1957), observation of heart activity being made through the almost transparent shell of young individuals. Hers (1943) etched away the outer shell layers of older Anodonta with acid and viewed the heart through the thin, transparent, inner nacreous sheet. Although these modi- fications of the “shell hole” technique might reduce interference with the animal, the use of direct observation is time consuming and impractical in the field.

Monitoring of heart activity using the impedance technique developed by Trueman (1967) requires the positioning of electrodes through the shell on either side of the heart. Once the electrodes are properly positioned, the animal need not be disturbed further and recordings can be made remotely, hence facilitating laboratory and field studies. Recordings of

106 H . B. AKBERALI A N D E. R. TRUEMAN

hydrostatic pressure in the pericardium and electrocardiographs have confirmed that the heart rate is satisfactorily recorded by the impedance technique (Helm and Trueman, 1967; Brand, 1976).

The impedance technique has been used, both in the laboratory and in the field, to record heart and valve activity of bivalve molluscs and other marine invertebrates in relation to a variety of environmental changes such as tidal exposure (Trueman, 1967; Helm and Trueman, 1967; Col- eman and Trueman, 1971; Coleman, 1972, 1974; Earll, 1975a), feeding (Thompson and Bayne, 1972; Widdows, 1973), temperature fluctuations (Segal, 1962; Trueman and Lowe, 1971; Lowe, 1974; Coleman, 1973, 1974; Parker, 1978; De Fur and Mangum, 1979; Dietz and Tomkins, 1980; Stone, 1980; Davenport and Carrion-Cotrina, 1981), environmental oxygen levels (Bayne, 1971; Lowe and Trueman, 1972; Badman, 1974; Brand and Roberts, 1973), salinity (Davenport, 1976, 1979; Akberali, 1978; Akberali and Davenport, 1981), pollution studies (Davenport, 1977; Man- ley and Davenport, 1979; Akberali and Black, 1980; Akberali et al., 1981, 1982b; Manley, 1983), and mantle cavity ventilation (Walne, 1972; Akberali and Trueman, 1979; Davenport, 1979). The relative size of the animals has also been shown to affect heart rate; in general, the heart beat in small specimens is faster than that of larger individuals in similar conditions (Pickens, 1965; Earll, 1975a).

Monitoring of the water-pumping activity of bivalves has also been improved with the development of thermistor flow meters (Heusner and Enright, 1966; Brand and Taylor, 1974; Earll, 1975a,b; Brand, 1976; Fos- ter-Smith, 1976; Akberali, 1978; Parker, 1978; Stone, 1980), although these have yet to be adapted for field recordings. This technique was, however, a great improvement on previous attempts at measuring the water flow through the mantle cavity involving the direct determination of water volumes by the insertion of tubes into the mantle cavity (Galtsoff, 1926, 1928) or the attachment of “rubber aprons” (Loosanoff and Engle, 1947) which considerably disturbed the animal being monitored. An alternative approach is the use of indirect particle filtration techniques which have posed a number of problems in the interpretation of the results, for these depend on several assumptions, any of which may not be met during the course of an experiment (Coughlan, 1969). In Pecten irradians after initial clearance on the gills, phytoplankton such as Nitzschia and Chlarny- dornonas may be returned to the suspension (Chipman and Hopkins, 1954). Further doubt on the accuracy of plankton filtration studies has been cast by Hildreth (1980), who observed that considerable amounts of particulate material were produced from the faeces of M . edulis, which may be added to the suspension, resulting in an underestimate of filtration rate on the order of 35%. Furthermore, the flow rate of water across the

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS I07

gills can vary enormously, depending upon experimental procedure (Jergensen, 1966).

A laser diffraction technique has recently been employed to measure the shell growth of Mytilus when subjected to fluctuating salinity regimes (Gruffydd et al., 1984) or the presence of copper or zinc in the sea water (Manley et al., 1984). This technique appears to be particularly suitable for future laboratory studies of the effect of stressors on shell growth.

The use of electronic techniques has given further insight as to what constitutes “activity” in bivalve molluscs. The isolated observation of valve movements or indirect filtration methods led to a widely held interpretation of bivalve behaviour as a “steady state” of continuous feeding and activity (Loosanoff, 1939; Jgrgensen, 1966; Purchon, 1968). Recently, with the development of electronic techniques, this view has been ques- tioned, for many examples exist in the literature which indicate differing levels of pumping, feeding, valve movements, and heart rate when bivalves are in the “active” state (Earll, 1975a,b). The results of recent studies (Purchon, 1971; Morton, 1973) make a “steady state,” or continuously active, concept difficult to apply, especially in an environmental context, for example, littoral bivalves are subject to tidal rhythms. Irregu- lar discontinuity in pumping rate has also been observed in the sublittoral bivalves Zsognomon alutus (Trueman and Lowe, 1971) and Arctica islan- dicu (Brand and Taylor, 1974). Morton (1973) has put forward the view that in the long term feeding is discontinuous, exhibiting a distinct pro- gression of events often closely synchronized with environmental rhythms. The concept of activity put forward by Loosanoff (1939), Jergensen (1966), and Purchon (1968) conceals the possibility of different levels of activity in “active” animals. The description of “active,” “rou- tine,” and “standard” levels of metabolism in M . edulis, as defined by Bayne et al. (1973), would all fall into the category of animals described as “active” using Loosanoff s criteria (Earll, 1975a). Many other examples exist of bivalves which show differing levels of pumping, feeding, valve movements, heart rate, and shell deposition when they are in an “active” state (Earll,\,l975a; Coleman, 1974; Morton, 1969, 1970, 1971, 1973; Sa- lAnki, 1966,a,b; Stone, 1980). In M . edulis, Bayne et ul. (1973) have suggested that, in the laboratory, once a period of behavioural adaptation has elapsed the main activities are those concerned with the maintenance of pumping for both feeding and respiratory purposes. Earll (1975a,b) de- fines “activity” as the maintenance of the pumping current for feeding, respiration, and excretion and “inactivity” as the cessation of pumping. However, most bivalves can show short-term discontinuous pumping patterns while still active (Earll, 1975a,b; Brand, 1976; Akberali, 1978; Parker, 1978; Stone, 1980), and accordingly Earll has defined inactivity as

I

108 H . B . AKBERALI A N D E. R . TRUEMAN

a period of prolonged cessation of pumping (exceeding an arbitrary period of 1 h) to differentiate between a short cessation of pumping during activity .

The effects of environmental variables on bivalve molluscs have been studied at the organismal, tissue, and cellular level. In the past, the main approach adopted has been the “steady state” or “direct transfer” experiments where either a particular response or a multitide of responses of the animal is monitored in relation to experimental variation of a single environmental variable, e.g., salinity, temperature, or pollutant, while maintaining other factors as constant as possible. Although such studies provide a less complicated basis for understanding interactions with a particular environmental variable, doubts have been recently expressed as to whether animals in their natural habitat will ever encounter changes in a single environmental variable. For example, salinity variation in the natural habitat may be expected to alter other related factors such as temperature or oxygen solubility. Further doubts have been raised as to whether changes in the media reach a steady state in the natural habitat, especially with respect to intertidal bivalves (Davenport, 1982). This has led to the design of multivariate dynamic experiments in an attempt to simulate naturally variable conditions. Regimes such as salinity-temperature, salinity-pollutant, temperature-pollutant, and temperature-oxygen have been attempted. It is not intended to review the numerous papers on this subject here, but the following should be consulted: Stickle and Ahokas (1975), Davenport et al. (1975), Shumway (1977), Widdows (1978), Cawthorne (1979), and Manley (1980), together with a review written by Davenport (1982) on the subject of environmental simulation experiments on marine and estuarine animals.

II. Behavioural Responses to Stress

A. Valve Closure as a Protective Mechanism

Bivalve molluscs can isolate their tissues from the external environment by closing their valves, and some have been shown to minimize desiccation by valve adduction during periods of emersion at low tides. In M . edulis (Helm and Trueman, 1967; Coleman and Trueman, 1971), Cardium glaucum, and C. edule (Trueman, 1967; Boyden, 1972a) bradycardia occurs in response to littoral exposure when the valves become closed; activity is depressed, resulting in the animal respiring at lowered levels. In I . alatus (Trueman and Lowe, 1971) and Modiolus demissus (Lent, 1969), controlled valve opening exposes a film of water between the mantle


margins when exposed to air. This may serve both for oxygen exchange and as a source of moisture for evaporative cooling (Lent, 1968). When the level of water in an experimental tank was lowered beneath S . plana, bradycardia occurred and the valves closed (Earll, 1975b). Similarly, when Scrobicularia were transferred from normal sea water to a lowered salinity, the valves tended to remain closed; the’greater the drop in salinity, the greater the tendency to remain closed (Freeman and Rigler, 1956; Akberali, 1978). The activity of both intertidal and subtidal species of Modiolus, as measured by valve movements, was also reduced when the animals were exposed to either a high or a low salinity. The valve movements appeared to return to normal after a period of time the duration of which was dependent on the magnitude of the salinity change (Pierce, 1971). Valve closure in response to the presence of heavy metal pollutants has also been reported in a number of bivalve molluscs (Davenport, 1977; Manley and Davenport, 1979; Akberali and Black, 1980; Akberali et al., 1981).

It has also been shown that low temperatures induce “hibernation” in oysters (Galtsoff, 1928). At low temperatures Mercenaria (Venus) mercenaria may remain completely closed for a period of several days; one clam was closed for a period of 18 days (Loosanoff, 1939). Valve closure response in M. edulis has been shown to operate at about -1.5”C when subjected to simulated subarctic conditions, which ensures that the tissues of mussels are exposed to little or no osmotic stress (Davenport and Carrion-Cotrina, 198 1). Furthermore, Loosanoff (1942) has also reported that M. edulis remained open more than 75% of the total time at temperatures as low as -1.O”C. The freshwater clam Pleurobema coccineum showed a continuous level of activity under well-aerated conditions, whereas in the absence of oxygen a remarkable activity shift occurred, consisting of prolonged shell closure interrupted by periods of intense valve activity (Badman, 1974).

Bivalves respond to the presence of predators either by taking avoiding action or by closing the shell as a protective measure. The swimming of scallops by the rapid flapping of the valves in response to starfish is well known (Brand and Roberts, 1973), and the leaping movements of two species of the Asaphidae by flexure of the foot have been observed by Ansell (1967). Simple experiments (A. Jenkins and A. R. Brand, personal communication) during investigations of the feeding behaviour of the starfish Astropecten, using Venus striatula and Tellina fabula, indicate that the former may survive ingestion by the starfish for periods of longer than 1 week with its valves closed, but that Tellina appears to be unable to close its valves for more than brief periods and is fairly rapidly consumed after ingestion. Astropecten is only able to consume T. fabula when placed in aquaria without sand, for a shallow layer of sand allows this

110 H. B. AKBERALI A N D E. R . TRUEMAN

species to escape. This suggests two alternative strategies by which bivalves may respond to predator stress, either by rapid locomotion and escape or by the closure of thickened valves with the ability to remain quiescent utilizing anaerobic respiration (Section IV,C). Further experiments with a range of sessile and active infaunal bivalves are required to elucidate this (Trueman, 1983b).

Valve closure of bivalves prevents drastic changes in osmotic concentration of their body fluids when exposed to short-term fluctuations in salinities (Shumway, 1977). With long-term exposure to salinity change, acclimatization of the internal body fluids does occur (Akberali, 1980a), suggesting that the valve closure mechanism allows the animal a period of grace and thus prevents osmotic shock. In M. edulis, Davenport (1979) showed that the valve closure response to short-term gradual decline in salinity is partially dependent upon rate of change of salinity. This led him to the discovery that the isolation of the mantle cavity fluid is not simply produced by valve closure, but comprises a three-part sequence. The first part of this sequence is the closure of the exhalant siphon, which effectively ceases the irrigation of the mantle cavity. Further decline in external salinity results in the closure of the inhalant siphon, which is then followed by valve closure. A similar sequence has also been reported in S . plana (Akberali and Davenport, 1981). Although “shell closing” in bivalve molluscs may help the organism to withstand transient adverse changes in the environment, it cannot contribute to its long-term survival in situations where a change in the environment may be of a permanent or recurrent nature. This is because during any period of valve closure the animal incurs penalties related to feeding, reproduction, or exchanges of gases and metabolites.

The term “valve closure” does not necessarily imply that there is no contact between the tissues and the media. In the strictest sense, valve closure means the complete sealing of the ventral margins of the shell. Studies have shown that during valve closure some exchange or contact with the environment is often maintained. In M. edulis, for example, during exposure at low tides, there is a reduction in valve gape and apposition of the mantle margins but usually a small gape remains (Coleman and Trueman, 1971). Low salinity stress produces a similar feature in Donax denticulatus (Trueman, 1983a), and Perna perna has been observed to respond to copper ions by incomplete valve closure where the periostracal margins of the two mantle lobes are brought into contact so as to seal the shell from the exterior (Hodgson, unpublished). Further- more, bivalves such as Mytilus californianus, Cardium edule, M . edulis, and S . plana, with the valves forcibly clamped together show a greater drop in the oxygen tension or increase in calcium concentration of the


mantle cavity than unclamped clams (Moon and Pritchard, 1970; Boyden, 1972a; Coleman, 1972; Akberali et al., 1977; Akberali and Trueman, 1979). When a bivalve is exposed to salinity or pollutant stress, the maintenance of some slight tissue contact with the media which may be intermittent is advantageous, for it allows the organism to monitor the situation and become active once the prevailing conditions become favorable (see Section 11,B). The animal must rely on the registering of chemical changes by a slight tissue contact with the media. This is in contrast to bivalves when exposed at low tide, for these may register vibrations due to water movement or temperature changes at reimmersion.

B. Relationship between Heart Rate, Valve Movement, and Pumping Activity

The heart rate of bivalves is a useful physiological rate function since it may easily be monitored together with valve movements and pumping activity (Trueman et al., 1973). Amplitude of heart beat is not, however, easy to monitor and interpret successfully using impedance techniques. A relationship between heart rate and valve movements was demonstrated many years ago by Koch (1917), who showed in Anodonta that the heart rate is reduced during periods of valve closure. More recently this phenomenon has been reported in a number of other bivalve molluscs (Bayne, 1971; Coleman and Trueman, 1971; Coleman, 1974; Earll, 1975a,b; Brand, 1976; Akberali, 1978; Dietz and Tomkins, 1980; Akberali et al., 1981). In bivalve molluscs high levels of heart rate are associated with periods of activity as indicated by valve gape, high rates of respiration, and filtering/pumping activity, whereas periods of inactivity are associated with a low heart rate (Fig. 1) . Such marked changes in heart rate accompany different activity levels in bivalves, such as those which occur when pumping ceases and the valves close.

Studies of some species, e.g., M . edulis, Anodonta anatina, M. arenaria, and Ostrea edulis, have demonstrated that when kept in constant conditions, bivalves show a particular activity level with relatively little change in the heart rate. The heart rate of these bivalves during activity is largely independent of gross changes in pumping activity. Depending on the species, heart rate might be expected to vary by 2, 3, or 4 beatslmin over a period of hours (Coleman, 1974; Earll, 197%). By far the most marked changes of heart rate occur when animals become inactive, for in these situations pumping ceases and in some species the valves are often completely closed (Fig. 1). This inactive period is associated with a marked slowing of the heart or bradycardia. Bradycardia is a term usually applied to describe major changes of heart rate in bivalves, such as be-

1 I2 H. B . AKBERALI AND E. R. TRUEMAN

L

c L

m a I

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/--- -I 2 A

lot - \

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FIG. 1. Analysis of events during experiments on the effect of exposure on the heart rate and valve gape of M. edulis (A), and the effect of exposure on the heart rate of Modiolus modiolus (B). The rate is measured as beatshinute, and gape in degrees. The period of exposure is indicated by the horizontal line above each (from Coleman and Trueman, 1971).

tween different activity levels, rather than to small fluctuations in rate during a particular activity level in the sense used by Boyden (1972a). Following periods of inactivity, pumping recommences and the heart rate increases markedly, often to a level greatly exceeding the heart rate of continual activity. This is commonly referred to as an “overshoot” effect. Within an hour of the onset of activity, the heart rate usually falls to a rate which is similar to that shown throughout the rest of the period of activity (Trueman, 1967; Helm and Trueman, 1967; Coleman and Trueman, 1971; Brand and Roberts, 1973; Coleman, 1974; Earll, 1975b; Brand, 1976; Tay- lor, 1976a,b; Akberali, 1978; Parker, 1978).

Pumping, associated as it is in bivalves with both feeding and respiration, has been considered of primary importance in studies of activity. While recognizing the importance of pumping, it is often more convenient to obtain an indication of pumping activity by monitoring valve movements. Since pumping activity usually occurs when the valves are gaping, it is possible to obtain some indication of pumping activity from valve movements, but in some species these may be at a superficial level and lead to wrong conclusions. For example, in M . edulis, although the valves may be gaping and at the same time be exhibiting a high heart rate, the mussel need not necessarily be actively pumping (Bayne et al., 1973).


Davenport (1979, 1982) showed the closure of the exhalant siphon, thus preventing pumping, was the crucial event which largely isolated the mantle cavity of the mussel from falling external salinities; shell valve closure occurred at rather lower salinities to produce virtually complete isolation. Akberali and Davenport (1981) have shown that closure of the exhalant siphon is even more important to Scrobicularia, in which abso- lute valve closure is impossible, and in which valve adduction is delayed until low external salinities occur. This is reasonable for an infaunal estuarine species since the interstitial salinities of estuarine muds tend to be high and relatively stable (Kinne, 1971). Scrobicularia would hardly be exposed to the rigours of the salinity fluctuations in the estuarine water column except in respect of water flowing through the mantle cavity.

In some other species of bivalves, variations of heart rate during activity in response to large changes in pumping level seem either dispropor- tionately small, e.g., 0. edulis (Walne, 1972), or nonexistent as in M. edulis (Thompson and Bayne, 1972). A “regular” heart rate is maintained in M. arenaria during activity while its pumping is essentially a discontinuous process, being interrupted for varying periods by closure of the exhalant siphonal aperture (Earll, 1975a) (Fig. 2). In Scrobicularia plana, pumping during activity is also a discontinuous process but the heart rate is sensitive to changes in pumping level (Earll, 1975a,b; Akberali, 1978). During activity, S . plana shows a high heart rate and trace amplitude so

1 min h

I I I I I I I I I I I I , I 1 1 I

FIG, 2. M . arenaria: simultaneous records of pumping activity (exhalant current speed H, high and 0, zero pumping level), impedance heart trace and midventral valve movement (dc impedance). Abbreviations: imp, impedance; 0, open; C, closed. Discontinuous pumping is not related to the heart record. A burst of water from the exhalant siphon (V) coincides with the elimination of faeces (from Earll, 1975a).

FIG. 3. S. plana: simultaneous records of pumping level, heart activity, and shell movement during short-term variation of heart activity; open (0) and closed (C) (from Earll, 1975a).


long as pumping is maintained (Fig. 3). When pumping is interrupted during activity, however, a gradual fall occurs in the heart rate and the amplitude of the heart trace falls rapidly to a low level (Fig. 3). Earll (1975a,b) has broadly defined the short-term variation in the heart pattern of Scrobicularia plana as a repeating cycle of heart activity beginning with a transition to a high rate. This is then followed by a slowing down in a variable manner until a period of lowered heart rate is reached, which precedes the transition to the high heart rate once again (Fig. 4). In Scro- bicularia, the heart rate and heart amplitude coincide significantly with changes in pumping level, suggesting that heart function is more sensitive to the cessation of pumping. This close relationship of heart rate and

0 20

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Time fh)

I 30 60

T i me (min)

FIG. 4. Heart activity patterns of M. arenaria (A) and S. plana (B) when constantly submerged at 10°C: heart rate in Mya during behavioural inactivity shown by the continuous line (-); in Scrobicularia during activity, a cycle of heart rate variation as shown by the broken line (----) between transition, T, from low to high rates (from Earll, 1975b).

I16 H. B. AKBERALI AND E. R. TRUEMAN

pumping during activity, described by Earll (1975b), and confirmed by Akberali (1978), is uncommon in other bivalves which have been studied (Coleman, 1974; Earll, 1975b). Another example occurs in Donax serra, buried normally in sand, where valve adductions may be observed when the exhalant current stops flowing and the heart beat immediately ceases (Trueman, unpublished).

Replacement of stressful change in environment, e.g., polluted, salinity, aerial exposure, with clean sea water is associated with a rapid resumption of activity (Fig. 5). An initial series of valve adductions, which may be associated both with testing behaviour and hyperventilation of the mantle cavity, is usually evident within 5-10 min, which is then followed by the progressive opening of the valves and extension of siphons. Ac- companying this is a marked increase in heart rate, often beyond normal activity rates (Figs. 7, 10). This “overshoot” response is more marked in clams subjected to severe environmental changes and is comparable to the “overshoot” response observed in a number of studies with bivalve molluscs (see Section 11,B). The greater the change in the environment from normal conditions, the longer are the inactivity periods resulting in the cessation of pumping activity, isolation of the clam from the media, and pronounced bradycardia. The longer the inactive period the greater the “overshoot” response to compensate for the physiological needs in terms of respiration, feeding, and excretion.

MINUTES

FIG. 5. Examples of recordings of heart beat and valve movements of S. p l u m at 10°C in control (A) and animal exposed to 10 ppm added zinc (B) for 6 h when test solutions were replaced by fresh sea water; c (J) and e (4) indicate removal of test solutions and d (1) subsequent replacement with fresh sea water (op, open; cl, closed). (From Akberali et al., 1981).


C. Behavioural Response to Some Pollutant Stressors

Since the heart rate in S . plana is so closely related to pumping, the heart rate may be utilized as a means of monitoring interactions with environmental stress (Akberali and Black, 1980). Whenever a high heart rate is recorded from this clam, pumping is indicated, thus implying interaction with the particular environment, and conversely.

An example of this is found in the response of Scrubicularia to sea water with added copper to final concentrations of 0.01-0.5 ppm. The immediate response is valve closure, but the timing and duration of this varies with the copper concentration (Fig. 6) and is associated with an

A a

L 20 40 60

C

o % r G - - % - Minutes

B

0- 20 40 60

D

0- Minutes

FIG. 6. Heart rate (a) and valve movements (b) of individual Scrobiculuriu during the first hour when subjected to various copper concentrations. A, 0.5; B, 0.1; C, 0.05; and D, 0.01 ppm copper concentrations in sea water (S, 31%0). Arrows indicate addition of copper solution. Heart rate (HR) sampled by counting heart beats per minute for each minute. Valves open (o), closed (c) (from Akberali and Black, 1980).


immediate drop in the heart rate indicating a cessation in pumping activity (Akberali and Black, 1980). In 0.01 and 0.05 ppm, heart rate subsequently increased, while in contrast, the heart rate in 0.1 and 0.5 ppm kept falling during the first hour of recordings. Over the 6-h exposure period to 0.5 ppm, the heart rate was maintained at about 6-8 beatdmin and the valves remained closed (Fig. 7). After 2-3 h in lower concentrations, there was an increase in heart rate and intense valve activity was observed, indicating interaction with the polluted sea water (Figs. 7 and 8).

Akberali er ul. (1981) have also used heart rate and valve movements to examine the behavioural response of S. p l u m to various zinc concentrations. At concentrations of 0.1-0.5 ppm, the clams did not behave differ- ently from the controls. At higher concentrations, however, the siphons were withdrawn and valves remained closed, with a marked lowering of

A B

O i 1 2 3 4 5 6 7 8 o1 1 2 3 4 5 6 1 8

C D

L - r r I l oL 1 2 3 4 5 6 7 1 1 2 3 4 5 6 1 8

Hours Hours

FIG. 7. Heart rate (HR) of Scrobicularia subjected to various copper concentrations over a 6-h exposure period. A, 0.5; B, 0.1; C, 0.05; and D, 0.01 ppm copper concentrations in sea water (S, 31%0). Addition of copper solution indicated by J, replacement with normal sea water by t. HR of each Scrobiculariu over the 6-h exposure period sampled by averaging 5- min periods. Horizontal bars (-) refer to increased activity in heart rate and valve movements during the 6-h exposure period. Each point is a mean of four animals recorded in each concentration. Vertical bars (I) represent the range of individual variation (from Akberali and Black, 1980).

EFFECTS O F STRESS ON MARINE BIVALVE MOLLUSCS I19

A

B

C

D

U I

5 min

FIG. 8. Examples of recordings of heart activity (HA) and valve movements (VM) of Scrobicularia 2-3 h after subjection to various copper concentrations and corresponding to the state of increased activity referred to in Fig. 7 by horizontal bars (-). A, 0.5; B, 0.1; C, 0.05; and D, 0.01 ppm. Copper concentrations in sea water (S, 31%0). Valves open (o), closed (c) (from Akberali and Black, 1980).

heart rate (Fig. 9). Concentrations of 1-5 ppm zinc resulted in a partial resumption of activities in some clams, while animals exposed to 10 ppm zinc maintained valve closure over the experimental period, accompanied by pronounced bradycardia (Fig. 10). At lower zinc concentrations, the frequency and duration of interaction with the environment became greater over a 6-h period, as reflected by increase in heart rate. Similar behavioural responses have been reported in S. plana exposed to abrupt salinity changes or the first hydrolytic product (1-naphthol) of the insecticide Sevin (Akberali, 1978; Akberali et al., 1982b).

Lethal toxicity tests based on a fixed exposure period, e.g., 24-h LCso,

I 20 H. B. AKBERALI A N D E. R. TRUEMAN

0.5pprn Zn

- --- open,

C l o s e d l

I lppm Zn

~ : : i : i : : : l i . : : ; : ; : : : : : ; : ; : ; ; : i : : : : : , , ; :

30 MINUTES

0 10

FIG. 9. Examples of recordings of heart beat and valve movements of S. plana at 10°C on addition of various concentrations of zinc as described in the original paper. AJ, addition of stock zinc solution; BJ, attainment of final zinc concentrations. Valves open (op), closed (cl) (from Akberali et al., 1981).

48-h LCso, or 96-h LC50, may lead to erroneous interpretation of results in terms of toxicity threshold. In some bivalves, e.g., S . plana, it has been shown that the clam can withstand valve closure for periods of 5-7 days (Akberali et al., 1977; Akberali, 1978) while effectively isolating its tissues from the stress environment. In such animals, care should be taken in interpreting toxicity results based on fixed exposure periods. In these bivalves, median lethal time exposures would be more meaningful than fixed exposure periods. For example, when M. edulis (Davenport, 1977) and S. plana (Akberali and Black, 1980) were subjected to short-term (6- h) 0.5 ppm copper concentration they suffered no mortality (Table 11) since they closed their valves immediately. Similar observations have


4’ I~~~~~~~~~ a l ~ , ~ H o u r s

0 1 2 3 4 5 6 FIG. 10. Effects of various levels of added zinc to normal sea water on the heart rate of S.

plana at 10°C. Arrow (4) at 0 h indicates application of test concentration of zinc, while f at 6 h indicates replacement with fresh sea water. Each point is a mean of 4-8 animals. Vertical bars represent the standard error. W-H, 0.5 ppm zinc ( N = 4); A-A, 1 ppm zinc ( N = 7); A- -A, 5 ppm zinc ( N = 5 ) ; 0-0, 10 ppm zinc (N = 8). 50 ml normal sea water (1) applied to controls (0- -0, N = 5) . Treatment for controls after 6 h as in test solutions (from Akberali et al., 1981).

been made in Scrobicularia subjected to short-term (6-h) 5-10 ppm zinc (Akberali er al . , 1981) or 1-naphthol (Akberali et al., 1982b). A longer exposure period, however, resulted in an increase in mortality, with a median lethal time of 2-7 days (Table 11). Akberali and Black (1980) and Akberali et al. (198 1) have suggested that there is a limit to the duration of anaerobic respiration, since the clam must eventually eliminate its accumulated wastes. When this occurs, the tissues then come into contact with the media, poisoning takes place, and death results.

D. Relevance of Valve Closure in Epifaunal and Infaunal Species

Not all bivalve molluscs are capable of complete valve closure; e.g., infaunal species such as S. plana and M . arenaria show a reduction in heart rate indicative of a period of reduced activity which can frequently be related to the closure and the partial or complete retraction of the siphons into the burrow (Coleman, 1974; Earll, 1975a,b; Akberali, 1978; Akberali and Davenport, 1981). In littorally adapted epifaunal M . edulis, water is retained in the mantle cavity during exposure at low tide by the apposition of the mantle margins coupled with reduction of valve gape, but usually an opening remains (Coleman and Trueman, 1971). The retention of mantle cavity water reduces the danger of desiccation from aerial exposure or from osmotic shock during a decline in external salinity.

TABLE 11. ADULT BIVALVE MORTALITY IN RELATION TO THE PERIOD OF EXPOSURE TO POLLUTANT STRESS'

Pollutant Concen- Duration of Observed Species stressor tration exposure response Reference

M . arenaria Copper

M . arenaria Zinc

M . edulis Copper

M . edulis Copper

5 mg/l 48 h 39 Fg/l 96 h 35 P d l 168 h

52 mg/l 48 h 5.2 mg/l 96 h 1.6 mgA 168 h

LCJO Eisler (1977) LCSO LCSO

LCso Eisler (1977) LCSO LCSO

300 pg/l Continuous LCSO Scott and Major (1972); exposure Martin e f al. (1975) for 7 days

500 c~gf l 6 h No mortality Davenport (1977) 250 pg/l Continuous MLT

500 pg/l Continuous MLT exposure 4-5 days

exposure 2 days

M. edulis

S. plana

S. plana

S. plana

Zinc

Copper

Zinc

1-Naphthol

> 5 mgfl

0.1-20 mg/l 10 mgA

20 mg/l

1-10 mg/l 5 mgA

10 mg/l

Continuous exposure for 7 days

6 h Continuous

exposure

6 h Continuous

exposure Continuous

exposure

6 h Continuous

exposure Continuous

exposure

No mortality MLT

5-7 days

No mortality MLT

MLT 6 days

5 days

No mortality MLT

15 days MLT

9 days

Martin et al. (1975)

Akberali and Black (1980)

Akberali et al. (1981)

Akberali et al. (1982b)

Abbreviations: MLT, median lethal time for 50% mortality; LCs0, lethal concentration for 50% mortality.


In the M. edulis, a 50% fall in heart rate occurs within 20 min of being exposed at low tides (Coleman and Trueman, 1971; Coleman, 1972, 1973). The behaviour of M . edulis is similar to M. californianus (Moon and Prit- chard, 1970; Bayne et al., 1976b) in that the valves are gradually closed at the onset of aerial exposure to retain water in the mantle cavity. Oxygen from the air, however, diffuses into the mantle cavity, leading to a higher oxygen tension in the mantle cavity than if the valves had been tightly closed (Moon and Pritchard, 1970). M . edulis respires during exposure at a level which approaches the lowest levels animals show when immersed (Widdows et al., 1979). Oxygen uptake, however, is erratic, and it appears to depend on an occasional opening of the valve (Coleman, 1973; Bayne and Livingstone, 1977). In the epifaunal M . demissus (Kuenzler, 1961; Lent, 1968), some water is expelled from the mantle cavity at the onset of exposure and the valve gape is reduced to about half that found when the animals are pumping actively. This enables air to enter the mantle cavity, so allowing aerobic respiration (Table 111). In M. edulis and Mytilus galloprovincialis, an aerial rate of between 4 and 17% of the rate of oxygen consumption in water occurs, and in C . edule and M . demissus between 28 and 78% has been reported (Widdows et al., 1979), the species differences being related to the degree of shell gape during exposure (Table 111). In C . edule, as in M . demissus (Kuenzler, 1961; Lent, 1968), some water is also expelled from the mantle cavity at the onset of exposure (Boyden, 1972a,b), and the heart rate of Cardium initially rises on exposure and then falls, but is maintained at a relatively high level throughout exposure (Trueman, 1967; Boyden, 1972a,b). I . alatus, a tropical epifaunal species, maintains a small valve gape during exposure, and it has been suggested that this species also respires aerobically (Trueman and Lowe, 1971).

Another behavioural pattern is adopted by infaunal species such as C . glaucum, S. plana, and M. arenaria (Boyden, 1972a; Earll, 1975a,b). In these species, for example, little or no valve movement occurs during exposure, a pattern in contrast with M . demissus, C . edule, and M . edulis, which show a reduced but regular sequence of valve activity and the ability to utilize atmospheric oxygen during exposure at low tides (Table 111). For example, C . glaucum (Boyden, 1972a) seals its valves tightly, whereas M . arenaria (Dicks, 1972) and S. plana (Earll, 1975b) close the siphonal apertures and withdraw the siphons into the burrow. Neither S. plana, C. glaucum, nor M . arenaria has been reported to utilize atmospheric oxygen during exposure at low tide, and oxygen comsumption in these species is very low during exposure and difficult to demonstrate (Collip, 1920, 1921; van Dam, 1935; Boyden, 1972a; Dicks, 1972). Fur- thermore, in these infaunal species a complete cessation of heart rate and


valve movement occurs during exposure, and it is possible that they are entirely dependent upon anaerobic respiration during exposure at low tides (Table 111).

Sublittoral species and those which live at low intertidal levels show erratic behaviour during experimentally simulated tidal exposure. This in part accounts for the greater variability of the heart and valve movement records found in these species during aerial exposure at low tides, e.g., M . modiolus (Coleman and Trueman, 1971; Coleman, 1972) and Pecten maximus (Brand and Roberts, 1973). Modiolus and Pecren appear unable to regulate either valve movements or the retention of water in the mantle cavity during aerial exposure.

The behavioural differences and the subsequent biochemical and physiological adaptations between littoral epifaunal and infaunal bivalves pose a few interesting questions. Although the respiratory system of bivalves is characteristic of aquatic life, several epifaunal bivalve species, such as M . edulis, M . demissus, M . californianus, M . galloprovincialis, and also C. edule, are capable of utilizing atmospheric oxygen and may, during aerial exposure, obtain part of their energy requirement by aerobic respiration (Moon and Pritchard, 1970; Boyden, 1972a,b; Coleman, 1973; Bayne el

al., 1976a,b; Bayne and Livingstone, 1977; Widdows et al., 1979). In infaunal species such as C. glaucum, S. plana, and M . arenaria, the opportunity for aerial respiration is reduced by the restricted atmospheric contact and the potentially anoxic conditions prevailing in the interstitial mud (Brafield, 1964). In these species there will thus be a far greater need for anaerobic respiration to sustain basal metabolism, either during exposure at low tides which results in the cessation of pumping activity or when exposed to other environmental stress conditions, such as saiinity or pollutants. These may induce valve closure and anaerobiosis in all bivalves, except those such as M . modiolus which appears unable to maintain valve closure. Moreover, among species shown to utilize atmospheric oxygen during exposure at low tides, differences in biochemical and physiological adaptations do exist in response to exposure at low tides. For example, M . edulis responds to aerial exposure by maintaining a tighter control over its valve gape than C . edule and M . demissus (Kuenzler, 1961; Coleman and Trueman, 1971; Boyden, 1972a,b; Col- eman, 1973; Widdows et al., 19791, and this is reflected in the increase in aerial rate of oxygen uptake in Cardium and Modiolus compared with Mytilus (Table 111). This is also associated with greater need for utilizing anaerobic pathways in M . edulis, with subsequent greater accumulation of end products in the tissues than in C. edule (Widdows et al., 1979). The accessibility of the tissues of Cardium and Myrilus to atmospheric I4CO2, and hence to atmospheric oxygen during air exposure, has been investi-

TAESLE 111. COMPARISON OF BEHAVIORAL RESPONSES AND ABILITY TO UTILIZE ATMOSPHERIC OXYGEN DURING EXPOSURE AT Low TIDE IN

SOME BIVALVE MOLLUSCS~

Valve Oxygen Aquatic Anaerobiosis Species Zone Habitat activity utilization rate demonstrated Reference

M . edulis

M . californianus

M . galloprovincialis

M. demissus

Littoral Epifaunal

Littoral Epifaunal

Littoral Epifaunal

Littoral Epifaunal

Closure with a small gape



Closure with a controlled wide gape

+ 4-6% + Coleman and Trueman (1971); Coleman (1974); Bayne and Livingstone (1977); Widdows et al. (1979)

+ 74% + Moon and Pritchard (1970); Bayne et al. (1976b)

+ 11-17% + Widdows et al. (1979)

+ 56-65% + Kuenzler (1961); Lent (1968, 1969); Widdows et al. (1979)

I. alatus Littoral

M . modiolus Sublittoral

C . edule Littoral

C. glaucum Littoral

M . arenaria Littoral

S. plana Littoral

Epifaunal

Epifaunal

Surface infaunal

Surface infaunal

Deep infaunal

Deep infaunal

Closure with a controlled wide gape

Unable to maintain valve closure

Wide shell gape

Closure

Closure

Closure

t +? Trueman and Lowe (1971)

Coleman and Trueman (1971); Coleman (1976)

+ 28-78% + Boyden (1972a,b); Widdows

- + Boyden (1972a,b)

et al. (1979)

+? Collip (1920, 1921); Dam (1935); Dicks (1972); Earll (1975a)

Earll (1975a,b); Akberali er al. +? (1977)

Symbols: +, shown to occur; -, shown not to occur; +?, not shown, but likely.

128 H . B . AKBERALI A N D E. R. TRUEMAN

gated by Ahmad and Chaplin (1977). They showed that C. edule is more efficient than M . edulis at incorporating 14C02 into its tissues, which is in part a reflection on the wider shell gape of Cardium during exposure to low tides. Furthermore, in M . edulis a greater proportion of the total radioactivity was recovered from anaerobic end products such as succinate than in C . edule (Ahmad and Chaplin, 1977). These authors concluded that Mytilus uses anaerobiosis to a greater extent than Cardium. At present, it is not known whether such differences exist between epifaunal and infaunal species in their capacity for anaerobic respiration.

E. EfJPct of Subthreshold Levels on Behaviour

Marine bivalve molluscs are frequently exposed to a range of sublethal levels of environmental stresses such as salinity, temperature, oxygen, and pollutants. The adverse effects of environmental stress on aquatic organisms including bivalve molluscs have been generally identified with their acute and lethal impact. Mortality is an end point that can be readily recognized and quantified; hence the standard assay for acute toxicity testing of pollutants in aquatic organisms measures the particular stress condition or concentration that causes 50% mortality over a standard period of time (LCso). It is evident that death is a very crude index of stress in the environment, and that sublethal effects can be induced at much lower levels than the LC5,,. While not directly resulting in death, sublethal effects can affect survival through effects on behaviour, growth, physiology, and reproduction (Bayne et al., 1978, 1979, 1981; Viarengo et al., 1980b; Lowe et al., 1982; Calabrese et al., 1984). The ultimate test of significance of a sublethal effect of environmental stress is whether it has an impact on the propagation of a species and on its population (Waldi- chuk, 1979; Bayne et al., 1979, 1981). However, as Perkins (1979) has pointed out, the demonstration of a sublethal effect is often of limited use because the ecological significance of a change in the measured parameter is usually not established. The lower the level at which the effect is demonstrated, the more difficult it is to translate it into a meaningful ecological observation.

Behavioural modification is one of the most sensitive indicators of environmental stress and may directly affect survival (Eisler, 1979). Available literature on bivalve behavioural response to stress is limited, but studies (Perkin, 1979; Eisler, 1979; Olla et al., 1983) carried out in the recent past indicate that sublethal effects on bivalve behaviour may give some insight into the observed physiological, biochemical, and reproductive responses.

EFFECTS O F STRESS ON MARINE BIVALVE MOLLUSCS 129

A behavioural avoidance mechanism to adverse environmental conditions has been shown to occur commonly in bivalves, and existing evidence indicates that below the sublethal threshold level the animal is capable of interacting with the environment. For example, it has been shown that siphonal and valve closure in M . edulis and S . plana is triggered at salinities of 25 and 20%0, respectively (Davenport, 1979; Akberali and Davenport, 1981). This implies that a drop from the normal salinity (32%0) to the respective salinities in these two species can be described as sublethal. Similarly, Akberali and Black (1980) and Akberali et al. (1981) have shown that S . plana interacts with 0.01-0.05 ppm copper and 0.1- 0.5 ppm zinc in sea water. It is only when the organisms are subjected to a more extreme salinity or pollutant level that the behavioural avoidance mechanism is mediated.

The present evidence indicates that during sublethal exposure, bivalve molluscs interact with stressors which may have long-term effects on metabolic processes. For example, sublethal stresses have been demonstrated to affect the behavioural and metabolic processes of bivalves in various ways. Both depth and rate of burrowing ofjuvenile hard clam M . mercenaria was affected by oil-contaminated sediments (Olla et al., 1983). These authors suggest that such effects indicated avoidance behaviour rather than oil-induced debilitation and may increase the vulnerabil- ity of this species to predation. McGreer (1979) studied the burrowing behaviour of the estuarine clam Macoma balthica in response to sublethal levels of mercury and cadmium. A correlation was found between higher concentration levels and decreased burrowing speed which was attributed to a behavioural avoidance mechanism. Similar effects on burrowing behaviour in the clam Protothaca (Phelps et d . , 1983) and in Venerupis decussata (Stephenson and Taylor, 1975) have been reported for copper in the sediment, the burrowing time being increased logarithmically with greater sediment copper concentrations. It has also been reported that sublethal levels of heavy metals decrease filtration rates in bivalve molluscs (Watling, 1981). In M . edulis, a 50% reduction in filtration rate was found at concentrations of only 0.04 ppm mercury, 0.15 ppm copper, and 1.6 ppm zinc (Abel, 1976). The rate of oxygen consumption of excised gill tissue of Crassostrea virginica showed a significant increase when continuously exposed to sublethal levels of 50 and 100 ppb copper (Engel and Fowler, 1979). The most obvious difference occurred after 14 days' exposure to 100 ppb copper, by which time the tissue concentration of copper had reached 0.8 pg/mg (dry wt). Robinson et al. (1984) have shown that in the surf clam Spisula solidissima, turbidity levels > 100 mg/l of attapulgite clay resulted in a significant increase of pseudofaecal production and a


decrease in the amount of algal food actually ingested. They have concluded that anthropogenic turbidity-producing discharges at low levels can possibly cause adverse effects on the energetics of surf clam populations. It has also been reported that continuous exposure to sublethal levels of copper and zinc suppresses gametogenesis in adult M . edulis, with copper being more toxic (Maung-Myint and Tyler, 1982).

F. Effect of Temperature on Heart Rate

It is apparent in many investigations (Pickens, 1965; Trueman and Lowe, 1971; Lowe and Trueman, 1972; Coleman, 1972, 1974; Davenport and Carrion-Cotrina, 1981) that heart rate is markedly affected by temperature. Increase in temperature leads to a rise in heart rate and a decrease in temperature results in a fall in heart rate in an intact M . edulis (Fig. 11). There is an almost linear relationship with a heart rate of 30 beats/min at 10°C decreasing to 3 beats/min at - 13°C (Davenport and Carrion-Co- trina, 1981). In the bivalve S. solidissima, an increase in temperature is associated with an increase in heart rate and vice versa (De Fur and Mangum, 1979). Similarly, in the freshwater Ligumia subrostrata the heart rate has been shown to be an exponential function of temperature (Dietz and Tomkins, 1980). In the bivalves M . arenaria and Crassostrea gigas, rapid temperature change also brings about an immediate response

I 0

n J

' 2 0 *10 0 - 5 Temperature VC)

FIG. 11. M. edulis: effect of temperature on heart rate. Norwegian mussels (O), Welsh mussels (0) (from Davenport and Carrion-Cotrina, 1981).


in heart rate (Lowe, 1974). In both these species, heart rate is dependent on the temperature of the bathing fluid, but during sudden changes of temperature there is significant relationship between heart rate and mantle cavity temperature, and it has been suggested by Lowe (1974) that thermoreceptors, possibly in the mantle tissue, play an important role with respect to the immediate response to temperature change.

A number of examples of perfect, partial, and nonexistent acclimation of seasonal variation in heart rate have been described in the mussels M. californianus and M . edulis by Pickens (1965). He suggested that these results might be accounted for by considering the effects of condition, in particular the reproductive state and food availability on a seasonal basis. Widdows (1973), in a closely controlled experiment, confirmed many of the results obtained by Pickens (1965). Using M. edulis he studied the effects of acclimation on heart rate, oxygen consumption, and pumping rates, and the effect of starvation. M. edulis acclimated at a set temperature was transferred to higher or lower temperatures and showed examples of both partial and complete acclimation in terms of oxygen consumption and pumping rate. Bayne et al. (1973) have also reported that the acclimation of these functions varies consistently on a seasonal basis. Heart rate, however, shows no acclimation in the long term and remains dependent upon ambient water temperature.

Heart rate has been used as a measure of temperature acclimation in a number of poikilothermic animals such as M. edulis and M . californianus (Pickens, 1965; Widdows, 1973). Perfect acclimation of heart rate to temperature alone was not evident in either of these species and consequently seasonal changes in heart rate were attributed to other factors. Widdows (1973) found that starvation of M. edufis produced a 35% reduction in heart rate over 9 days, emphasizing the point that in the field, heart rate may depend not only on the ambient water temperature but also on the nutritive and reproductive state of animals. In addition, temperature has been shown to affect burrowing behaviour in the tropical surf clam D . denticulatus (Trueman, 1971, 1983a; Ansell and Trueman, 1973) and in D . serru (McLachlan and Young, 1982). Environmental variables, especially temperature, do affect patterns of activity on a seasonal basis, and some species, such as M . mercenaria, show a reduction in the duration of active periods (Loosanoff, 1939) and shell deposition (Panella and Mc- Clintock, 1968; Jones et al., 1983) with seasonal fall in temperature. A similar reduction in activity was found in A. cygnea (Salanki et al., 1974; Parker, 1978), although short-term fluctuations in temperature, which were equivalent to natural daily variation, appeared to have a minimal effect on periodic activity.


111. Detection of Stress

A. The Signijicance of Registering Changes in the Environment

Many bivalve molluscs, when exposed to lethal levels of environmental stress, rely on behavioural mechanisms which enable them to avoid contact with such conditions (see Section 11, Behavioral Responses to Stress). Mobile species have the ability to move away from regions of potentially harmful stress conditions, whereas sedentary species possess behavioural mechanisms such as burrowing into the substratum, retract- ing into existing burrows, or closing of valves. In employing such mechanisms for osmotic control in response to salinity fluctuations and in avoiding harmful effects of pollutants, bivalve molluscs must therefore be capable of detecting changes in the environment and implementing the appropriate response.

The immediate detection of environmental changes is essential for the success of any protective response. This applies at both the commencement and the termination of stress so as to allow feeding to be resumed immediately on the removal of the stress. Detection of changes in the environment by bivalves is particularly important with respect to distin- guishing between sublethal and lethal levels and eliciting appropriate behavioural responses to lethal levels. Occurrence of environmental stress is often intermittent, being affected by the duration of tidal cycles, cur- rents, and variable freshwater runoff. These factors are particularly relevant in intertidal and estuarine conditions. Reference should be made to the detailed review by Davenport (1982) on environmental simulation experiments.

Bivalve molluscs such as S . plana, M . edulis, C. edule, and 0. edulis have been shown to accumulate heavy metals in their tissues far in excess of the environmental levels (Phillips, 1977; Bryan, 1979; Bryan and Gibbs, 1983; Viarengo et al., 1981; Calabrese et al., 1984). Tissue concentrations in S . plana at Restrongnet Creek (Cornwall) show a wide variation in metal ion concentration. The highest concentrations of 7270, 101, and 25 pg/g (dry wt) for zinc, copper, and manganese, respectively, occurred in the digestive gland, whereas highest iron concentration of 2051 pg/g (dry wt) was found in the mantle and siphons (Bryan and Gibbs, 1983). Such differences in tissue concentration in relation to various heavy metals and also between different localities have been reported in numerous studies involving bivalve molluscs.

Akberali and Black (1980) and Akberali et al. (198 1) have shown that S . plana avoids levels of 100-500 pgfl (0.1-0.5 ppm) and > 5000 pg/l (> 5 ppm) of copper and zinc concentration, respectively, by prolonged valve

EFFECTS OF STRESS ON MARINE BlVALVE MOLLUSCS 133

closure and thus protects the tissues from the presence of these heavy metals. However, at concentrations below these levels the clams continue to interact with the pollutant after an initial valve closure response. This response to lower concentrations in S. plana, which is probably similar to other bivalves, implies that bivalves may discriminate between toxic and nontoxic effects of heavy metals. It is important to emphasize that the high tissue levels of heavy metals, for example copper and zinc, in Scrobi- cularia could only have accumulated from heavy metals associated with particulate food or in solution in the ambient medium at subthreshold levels. The temporary incidence of lethal levels could be avoided by valve closure and would not lead to accumulation in the tissues. It has been rightly pointed out, in Scrobicularia for example, that bivalves would avoid the worst of conditions by deposit feeding when sediment metal contamination is low and suspension feeding when dissolved metal level is low (Bryan and Gibbs, 1983).

It has also been shown that osmoconforming bivalves such as M . edulis and S. plana minimize osmotic stress when exposed to low external salinities of short-term duration by isolating their tissues and body fluids from the water to a considerable extent (Shumway, 1977; Davenport, 1979; Akberali and Davenport, 1981). In M . edulis this is achieved by siphon and shell valve closure at a salinity of about 25%0, while S. plana closes and retracts the siphons at about 20%0 (Fig. 12). This implies that the behavioural avoidance mechanism was triggered at a lower external salinity level, the difference being 7 and 12%0 in M . edulis and S. plana, respectively, indicating that a fluctuation in the media of this magnitude

Minutes

FIG. 12. Shell valve recordings from anterior (a) and posterior (b) part of normal Scrobicu- laria subjected to a decline and rise in salinity of the external medium (graph below). Op, open, C1, closed (from Akberali and Davenport, 1981).

I34 H. 0. AKBERALI A N D E. R. TRUEMAN

can be tolerated without serious consequences. In both M. edulis and S . plana, closure of the exhalant siphon, which prevents pumping, was the crucial event in largely isolating the mantle cavity from falling external salinities, while shell valve closure occurred at rather lower salinities, so producing almost complete isolation (Davenport, 1979; Akberali and Dav- enport, 1981). Even when apparently isolated, bivalves can detect favorable changes, since they respond within a short period of time, resulting in valve opening and commencement of pumping (Figs. 5 and 12).

The foregoing account suggests that bivalves may distinguish between lethal and sublethal levels of pollutant or salinity stress. During periods of valve closure the animal incurs penalties, and unnecessary valve closure at sublethal levels of pollutant or salinity would be of little survival value.

B. Sites of Reception

In the class Bivalvia, envelopment of the body by paired valves has resulted in the reduction of the head and the role of sensory perception has been taken over by the mantle margin and siphons, which are the main sites of contact with the external environment (Dakin, 1910; Bullock and Horridge, 1965). The structure of the mantle of a number of bivalves has been described and the sensory structures developed on the middle mantle fold discussed (Yonge, 1949, 1957; Kawaguti and Ikemoto, 1962; Gilmour, 1963; Beedham and Owen, 1965; Barber et al., 1967; Land, 1968; Petit et al., 1978). Tactile sensitivity in the mantle, which is probably a feature of all bivalve mantles, has been demonstrated in M. arenaria (Pumphrey, 1938), S . solidissima (Wilson and Nystrom, 1968), Lima scu- bra (Stephens, 1978a), Chlamys opercularis (Stephens and Boyle, 1978), and S . plana (Hodgson, 1982; Black, 1983). In the mantle of bivalves P . maximus (Thomas and Gruyffyd, 1971) and L. scabru (Stephens, 1978a) chemical sensitivity has also been reported. The application of extract from a predatory starfish onto the mantle in two species of Asaphidae (Ansell, 1967) triggered a violent escape response and caused leaping movements.

The formation of siphons in bivalve molluscs involves partial or complete fusion of one or all of the mantle folds (Yonge, 1948). In some bivalves, such as C. edule, siphonal tentacles bearing eyes have been observed (Barber and Wright, 1969). Furthermore, tactile and chemical sensitivity in response to stimuli has been reported in bivalve siphons, e.g., touching the siphons of Ensis (Trueman, 1966a), Spisula (Mellon, 1965; Prior, 1972), and Scrobiculuriu (Hodgson, 1982; Black, 1983). The isolated and in situ siphon in S . plana responds to salinity decline and


presence of pollutants (Akberali, 1981; Akberali et al., 1981, 1982a,b; Akberali and Davenport, 1982). Photosensitivity of the siphon has also been reported, exposure to high light intensities causing withdrawal (Light, 1930; Kennedy, 1960).

In M. edulis it has been shown that salinity detection is carried out peripherally by salt-sensitive receptors on the tentaculate portion of the inhalant siphon (Davenport, 1981). In S. plana the sensory structures are more deeply situated, and it is thought that salinity or zinc detection is mediated by the central nervous system rather than by any peripheral neural network within the siphon (Akberali et al., 1981; Akberali and Davenport, 1981,1982). In none of these studies, however, have the sense organs been located or described structurally, and the areas of sensitivity are based on experimental studies related to observations, ablation, or isolation of likely sensory sites, and the recording of behavioural response to a particular environmental stress.

In a number of bivalves, different types of ciliary tufts occur on the mantle and siphons (Moir, 1977; Owen and McCrae, 1979; Frenkiel and Moueza, 1980; Hodgson et al., 1982; Black, 1983); they show many characteristics of sensory receptors as described by Laverack (1968). It is difficult to assign functions for the receptors from their morphological features alone, but it is possible to assign some function by comparison with receptors whose functions have been identified, or by correlating receptor density in an area showing sensitivity to a particular stimulus (Zylstra, 1972). It has been shown in S. plana that the siphons have a higher density of ciliary tufts than the mantle, and this may explain a greater response of the inhalant siphon to chemical stimulation than the mantle (Black, 1983). It is likely that, in the mantle and siphons of bivalves, at least some of the different types of ciliary tufts are sensory and are the mechano- and chemoreceptors.

The possibility of the cruciform muscle complex in S . plana functioning as a chemoreceptor has been suggested by Odiete (1978), who recorded a burst of electrical activity from this structure when the clam was exposed to “foul water.” There is some evidence that the cruciform muscle complex in Scrobiculuria is involved in the siphonal withdrawal response to changing salinity (Akberali and Davenport, 1982) and zinc ions (Akberali el al., 1981). Ablation of the cruciform muscle complex in Scrobicularia when exposed to changing salinity or zinc ions resulted in the siphonal withdrawal response being delayed or weakened (Figs. 17, 21, and 22). The withdrawal response was not, however, totally abolished, suggesting that receptors in other areas, such as mantle and siphons, were still functioning. In other Tellinacean bivalves, such as Donax trunculus (Moueza

136 H . B . AKBERALI A N D E. R . TRUEMAN

and Frenkiel, 1974; Frenkiel, 1980), it has been reported that the cruciform muscle complex functions not as a chemoreceptor but as a vibration receptor (Pichon et al., 1980).

Studies using electrophysiological techniques have shown tactile and chemical sensitivity in the mantle edge of the bivalves L . scabra and Aequipecten (Stephens, 1978a,b). Afferent impulse activity in nerves in- nervating peripheral sensory regions of S. plana in response to tactile and chemical stimulation has also been demonstrated (Hodgson, 1982; Black, 1983). The visceral ganglion in Scrobicularia is involved in controlling adductor muscle rhythms (Odiete, 1976a,b; Black, 1983) and also in the withdrawal of the in situ siphon in response to decline in salinity (Fig. 17). Activity generated in the posterior adductor muscle nerve in response to tactile or chemical stimulation of peripheral regions may lead to valve adduction in the intact clam. Furthermore, Black (1983) has shown that, in Scrobicularia, the mantle and siphons possess chemoreceptors that respond to the presence of zinc. In mantle nerves, the electrical response to zinc or to tactile stimulation was less than that of the siphons, and she suggested that this may be either related to the mantle nerves being of smaller diameter than the siphonal nerves, to the number of chemoreceptors present in each site, or to their firing threshold. Black (1983) also made the important observation that the threshold for response in the lower mantle nerve was 2-3 ppm zinc, whereas for the inhalant siphon nerve it was less than 1 ppm. This finding is in close agreement with previously reported studies on the behavioural and inhalant siphon responses of S . plana to zinc, where 1 ppm zinc was the lowest concentration tested to cause a temporary reduction in heart rate, siphon contraction, and valve closure (Akberali et al., 1981). The thresholds for the exhalant siphon and mantle were higher than the inhalant siphon, which may reflect the lesser importance of these sites for chemoreception in the natural habitat (Black, 1983).

Valve closure provides a useful behavioural avoidance mechanism during exposure to adverse environments (see Section II,A), but the site of perception has been less thoroughly investigated. In a few bivalve molluscs, including S . pluna, ciliated tufts on the mantle and siphons resem- ble sensory receptors and are probably involved as mechano- or chemoreceptors. Their stimulation may then elicit siphonal withdrawal and valve closure as a general stress avoidance response.

C. Detection and Response to Environmental Changes

There have been few studies on mechanical and chemical detection in bivalve molluscs. From the literature, it is apparent that among marine


invertebrates there is no common mechanism for registering environmental changes. Studies on certain mobile species such as gastropod molluscs (Blandford and Little, 1983), crustaceans (Gross, 1957; Lagerspetz and Mattila, 1961; McLusky, 1970; Thomas e? al., 198l), and annelids (Jan- son, 1962) indicate that, when subjected to a choice of different salinities, these organisms are capable of detecting and discriminating salinity levels. In sedentary species such as bivalve molluscs, the underlying basis of salinity detection has been investigated by exposing animals to artificial sea waters of differing ionic and osmotic composition and observing the effect on behaviour (Davenport, 1979, 1981 ; Akberali and Davenport, 1981, 1982). In both mobile and sedentary species, salinity detection de- pends on the concentration of particular ions (Barnes, 1939, 1940; Daven- port, 1981; Akberali and Davenport, 1982; Black, 1983), the osmotic pressure of the medium (Davenport, 1972; Bettison and Davenport, 1976; Blandford and Little, 1983), or to the combination of both (Barnes and Barnes, 1958).

S. plana is an osmoconforming bivalve in which valve closure is mediated by detection of change in osmotic pressure, and not by measurement of any ionic constituent (Freeman and Rigler, 1956). Akberali and Daven- port (1982) showed that siphon withdrawal is triggered by a change in groups of ions such as sodium, magnesium, calcium, and chloride (Fig. 13) rather than by changes in the gross osmotic pressure (Figs. 14 and 15)

- i

MINUTES

FIG. 13. The effects of exposure to NaC1, MgCI2, and CaClz followed by a salinity regime (graph below) on (a) an in siru Scrobicutnria siphon preparation in which the downward deflection of the trace represents isotonic siphon contraction [note the effect of a mechanical stimulus (s) applied by pinching with forceps] and on (b) intact mussels in which the horizontal bar represents activity: open sections, gaping; closed (black) portions, closed valves. The “ionic test medium” regime description represents an alteration from 0% (when animals were supplied solely with sea water of salinity 32%0) to 100% (when animals were supplied with pure “ionic test medium”) (from Akberali and Davenport, 1982).

138 H. B . AKBERALI AND E. R . TRUEMAN

a- -

b 6 l b 1’5 2 b 25 3b 35 do 45 50 i

MINUTES

FIG. 14. The effects of exposure to two test media, the first containing NaCI, MgC12, and CaCI2, the second containing NaCl alone: response of (a) an in situ Scrobiculan‘a siphon preparation and (b) of intact mussels: details as in Fig. 13, except that the “NaCI only” regime description represents an alteration from 0% (when animals were supplied solely with “ionic test medium” regime) to 100% (when animals were supplied with pure “NaCl only”) (from Akberali and Davenport, 1982).

or in the concentration of a single ion (Fig. 14). Similarly, in M . edulis, both siphon withdrawal and valve closure (Akberali and Davenport, 1982) and the opening reaction (Davenport, 1981) are mediated by a group of ions such as sodium, magnesium, and chloride rather than by changes in the osmotic pressure or in the concentration of a single ion (Figs. 13-15). Siphon withdrawal and valve closure in S . plana and valve closure in M . edulis are different with respect to the presence of one ion. In order to prevent siphon withdrawal and valve closure in Scrobiculariu, the presence of sodium, magnesium, calcium, and chloride is necessary, whereas in M . edulis valve closure can be prevented by the presence of only sodium, magnesium, and chloride (Figs. 14 and 15). The reasons underlying these differences in calcium ion specificity are not clear. They may be due to differences in the type of habitat occupied or to a variety of ionic and osmotic mechanisms of salinity detection of animals within the same class (Bettison and Davenport 1976). There is as yet no sign of coherent evolutionary or physiological pattern.

This calcium dependency requirement for the closing response in S . plana with respect to salinity changes, and its absence from M . edulis, may reflect an additional physiological requirement in the former species. Both species use behavioural mechanisms to avoid salinity variations (Davenport, 1979; Akberali and Davenport, 1981). S. plana, however, has two long, mobile, and highly extensible siphons, whereas in the epifaunal M . edulis the siphons are of negligible length. In Scrobiculariu siphonal contractions are dependent on the presence of calcium ions in the external


4 7 0 m M NaCl a

h

NaCI.CaCI 9

CholineCI.CaCl2. MgC12

b h

010 d

MINUTES

FIG. 15. The effects of various test media on in situ Scrobicukuriu siphon preparations (a) and intact mussels (b); details as in Fig. 13. The graph at the bottom indicates percentage concentration (CONC.) change for ail media (from Akberali and Davenport, 1982).

medium (see Section VI,B), and this may reflect the requirement of calcium ions for detection of falling external salinities. The opening response of S. p l u m to rising salinities has been shown to be dependent on sodium, magnesium, and chloride ions, but the presence of calcium is not required (Black, 1983). In M. edulis, both the closing response to falling salinities and the opening response to rising salinities rely on the presence of the identical ions (Davenport, 1981; Akberali and Davenport, 1982). In M. edulis the salinity-sensitive receptors lie on the tentaculate portion of the inhalant siphon (Davenport, 1981). Therefore, when the mussels are open

140 H. B . AKBERALI A N D E. R. TRUEMAN

and pumping, the water entering the mantle cavity will be continuously monitored near the margins of the valves so that any behavioural reaction to adverse media will result in negligible exchange between the mantle fluid and the external medium. It has been reported that the opening response to rising salinities is mediated by changes in electrochemical gradients due to the diffusion of salts to the tentaculate portion of the inhalant siphon and not to any other portion of the mantle edge or to any more deeply located structures (Davenport, 1981). Such a diffusion of salts to the salinity-sensitive region can easily be achieved in an epifaunal species even when the valves are closed, since valve closure does not always provide complete isolation. The existence of fine passageways between the mantle margins may allow salt diffusion. An epifaunal bivalve mollusc can thus continuously monitor the environment.

In the infaunal S. plana when open and pumping, as in M . edulis, the water entering the clam will be registered by the salinity-sensitive region and accordingly trigger the behavioral avoidance mechanism when conditions become unfavourable. Once siphons are retracted and valves closed, an infaunal species cannot rely on salt diffusion gradients from interstitial water. These would not give a realistic assessment of conditions in the water column, since interstitial salinities of estuarine muds tend to be high and relatively stable (Kinne, 1971). In the natural habitat during exposure at low tide Scrobicularia does not withdraw its siphons into the shell, nor are the valves completely closed; the siphons are just withdrawn to the entrance of the burrow and the clam stops pumping (Earll, 1975a,b). It is possible that this behaviour allows periodic sampling by opening the siphonal apertures and drawing in small amounts of the external medium. In doing so the clam is at risk by the introduction of the stressor into the mantle cavity, and whether such a strategy is actually adopted by infaunal bivalves is not yet clear. Another alternative would be to locate sensory structures at the siphonal tips and along the entire length of the siphon so that loss of the tips by predation (Edwards and Steele, 1968) would not deprive the organism of its sensory structures. The isolated inhalant siphon of S. plana (Fig. 16) does not respond to dilute sea water, indicating that salinity detection either does not occur in the siphon or is mediated by the central nervous system (Fig. 17) rather than by any peripheral neural network.

The dependency of the closing and opening response of M . edulis to sodium, magnesium, and chloride ions (Davenport, 1981 ; Akberali and Davenport, 1982) is hardly surprising since these ions make up the bulk of the salt content of sea water. It is possible that adequate concentrations of sodium and magnesium for ATPase activity, probably involved in the neural control of the gaping response, are also important. In S. plana;


ISOLATED SIPHON

OJ I

0 5 Ib ;5 $0 215 do MINUTES

FIG. 16. Effects of salinity decline (graph below) on an isolated and an in situ pieparation of Scrobiculuria: the downward deflection of the traces represents isotonic siphon contraction; mechanical stimulus (s) applied by pinching with forceps (from Akberali and Daven- port, 1982).

calcium is an added requirement for the closing response (Akberali and Davenport, 1982), but it is not required for the opening response (Black, 1983). The reason for this difference may be related to the dependence of the siphonal contraction on this ion (Akberali et ul., 1982a). Future work is required on other infaunal and epifaunal bivalve molluscs to elucidate this interesting phenomenon and to indicate whether calcium ions are an added physiological requirement in infaunal species for the contraction of the long extensible siphons. It must be understood throughout that the metal ions referred to in the text are those forms of inorganic metallic salts tested in sea water without taking into account the form of metal ions in solution. Behavioural avoidance mechanism in response to the presence of copper and zinc have been reported in both S. p l u m (Akberali and Black, 1980; Akberali ef a/., 1981) and M . edulis (Manley and Davenport, 1979; Davenport, 1977). These studies have also shown how quickly the animal responds to the removal of the pollutant, by the valves opening and pumping commencing (Fig. 5).

The first visible response to changes in salinity or presence of pollutant in S. plana and M . edulis is the closure of the siphonal aperture, followed

I42 H . B. AKBERALI A N D E. R. TRUEMAN

VISCERAL GANGLFIO ~~~ ~ . - ~-

'a CRUCIFORM MUSCLE

FOOT

? 3 2 ] - v El6 (I)

0 I I I 1 @

0 5 10 15 20 25 30

MINUTES

FIG. 17. The effects of ablation of the visceral ganglion, cruciform muscle complex, foot (including pedal ganglion), or gills upon the response of in situ Scrobiculuviu siphon preparations to a fall in salinity; for other details see Fig. 16 (from Akberali and Davenport, 1982).

by siphonal withdrawal and valve closure. This led Akberali (1981) to examine the direct effects of copper on the isolated inhalant siphon of S. plana to investigate the possibility that the behavioural response observed in the intact clam may be due to the detection of copper by the siphons. The isolated siphon reacts to the presence of copper with a series of spontaneous contractions (Fig. 18). The copper concentrations were close to those found to trigger the behavioural avoidance response in the intact clam. Similarly, Akberali et ul. (1981) showed that the intact clam avoids lethal zinc concentrations by behavioural avoidance mechanisms and that zinc has no effect on the isolated siphon (Fig. 19). In an in situ siphon


0 10 20 30 40 50 60

Time (mid

FIG. 18. S. plana: Effects of addition of copper on an isolated inhalant siphon preparation. Upward deflection of the trace indicates isotonic siphonal contraction. The following experimental protocol was carried out in sequence: appropriate volume of stock copper solution (10 ppm copper nitrate) in normal sea water was added to give 0.25 ppm at (a) and 0.5 ppm at (b); removal of sea water containing 0.5 ppm copper (c), followed by washes (w) and replacement with normal sea water (d). Copper was added again to give a final concentration of 0.25 ppm (a’) and 0.5 ppm (b’) (from Akberali, 1981).

preparation where the clam is intact except for removal of one shell valve, the siphon reacts in a manner similar to that in the intact clam (Fig. 20). This led to the suggestion that zinc is registered by sensory structures on the mantle and ablation of the sensory cruciform muscle complex partially abolishes the in situ siphonal withdrawal response (Akberali et al., 1981) (Fig. 21). The possibility that the cruciform muscle complex in Scrobicu- laria has a chemoreceptive function has been suggested by Graham (1934), Yonge (1949), Moueza and Frenkiel (1974), and Odiete (1978); it appears to have a multiple function, facilitating siphonal movement vibration reception and acting as a chemoreceptor. However, as shown by Akberali et al. (1981) and Akberali and Davenport (1981, 1982), it is evident that ablation of the cruciform muscle complex affects the behavioural avoidance mechanisms to some extent but does not completely abolish the response to salinity decline (Figs. 17 and 22) or to the presence of zinc (Fig. 21). This suggests the possibility that the cruciform muscle complex together with other parts of the mantle lobes or siphons or ganglia may be involved.

t-httrmii i i i i i : : , , : < < i t i i i i i j t i i i i i i i : i i : i : , , i : i i i i i : : i : : i i b l’t-ti, 4 - f t 1 I 1 + +

0 10 20 30 40 50 60 70 80

Minutes

FIG. 19. Effects of various zinc and copper concentrations on an isolated inhalant siphon preparation of S. plann. Upward deflection of the trace indicates isotonic contraction of the inhalant siphon. The following experimental protocol was carried out in sequence: application of zinc at the final concentrations indicated (4) and removal of sea water containing 10 ppm zinc (C), followed by washes (w) and replacement with normal sea water (D). Prior to copper applications (J), the siphonal response was tested by a mechanical stimulus (s) (from Akberali et a / . , 1981).


0 10 20 30 40 50 Minutes

FIG. 20. Effects of various zinc concentrations on an in situ inhalant siphon preparation of S. plana. Upward deflection of the trace indicates isotonic contraction of the siphon. The following experimental protocol was camed out in sequence: application of zinc at the final concentrations indicated (J) and removal of sea water containing 10 ppm zinc (C), followed by washes (w) and replacement with normal sea water (D), then mechanical stimulus (s) (from Akberali et al . , 1981).

S. plana and M. edulis clearly avoid lethal levels of copper and zinc solutions by employing behavioural avoidance mechanisms, and are capable of discriminating sublethal levels. This response in Scrobicularia has also been demonstrated recently using other metal ions (Black, 1983). Black showed that silver and mercury cause siphon withdrawal and complete valve closure, whereas partial valve closure was observed in the presence of nickel and cobalt. The behaviour was least affected when Scrobiculuria were exposed to manganese, cadmium, or chromium (tri- or hexavalent forms). Observed differences in the behavioural responses of

* 0 10 20 30

Minutes

Frc. 21. Effects of various zinc concentrations on in situ inhalant siphon preparations of S. plana. Arrows (1) mark the application of zinc stock solution to give the indicated concentrations. X, cutting of the cruciform muscle complex before addition of 10 ppm zinc; (A and B) Two examples of the recordings observed; s, mechanical stimulation (from Akberali et a / . , 1981).


CI I

O J I I I I I I I I I I I I

0 20 40 60 80 100 120 Minutes

FIG. 22. Shell valve recordings from anterior (a) and posterior (b) part of a specimen of S. plunu, with its cruciform muscle complex destroyed by making a median longitudinal incision through the muscle and then subjected to a decline and rise in salinity of the external medium (graph below). Op, Open; C1, closed (from Akberali and Davenport, 1981).

Scrobicularia can also be related to the toxicity of each metal ion over longer exposure periods. Black (1983) found that those metals which evoked closure responses were also acutely toxic to S . plana during prolonged exposures (median lethal time, ca. 1-7 days), whereas metal ions that did not cause complete valve closure had low toxic effects over a similar period of time.

The isolated siphonal preparation of S. pfana was also used to test the effects of copper, zinc, cadmium, nickel, manganese, chromium (tri- or hexavalent), silver, and mercury (Black, 1983). Only copper was effective in causing spontaneous contractions of the isolated siphon, which is dependent on the presence of calcium ions in the external medium (Akberali et al., 1982a). The possible mode of action of copper is discussed later (Section V1,D). At present, it is not clear why copper, of all the metal ions studied so far, has this ability to induce contractions in the isolated inhalant siphon. Recent studies, using isolated myogenic heart of the land snail Helix aspersa, have shown that low copper concentrations induced continuous tonic contraction, whereas zinc does not have this effect (Akberali, unpublished). It is not known whether the direct effect of copper on the isolated siphon forms the basis of siphon withdrawal in the avoidance response of the intact animal or whether it acts in the same manner as other metal ions, through sensory reception sites. The evi-

146 H . B. AKBERALI AND E. R . TRUEMAN

dence so far indicates that the bivalve Scrobicularia is able to detect the presence of metal ions and discriminate potentially lethal levels.

IV. Respiratory Physiology during Stress

A. Valve Closure and Cessation of Aerobic Processes

Periods of valve closure are commonly associated with behavioural inactivity and cessation of pumping activity. The most obvious effects of this are limitation of time available for feeding and consequent reduced growth potential, cessation of aerobic respiration, and accumulation of excretory products. Despite these penalties, the behavioural avoidance strategy of bivalve molluscs is of great survival value. There is, however, a limit to the time an animal can remain isolated from the environment, depending on its overall physiological and biochemical condition and the adaptive mechanisms to sustain the basal metabolic processes. Such mechanisms will include the utilization of anaerobic respiration to sustain basal metabolism, availability of stored food reserves, and the ability to tolerate and accommodate levels of excretory products.

B. Relationship between Heart Rate, Valve Movements, PO?, and PC02

Numerous studies (Trueman, 1967; Coleman, 1974; Earll, 1975b; Brand, 1976; Akberali er al., 1981) have shown that the effect of valve closure on heart rate in bivalve molluscs is bradycardia, which may be associated with the depletion of oxygen tension in the mantle cavity. Oxygen depletion in the mantle cavity of M. californianus is rapid when the valves are clamped shut, falling from 115 mmHg to 20 mmHg O2 in 20 min or less (Moon and Pritchard, 1970). Similar rapid depletion of the oxygen in the mantle cavity of A . islandica (Brand and Taylor, 1974), S . plana (Akberali and Trueman, 1979), M. edulis (Davenport, 1979), and A. cygnea (Parker, 1978) has been reported. It is possible, as Bayne (1971) suggests, that the rapid fall of oxygen tension in the mantle cavity could account for the rapid bradycardia shown by M. edulis.

Earll (1975a,b) considered why S . plana, of all the bivalve species investigated, alone shows a marked temporal variation in heart rate which is dependent upon pumping (Fig. 3). A number of factors may be responsible. The heart rate is reduced when the clam is subjected to reduced oxygen tensions. A high weight-specific oxygen consumption (Hughes, 1970) compared with a small mantle cavity volume would ensure an even


faster depletion of mantle cavity oxygen tension than occurs in species such as A. islandica (Brand and Taylor, 1974) or the large M. arenaria which requires an average of 30 min for the heart rate to fall after the cessation of pumping (Earll, 1975a). Akberali and Trueman (1979) have examined the pattern of short-term variation in heart rate in S. plana in relation to p 0 2 and p C 0 2 changes in the mantle cavity. Samples of water were withdrawn from the mantle cavity at known heart rates (Fig. 23) and indicated that a high heart rate (16-18 beatshin, indicative of pumping) was correlated with ap02 value of between 140 and 160 mmHg. Decrease in heart rate to 10-13 beatshin was associated with a p 0 2 level of 80-1 10 mmHg. During activity, the p C 0 , rarely exceeded 2 mmHg, but on cessation of pumping never fell below this value.

The factors causing suppression of heartbeat during inactivity are not clear. For example, Ptcsi and Salanki (1974), Brand (1976), and Parker

K I

I" E E

O ' ' 2 ' 4 ' 6 . e ' l o ' l z ' i 4

Individual Animal No.

FIG. 23. Mantle cavity pOz, pCOz, and heart rate (HR) of individual S. p l u m during active periods (0) and ventilatory pauses (0) when constantly immersed in sea water. Each point for the 14 animals during active periods is the mean of 6 determinations and each point for the 5 animals during ventilatory pauses is the mean of 4 determinations. Bars indicate SE; points for the same animal are joined by broken lines (from Akberali and Trueman, 1979).


(1978) showed that bradycardia in A . cygnea and A . anatina is not merely an affect of shell closure. Schleiper (1957) suggested that bradycardia in M . edulis may be effected by a build-up of carbon dioxide, whereas Bayne (1971) postulated that depletion of oxygen within the mantle cavity of M . edulis was responsible. The factors and mechanisms of suppression of the heart during valve closure are thus largely uncertain, and further investigations are needed.

C. Anaerobic Respiration during Valve Closure

It is well known that bivalves are able to withstand periods of shell closure and resultant lack of oxygen (Dugal, 1939; Trueman, 1967; Helm and Trueman, 1967; Crenshaw and Neff, 1969; Moon and Pritchard, 1970; Coleman and Trueman, 1971; Crenshaw, 1972; Akberali and Trueman, 1979; Davenport, 1979,1982; Widdows et al., 1979). The changeover from aerobic to anaerobic respiration in bivalves normally occurs when the oxygen tension of the mantle cavity falls to low levels, after the bivalve has closed its valves in response to environmental stress.

Early work on this subject, reviewed by von Brand (1946), recorded a variety of species able to survive long periods in the absence of oxygen. Most of the earlier work reported for anaerobic metabolism in bivalves is based on the observation that, on return to aerobic conditions, there is a rapid rise in the rate of oxygen consumption to levels above normal (Sch- lieper, 1957; Moon and Pritchard, 1970; Coleman, 1973, 1974). Other authors have reported a similar “overshoot” in heart rate (Trueman, 1967; Helm and Trueman, 1967; Coleman, 1974; Earll, 1975b; Brand, 1976; Akberali, 1978; Parker, 1978; Stone, 1980). Shell closure in A . islandica is followed by an exponential decrease in the oxygen tension of the mantle cavity water and is accompanied by an initial increase in heart rate, which is then followed at lower oxygen tensions by bradycardia (Taylor, 1976a,b). On return to aerobic conditions, both the heart rate and oxygen consumption are increased to a high level but then decline gradually to normal levels. When the freshwater bivalve P . coccineum is kept out of water for 5 days, the gill tissue exhibits a well-developed oxygen debt (Badman and Chin, 1973). Crassostrea (Ishida, 1935) and M . arenaria (Collip, 1921; van Dam, 1935) exhibit a higher rate of oxygen consumption after anaerobiosis than normal. These observations have frequently been interpreted as representing an increase in circulatory and respiratory demands, associated with the repayment of an “oxygen debt” incurred during the period of anaerobiosis.

In recent years, research carried out by a number of workers has led to a greater appreciation of the biochemical pathways operating in bivalve


molluscs during anaerobiosis, and has shown that these pathways differ in some ways from the classical ones described in vertebrates (Stokes and Awapara, 1968; Hochachka and Mustafa, 1972; De Zwaan and Zandee, 1972). It is now evident that many invertebrates are true facultative anaer- obes, capable of surviving indefinitely in the absence of oxygen and capable of active oxidative metabolism in its presence. In these organisms, as in primeval ones that possibly arose under reducing conditions in the absence of molecular oxygen (Wald, 1964), organic substrates, instead of oxygen, are the acceptors of electrons and protons. In intertidal bivalve molluscs, Hammen (1969) has shown that under aerobic conditions the catabolism of glycogen leads to pyruvate, which is then fully oxidized to carbon dioxide and water by the Krebs cycle reactions, a metabolic adaptation similar to that occurring in vertebrate muscle. Under anoxic conditions the breakdown of glycogen (or glucose) to the level of phos- phoenolpyruvate (PEP) is also similar to the process in vertebrates, but the breakdown products are different. Whereas vertebrates convert PEP to pyruvate and accumulate lactate, the main end products of anaerobic glucose catabolism in intertidal bivalve molluscs are succinate and alanine (Stokes and Awapara, 1968; Chen and Awapara, 1969; Hammen, 1969; De Zwaan and Zandee, 1972; De Zwaan and Marrewijk, 1973). Metabolic pathways for the conversion of glucose into succinate and alanine have been discussed by numerous authors (Stokes and Awapara, 1968; Chen and Awapara, 1969; Hochachka and Mustafa, 1972; Hochachka et al., 1973; De Zwaan et al., 1973, 1975; De Zwaan and Wijsman, 1976; De Zwaan, 1977; Newell, 1979). Anaerobic conditions are common for intertidal bivalves, and it has become clear that their ability to survive these stress conditions is connected to a remarkably high tissue glycogen content (De Zwaan and Zandee, 1972; Hochachka and Mustafa, 1972) together with certain adaptations of their intermediary metabolism (De Zwaan, 1977; De Zwaan et al., 1973; De Zwaan and Wijsman, 1976; Newell, 1979). For example, the glycogen and glucose concentrations of the clam P. coccineurn under anaerobic conditions decrease rapidly from 24 to 48 h, but level off after 48 h. Clams in low, but not zero, oxygen concentration appear to be unaffected (Badman and Chin, 1973). The ability to respire anaerobically also varies in bivalve tissues, depending on the easy accessibility of oxygen from the environment or circulatory system. For example, deeply located tissues have a greater tendency to respire anaerobically than superficially located tissues as a result of relative oxygen availability. It has been suggested that in bivalve molluscs, some tissues may be adapted to function anaerobically while others which are near to the sites of gas exchange may be primarily aerobic (Booth and Mangum, 1978). This may be of particular importance in epifaunal species


such as M . edulis, M . californianus, M . demissus, I . alatus, and C . edule during exposure at low tides when a small gape is maintained in their mantle margins during valve closure (Table 111). These bivalve molluscs have been shown to utilize atmospheric oxygen which diffuses into the mantle cavity fluid (Lent, 1968, 1969; Moon and Pritchard, 1970; Coleman and Trueman, 1971; Trueman and Lowe, 1971; Boyden, 1972a,b; Bayne et al . , 1976a,b; Widdows et al . , 1979). During valve closure, the oxygen obtained this way may sustain a slight level of aerobic respiration in superficially located tissues, e.g., mantle margins, gills, but it would be insufficient to meet the animal’s overall requirements. M . californianus is able to meet only part of its metabolic requirements by aerial oxygen consumption during the intertidal period, and approximately 25% of the minimal metabolic requirements are met in this animal by anaerobic pathways (Bayne ef al. , 1976b). Also relevant here are the observations made on other molluscan species. For example, cephalopod mantle, which is rich in mitochondria, has been shown to carry out aerobic respiration by superficial contact with aerated water both externally and in the mantle cavity; deeply situated muscle is, however, anaerobic (Bone et al . , 1981). In the squids Loligo pealii and Lolliquneula brevis the cardiac myofibre differs fundamentally from that of lamellibranchs in terms of mitochondrial and tubule density (Dykens and Mangum, 1979). The authors have concluded that these properties are correlated with a very high rate of oxidative metabolism in the intact animal, with little capacity for anaerobiosis. In the gastropod Bulliu, the foot, 1 mm thick, is entirely aerobic, whereas the deeply situated muscles, including the columellar muscle, may be anaerobic (Brown, 1982). In the large and actively burrowing bivalve D . serru, blood supply to the foot is probably cut off for 2-3 min while digging, and the respiratory demand of the pedal tissues is maximal (Trueman, unpublished). These observations suggest that anaerobic pathways play a significant role in molluscan respiration.

In contrast to conditions in the sublittoral zone, intertidal organisms are normally subjected to marked changes in oxygen availability. In coarse sediments, for example, interstitial oxygen concentrations tend to be high, but as the deposits become finer, the redox-potential discontinuity layer may approach the surface so that burrowing organisms are effectively surrounded by nearly anoxic conditions (Brafield, 1964). Therefore, infaunal bivalve species, for example, S . plana and M . arenaria, retain a connection to the oxygenated water at the surface of the sediment by means of siphons. There may be no oxygenated surface water available to draw over the gills at low tide, and future studies should consider differences between the behaviour of epifaunal and infaunal species in relation to their capacity for anaerobic respiration.

EFFECTS OF STRESS ON MARINE BIVALVE MOLLUSCS 15 I

D. Valve Activity and p H Changes

When the clam M . mereenaria was kept out of water for a period of days, dissolution of the inner layer of the shell occurred (Dugal, 1939). This was thought to buffer acid metabolites, principally lactic acid, but subsequent work has shown them to be largely succinic acid (Stokes and Awapara, 1968; De Zwaan et al., 1975). In Mercenaria measurements of calcium and succinic acid in the tissues and fluids indicate that succinic acid produced by the anaerobic metabolism is neutralized by the dissolution of previously deposited shell (Crenshaw and Neff, 1969; Crenshaw, 1972). The conversion of neutral substances into organic acids may cause a drop in the pH unless an animal has strong buffering systems at its disposal. In the absence of oxygen there is a rapid decrease in the glycogen content of the tissues and an increase in acid and carbon dioxide content. Evidence that the calcareous shell is involved in buffering acids has been found in various other bivalves when the animals are exposed to air or during the closure of the valves under normal or stress conditions (Collip, 1920, 1921; Dugal, 1939; Crenshaw and Neff, 1969; Crenshaw, 1972; Akberali et al., 1977, 1983; Parker, 1978; Akberali, 1980b; Akberali and Black, 1980). Crenshaw (1972) and Wijsman (1975) noticed that bivalves kept in aerated sea water closed their shells from time to time, with a concomitant drop in pH of the mantle cavity (Table IV). Measurements of oxygen tension in the extrapallial fluid of Mercenaria (Crenshaw and Neff, 1969; Crenshaw, 1972) revealed that the clam became completely anaerobic within 25 rnin of shell closure. Wijsman (1975) showed that the pH drop in aerially exposed M . edulis was even greater than in mussels kept in oxygen-free sea water (Table IV). The last group rarely kept their valves closed for longer than 5 h. When the valves of S. plana are closed (due to an osmotic shock), a significant increase in mantle cavity fluid calcium concentration occurs whereas clams in oxygen-free normal sea water keep their valves open with no noticeable rise in mantle cavity calcium concentrations (Fig. 30). It has been suggested that continuous water flow through the mantle cavity in the latter situation may explain the absence of a noticeable rise in calcium concentration, for under these conditions no anaerobic metabolites will accumulate, and in consequence little, if any, buffering will be required. In aerially exposed M . edulis, the lowest pH value recorded was 6.5, while in water-immersed “anaerobic” mussels it was 7.2 (Wijsman, 1975). A rapid rise in pH was noticed after opening the shell, suggesting that during exposure an increase in acidity is less harmful to Mytilus than the danger of desiccation.

A drop in pH of the mantle cavity fluid also occurs in the bivalve S . plana due to prolonged valve closure in normal sea water (Fig. 24) or

152 H. B. AKBERALI AND E . R . TRUEMAN

TABLE IV. SPECIES SHOWING A CHANGE I N pH OF THE MANTLE CAVITY OR

EXTRAPALLIAL FLUID DURING NATURAL OR ENFORCED PERIOD OF VALVE CLOSURE^

Period of valve

Species Condition closure pH change Reference

M . edulis

S. plana

M . mercenaria Aerial exposure

quiescence

quiescence

Natural

Natural

Aerial exposure

Ox ygen-free sea water

Natural

Aerial

Salinity

A . cygnea Natural

quiescence

exposure

change

quiescence

2 weeks

l h

4 h

30 h

3 h

8 h

6 h

24 h

24 h

7.48 -+ 7.25 (MC)

7.41 -+ 7.25 (EP)

7.59 -+ 6.91 (EP)

7.5 -+ 6.5 (EP)

7.5 -+ 7.2 (EP)

7.8 + 7.2 (MC)

7.8 --$ 7.65 (MC)

7.8 + 7.0 (MC)

7.8 -+ 7.45 (MC)

Dugal ( I 939)

Crenshaw (1972)

Crenshaw and Neff (1 969)

Wijsman (1975)

Wijsman (1975)

Original data

Original data

Original data

Parker (1978)

Abbreviations: MC, mantle cavity fluid; EP, extrapallial fluid.

0 2 4 6 8 1 0 1 2 Hours

FIG. 24. S. plana: pH of mantle cavity water, heart rate (HR), and valve movements during quiescence (- - -) and activity when constantly submerged in normal sea water (S, 31%0) at 10°C. Onset of activity, T; open, 0; closed, C. Continuous pH and HR recordings are represented as a mean of alternate 5-min intervals (original data).


salinity stress (Fig. 25). When prolonged valve closure occurs during inactivity, the heart rate is low and the pH gradually drops (Fig. 24). At the termination of quiescence, the heart rate first increases, then the valves open before pumping activity recommences. Only when pumping recommences does the pH of the mantle cavity water gradually return to normal. Immersion in 20% sea water (Fig. 25) results in the siphons being withdrawn, the valves being tightly closed, and the heart rate falling within 2 h. The pH of the mantle cavity shows a gradual stepwise drop, with intermittent rise and fall, but the cause of this remains obscure (Figs. 25 and 26A). The valves are still closed after 24 h in 20% sea water, and the pH falls to 7.00, while the heart rate remains at 6-8 beatdmin (Fig. 25). On replacing the low-salinity water with normal sea water, the heart rate and pH return to the previous recorded levels within I f h of the valves opening and recommencement of ciliary pumping. A common feature of recovery from quiescence in most intertidal bivalves (Coleman, 1974), including S. p l u m (Earll, 1975b), is the repeated sharp adduction and relaxation of the valves (see Fig. 5). This has been explained as effecting repayment of an oxygen debt or the “flushing out” of excretory products (Brand and Roberts, 1973). Resumption of activity in Scrobicu- luriu, either in normal sea water or after salinity or pollutant stress, is also characterized by rapid valve adductions. There is a corresponding drop in the p H of the mantle cavity water a short time after each of these adductions (e.g., see Fig. 26B) followed by a slow rise as water circulates through the open mantle cavity. This suggests release of an acidic sub- stance at each adduction which slowly diffuses out during the intervals between adductions. It is possible that acid metabolites, accumulated

FIG. 25. S . plana: pH of mantle cavity, heart rate (HR), and valve movements when transferred directly to S, 6.2%0 sea water (V) from normal sea water (S, 31%0) at 10°C. At the end of 24 h, 20% medium replaced with normal sea water (A). Open, 0; closed, C. pH and HR sampled as in Fig. 24 (original data).

I54 H . B . AKBERALI AND E. R . TRUEMAN

A

I, :;-----

78f ~- - 70

0

0 C

FIG. 26. S. piuna, examples of recordings of mantle cavity pH and valve movements: A, pH fluctuations seen in the mantle cavity water during a salinity stress (S, 6.2%0); B, rapid valve movements observed shortly after transference to normal sea water (S, 31%0) from a 24-h salinity stress; C, immersion in normal sea water (J) after a 6-h exposure period (original data, except for Fig. 26B, from Trueman and Akberali, 1981).

during anaerobiosis, are released from the tissues during valve adductions. Adduction of the valves causes the generation of simultaneous pressure pulses in both the mantle cavity and the tissues, resulting in rapid outflow of water from the mantle cavity (Trueman, 1966b). The pressure lasts longer in the tissues (1-2 s), however, than in the mantle and could well bring about the rapid flushing of metabolites from the renal tissues while normal ciliary pumping between adductions would remove these from the mantle cavity. During normal pumping activity, or when the pH drop in the mantle cavity is less, the pH is hardly affected by valve adductions (Fig. 26C); the feature shown in Fig. 26B is only observed after anaerobiosis and prolonged adduction of valves. Similar stepwise changes of p 0 2 and $ 0 2 levels in the mantle cavity of S . plana during recovery from anaerobiosis support this suggestion, for these changes imply a somewhat intermittent recovery compatible with hyperventilation of the mantle cavity by adduction of valves (Fig. 27). During 6-h emersion, S. plana showed a rather variable heart rate (12-22 beatdmin). In contrast to a drop in pH of the mantle cavity under normal conditions and salinity exposure (Figs. 24 and 2 3 , the pH in aerially exposed S. plana remained stable at 7.65 (Fig. 26C).

Valve closure in bivalves such as S. plana, M. edulis, and M. merce- nariu during short-term stress conditions or natural quiescence thus results in cessation of pumping, bradycardia, and the introduction of largely anaerobic respiration. The resulting accumulation of acid metabolites leads to the pH of the mantle cavity falling (Table IV). Resumption of

EFFECTS O F STRESS O N MARINE BIVALVE MOLLUSCS I55

0 40 80 120 Time (rnin)

FIG. 27. p o l , pCOz, and heart rate (HR) of S. plana transferred from normal sea water ( S , 31x0) to sea water (S, 6.2%0) for 24 h when they were replaced in normal sea water at 0 min. Each point for pOz and p C 0 2 is mean of seven animals, which were then discarded. Bars represent SE. Heart rate presented for one typical animal (from Akberali and Trueman, 1979).

activity is characterized by a rapid series of valve adductions whose probable function, at least in S . plana and possibly in other bivalves, is to hyperventilate the mantle cavity and probably to accelerate the removal of metabolites.

V. The Role of the Shell

A. Physical Protection and Isolation from Environmental Stress

Since most bivalve molluscs lead a relatively sedentary mode of life, it is essential that they have means of protection from predators and the prevailing physical and chemical environmental variables. This protection is provided by the shell. It is well known that molluscs, including bivalve

156 H . B. AKBERALI AND E. R. TRUEMAN

species, inhabiting exposed environments have stronger and thicker shells to protect their tissues from abrasive physical forces such as wave action than those inhabiting sheltered environments. Epifaunal species such as mussels and oysters have strong shells in order to protect their soft parts from predation and are also fixed to the substratum to prevent dislodgement. Infaunal species have different risks from predators, but may have thin slim shells to aid burrowing into the substratum. Apart from the use of the shell for protection from predation and physical factors, the shell can also provide valuable protection for the tissues from other environmental stress, e.g., salinity, pollutants, and aerial exposure. A sedentary mode of life restricts bivalves to a particular habitat where environmental stress cannot be avoided by migration, and under these circumstances the behavioural avoidance mechanism is of importance.

B. Shell Closure and Calcium Reabsorption

It has often been suggested that bivalve molluscs are able to withstand long periods of valve closure through a buffering of accumulated acid metabolites during anaerobiosis by dissolution of previously deposited shell material (Table V). This was first proposed by Collip (1920, 1921) for M . arenaria and has recently been reiterated by a number of other researchers working on A . cygnea (Dottenveich and Elssner, 1935; Parker, 1978), M . mercenaria (Dhgal, 1939; Crenshaw and Neff, 1969; Crenshaw, 1972), M . edulis, C. edule, Donax juliane and M . arenaria (Alyakrynskaya, 1972), Unio tumidus (Stone, 1980), and S . plana (Akberali et al., 1977, 1983; Akberali, 1980b).

The osmoconforming bivalve S . plana, when subjected to a long-term low-salinity stress, shows a gradual decline in the internal sodium, magnesium, chloride, and potassium concentrations becoming isosmotic with the external medium (Fig. 28). However, the calcium concentration in the body fluids examined shows an initial increase, reaching a maximum of 30 mM (about 3 times that in normal sea water) within 5-7 days, when it drops toward that of the external medium, stabilizing at a new low level at about 18 days (Fig. 29). To determine whether the increase of calcium over the first 7 days is related to anaerobic metabolism, clams were placed in oxygen-free sea water, where they exhibit only a slight rise in calcium levels (Fig. 30). It is only when the valves are closed by immersion in 20% sea water and the mantle cavity is not ventilated that calcium levels increase markedly. Forcible closure of the valves results in a rapid increase of calcium level over 2 days to about the same level as that reached by clams in 20% aerated sea water in 7 days (Fig. 29). A continuous flow of oxygen-free 100% sea water through the mantle cavity may explain the

TABLE V. ORGANISMS WHICH HAVE BEEN SHOWN TO MOBILIZE CALCIUM FROM THE SHELL DURING PERIODS OF VALVE CLOSURE^

Period of valve Increase in calcium

Species Condition closure concentration Reference

M . arenaria Aerial exposure 96 h

M . mercenaria Aerial exposure 13 days Natural quiescence l h Natural quiescence 4 h

S. plana Salinity change 5-7 days 0.5 ppm copper 6 h

U . tumidus Natural quiescence 30 h

A. cygnea Natural quiescence 48 h

a Abbreviations: MC, mantle cavity fluid; EP, extrapallial fluid.

8.9 + 60 mM (MC)

11.8 + 75.6 mM (MC) 21.1 + 24.6 rnEq/l (EP) 8.8 + 11 .O mM (EP)

11.7 + 30.0 mM (MC) 8.7 --$ 11.0 mM (MC)

3.1 -+ 5.5 mM (MC)

1.8+ 11.2mM(MC) Parker (1978)

Collip (1920)

Dugal(l939) Crenshaw (1972) Crenshaw and Neff (1969)

Akberali et al. (1977) Akberali and Black (1980)

Stone (1980)

I58 H . B . AKBERALI AND E. R. TRUEMAN

0 4 x 12 I6 20 24 2x

Time (days)

FIG. 28. Na+ concentrations in S. pfana transferred directly from 30%0 sea water (day 0) to 6%0 sea water in well-aerated conditions. (B-B), mantle cavity fluid; (8-0). blood from the ventricle; ( b a ) , medium. Each point is the mean of four determinations made on samples pooled from 12-14 individual S. plana selected randomly (from Akberali et al. , 1977; reprinted by permission from Nature (London) 266, No. 5605, 852; copyright 0 1977, Macmillan Journals Limited).

absence of a significant rise in calcium ions (Fig. 30A and B), for in this condition no anaerobic metabolites would accumulate and no buffering would be required. An increase in calcium concentration of the mantle cavity water has been shown in a number of bivalves during periods of valve closure arising from various environmental stressors or natural quiescence (Table V).

The increase in body fluid calcium concentration suggests mobilization from the shell. The calcium ions are derived from the interior of the valves by dissolution during stress, as shown by the use of 45Ca in S. plana (Akberali, 1980b) and M . mercenaria (Crenshaw and Neff, 1969). When Scrobicularia with 45Ca incorporated in its shell is subjected to a long- term salinity stress, 50% of the incorporated 45Ca is lost within the first

T 20

No 10 - !h 0 4 E 12 16 20 24 28

Time (days)

FIG. 29. Calcium concentrations of S. plana transferred directly from 100% sea water (S, 30%0) at day 0 to sea water (S, 6%0). Mantle cavity fluid (B-B); blood from the ventricle, (8-8); medium ( b A ) . Each point is the mean of four determinations made on samples pooled from 12- 14 individual Scrobiculnria selected randomly (from Akberali et a/ . , 1977; reprinted by permission from Nature (London) 266, No. 5605, 852; copyright 0 1977, Macmillan Journals Limited).


0 ' 8 ' ' " ' ~ ' ' ' ~ '

Time (days) 4 8 12 16 20 24

FIG. 30. Calcium concentrations of intact Scrobicuhia placed in oxygen-free normal (S, 30%0) sea water at A. After 6 days, the clams were additionally subjected to salinity stress, by immersion in oxygen-free, dilute (S, 6%0) sea water at B and C. After 7 days the valves of 20 animals were forcibly closed (D). Mantle cavity fluid, (W-W); blood from the ventricle (0-0); medium ( b d ) (from Akberali et a / . , 1977; reprinted from Nature (London) 266, No. 5605, 853; Copyright 0 1977, Macmillan Journals Limited).

24 h (Fig. 31). In contrast, clams left in normal sea water as a control show a fairly stable level of incorporated calcium in the shell. The rapid initial drop was also observed in clams exposed to a short-term (6-h) salinity stress where a rapid drop in 45Ca was caused in the 2 h after the onset of salinity stress (Fig. 32) and approached, after 24 h, levels similar to those of the previous experiment (Fig. 31). This suggests that the freshly deposited calcium is more labile than the remainder of the valve and is lost initially. This seems a relevant physiological adaptation; for example,

- *' $ lo: \

X f*- t*-*\*. 9 8

s 4-

2-

k- ' I 2 3 4 5 6 7 \ 1 4 Time (days)

FIG. 31. S. pluna with their valves covered with amyl acetate on the outside were left for 72 h in 45Ca-labelled sea water (5 pCi/l) for incorporation of labelled calcium in the valves on the inside, followed by placing those clams in unlabelled sea water for 48 h. The clams were then either subjected to a salinity stress at day 0 for 14 days (-) or placed in normal sea water (S, 31%0) as a control (----); values are presented as means of total 45Ca counts per minute (cpm) per valve (n = 8); bars represent SE (from Akberali, 1980b).

I60 H. B. AKBERALI A N D E. R. TRUEMAN

1

Time ( h )

FIG. 32. Loss of incorporated 45Ca counts per minute (cpm) per valve in Scrobicularia subjected to sea water (S, 6.2%0) for 6 h (-); clams in unlabelled normal sea water (S, 31%0) as control (----); treatment of animals and experimental procedure as in Fig. 31 (n = 8); bars represent SE (from Akberali, 1980b).

during exposure at low tides or under short-term stress conditions reflect- ing tidal duration when the valves are closed, the calcium most recently secreted is most likely to be utilized. The disappearance of the freshly incorporated 45Ca has also been shown using autoradiographic technique in S. p l u m by Akberali (1980b). This provides visual evidence of the disappearance of calcium from the valves during anaerobiosis under stress conditions. Further evidence of the dissolution of the inner shell surface has also been shown using scanning electron microscopy (Akberali et al., 1983). Under long-term, laboratory-induced salinity stress, the shell surface is markedly eroded in comparison with the control shells over the same period. This has also been investigated in the natural habitat by examining the effects of tidal immersion and exposure. There is no visible sign of erosion, and the inner shell surface is smooth at the end of the period of tidal immersion. In contrast, the shells that were collected toward the end of the exposure period at low tide show areas of dissolution in the form of eroded localized pits like those of long-stressed shells, though the pits are not as extensive (Akberali et ul., 1983).

The foregoing account shows the use of the shell in counteracting the harmful effects of both short-term and long-term environmental stressors. Although the shell may be weakened, this weakening is presumably a price worth paying for the survival of the organism during a short-term, unfavourable stress. A number of bivalve species utilize shell calcium in order to buffer acid end products of anaerobic respiration (Table V). Although both epifaunal and infaunal species mobilize shell calcium during anaerobiosis, it is likely that this phenomenon is more marked in


infaunal than epifaunal species. For example, the ability to utilize atmospheric oxygen during aerial exposure has been reported in a number of bivalves (Table 111), and evidence suggests that these species are not entirely dependent on anaerobic metabolism during aerial exposure. In contrast, such an option is not available to infaunal bivalve species, and this will result in a subsequent increase in demand for calcium to buffer the end product of anaerobiosis. Furthermore, in the infaunal bivalve S. plana valve closure may persist for 5-7 days during exposure to various environmental stressors such as copper and zinc, after which the neces- sity to open their valves results in pollutant poisoning and possibly death (Akberali and Black, 1980; Akberali et al., 1981). In the epifaunal bivalve M. edulis, continuous long-term exposure to 0.5 ppm copper results in an increase in mortality, with a median lethal time (MLT) of 2 days, whereas in Scrobicularia continuous long-term exposure to either copper or zinc results in a MLT between 5 and 7 days (Akberali and Black, 1980; Akberali et al., 1981). It is likely that this difference in MLT (Table 11) is due to the ability to survive varying periods of valve closure in these two species. The infaunal Scrobiculuria is probably better adapted for the prolonged use of behavioural avoidance mechanisms, and hence anoxic conditions, than the epifaunal M. edulis. Further work on habitat differences is necessary in order to enhance the understanding of the responses of bivalve molluscs to environmental stress.

C. Effect of Prolonged Stress on Shell Strength

Mobilization of calcium from the shell in order to buffer the end products of anaerobic respiration is a relevant physiological adaptation to environmental stress which allows the animal to protect its tissues by valve closure while sustaining basal metabolism. As mentioned in the previous section, during short-term environmental stress freshly deposited calcium is more labile than the remainder of the valve and is certainly utilized during the initial stress period. Under long-term or repeated short-term stress conditions, however, there will be a greater demand for calcium due to the more prolonged periods of valve closure (Akberali et al., 1977, 1981; Akberali, 1978; Akberali and Black, 1980). It is likely that the more tightly bound previously deposited calcium will then be mobilized, which could lead to an overall decrease in shell mass. The effects of increasing duration of continuous salinity stress on shell mass and shell strength in Scrobicularia have been examined and indicate that calcium in the mantle cavity fluid is directly related (or proportional) to a reduction in shell mass (Akberali et al., 1983). Such a reduction in shell mass will also include the loss of organic and other inorganic constituents. The shell


strength is reduced approximately in proportion to calcium dissolution from the valves and shows a general decrease with increased duration of the applied stress.

VI. Action of Heavy Metal Stressors

A. Accumulation of Heavy Metals

The capacity of bivalve molluscs to accumulate potentially toxic heavy metals in their tissues, far in excess of environmental levels, is well known and has become the focus of an increasing number of studies (Manley and George, 1977; Phillips, 1976, 1977; Bryan and Gibbs, 1983). Most of the evidence for absorption of metals and their radionucleotides from solution seems to involve passive diffusion of the metal, probably as uncharged soluble complexes, down gradients created by adsorption at the surface and binding by constituents of the surface cells, body fluids, and internal organs (Bryan, 1979; Carpene and George, 1981; Simkiss, 1983). However, this does not exclude, e.g., in higher organisms, the possibility of some movement of heavy metals by carrier systems used for calcium or magnesium transport (Bryan, 1976; Davies, 1978). The most important source of heavy metal bioaccumulation in bivalve molluscs is from suspended particles, in the case of suspension feeders, and from sediments in deposit feeders (Bryan, 1976, 1979). Tissue distribution of heavy metals as a result of bioaccumulation is typically uneven, and in some cases shows a high degree of organ specificity (Bryan, 1973; Bryan and Gibbs, 1983; George and Pirie, 1980; Viarengo et al., 1980a,b, 1981; Carmichael, 1980; Calabrese et al., 1984). The ability of bivalve molluscs to concentrate heavy metals at very high levels in the different tissues and yet to survive and apparently to reproduce normally indicates that they have evolved control or tolerance mechanisms at the cellular levels. Vari- ous systems for losing or immobilizing metals have been recognized in bivalve molluscs, apart from the possibility of diffusion. These include the immobilization of heavy metals in membrane-bound vesicles prior to their excretion from the kidney (Bryan 1973; George and Pirie, 1980; Carmi- chael, 1980; Viarengo et al., 1981; Simkiss et al., 1982; Calebrese et al., 1984) and by binding to wandering leucocytes, polysaccharides, amino acids, and proteins, e.g., metallothioneins (George et al., 1979; Bryan, 1979; Roesijadi, 1980; Viarengo et al., 1980a,b). A comprehensive review by Cunningham (1979) on the factors affecting accumulation, distribution, and loss of metals from the tissues due to intrinsic and extrinsic factors should also be consulted.


It has been suggested that the usefulness of bivalves such as M . edulis (Davenport, 1977) and S. plana (Akberali and Black, 1980; Akberali et al., 1981) as biological monitoring agents may be limited, since the bivalves may fail to register the transient or recurrent short-term presence of high pollutant levels as a result of their behavioural avoidance mechanisms (Figs. 6 and 9). It should be pointed out, however, that Scrobicularia shows behavioural avoidance of high concentrations of soluble zinc (5- 10 ppm) and copper (0.1-0.5 ppm) which will make it ineffective at the natural levels of between 1 and 52 pgll (ppb) zinc and 0.2-1.7 pg/l (ppb) copper currently recorded in coastal and estuarine water (Phillips, 1977; Coombs, 1980). In some polluted estuaries, copper and zinc levels in estuarine water may rise to only 0.1 and 2 ppm, respectively (Bryan and Gibbs, 1983). Even at such concentrations, bivalves such as Scrobicularia and Mytilus will remain active and interact with the medium, so that any determination of copper and zinc contained within the tissues will reflect the availability of metals in the environment. It should be stressed that quoted environmental levels of copper and zinc do not necessarily corre- spond with the concentration at the time of arrival of the pollutant, when the concentration may be far greater. During such an instance, if the environmental concentration exceeds the sublethal tolerance levels of the animal, it will result in the triggering of the behavioural avoidance mechanism.

B. Effects of Heavy Metals on Tissues

Bivalves can protect and isolate their tissues from adverse lethal effects of heavy metal pollutants by closure of the shell. The first visible response of S. plana to adverse environmental stress, e.g., copper and zinc, is to retract the siphons rapidly into the shell. Accordingly, an isolated inhalant siphonal tissue preparation was developed to examine the direct effects of heavy metals and pesticides (Akberali, 1981; Akberali et al., 1981, 1982a,b). Scrobicularia possesses extensible inhalant and exhalant siphons through which the clam makes contact with the overlying sea water. The inhalant siphon of Scrobicularia comprises longitudinal, circular, and radial muscles with six longitudinal nerves (Yonge, 1949: Chapman and Newell, 1956; Hodgson and Trueman, 1981). The posterior pallial nerves pass from the visceral ganglion to the siphonal nerves and inner- vate the muscle fibres. Longitudinal muscles are the major muscular com- ponent, and their stimulation causes the siphons to retract rapidly into the shell. In the isolated siphonal preparation, the nerve tracts are detached from central ganglia so that responses do not occur by means of the normal motor nervous system. Addition of copper at a final concentration


of 0.25 or 0.5 ppm causes the isolated inhalant siphon to go into a series of contractions (Fig. 18). Removal of copper from sea water terminates siphonal contractions, indicating that copper has a direct effect. Earlier studies (Akberali and Black, 1980) on the effects of copper on the behaviour of intact Scrubicularia revealed the complete withdrawal of siphons, followed by valve closure during a 6-h exposure period to concentrations of copper from 0.1 to 0.5 ppm (Fig. 6). The effect of copper on the isolated inhalant siphon of Scrobiculuria is comparable to its action on the behaviour of the intact clam and so the sensitivity of the clam to dilute copper solutions may be explained in terms of a direct effect of copper on the siphons. Additionally, it has been shown that the copper-induced contractions of the isolated siphon are dependent on the presence of calcium in sea water (Fig. 33). Such contractions may arise via the displacement of free calcium from intracellular reservoirs, resulting in a stimulation of the nerve/muscle system (see’ Section V1,D). In contrast, addition of 1-10 ppm zinc has no direct effect on isolated siphons (Fig. 19), whereas the same concentrations of zinc, when added to an in situ siphon preparation, caused marked contractions of the inhalant siphon (Fig. 20).

It is well known that heavy metals can inhibit the activity of many enzymes (Dixon and Webb, $967) and affect the function of several ceHu- lar constituents such as membranes (Rothstein, 1959), lysosomes (Moore, 1977), and mitochondria (Corner and Sparrow, 1956; Kleiner, 1974; Zaba and Harris, 1978; Akberali and Earnshaw, 1982a,c; Akberali et ul., 1984). With respect to tissues of marine organisms, and bivalves in particular (Table VI), it has been reported that heavy metals exert inhibitory effects on physiological processes, e.g., ciliary activity of the gills, oxygen consumption (Brown and Newell, 1972; Manley, 1983; Calabrese et al., 1984; Martin et al., 1984), heart rate (Scott and Major, 1972), byssus synthesis (Martin et al., 1975; Davenport, 1977), changes in ATP content, uptake of amino acids and protein synthesis following sublethal

ASW __L- h h h I I

At Bt CtwtD at bt €7 t----- - - + - - - - + - - - - i * P --t-*--l

0 20 40 60 80 100 120 140 MINUTES

FIG. 33. S. plana: effects of copper on an isolated inhalant siphon preparation in artificial sea water (ASW) (see original paper for formula). Upward deflection of the recording trace indicates isotonic contraction of the inhalant siphon. The following experimental protocol was carried out in sequence: application of copper at final concentrations of 0.25 ppm (A) and 0.5 ppm (B); removal of ASW containing 0.5 ppm copper (C), followed by washes (w) and replacement of Ca-free ASW (D); further application of copper at final concentrations of 0.25 ppm (a) and 0.5 ppm (b); and addition of CaCI2 to the organ bath to give a final concentration of 10 mM (E) (from Akberali et a / . , 1982a).

TABLE VI. SUMMARY OF SOME OBSERVED RESPONSES TO HEAVY METAL STRESSORS I N RELATION TO ALTERATION IN

BEHAVIOURAL/BlOCHEMICAL/PHYSIOLOGICAL PROCESSES IN MARINE BIVALVE MOLLUSCS

Stage of organization Heavy where stressor is metal Observed response

Species applied stressor to stressor -

M . edulis, M . modiolus, C . gigas, Anadara senilis, M . demissus

S . plana

M . edulis

M . edulis

M . edulis, C . gigas, C . margaritacea, P . perna, C . meridionalis

M . galloprovincialis

Adult Copper

Adult Copper, zinc, 1- naphthol

zinc, mercury

Adult Copper,

Adult Copper, zinc

Adult Copper, zinc, cadmium, lead

Adult Copper

Valve closure depending on concentration

Valve closure depending on concentration

Inhibition of byssal thread production

Inhibition of respiration

Inhibition of filtration rate

Reference

Davenport (1977); Manley and Davenport (1979); Manley (1983)

Akberali and Black (1980); Akberali et al. (1981, 1982b)

Martin et al. (1975); Davenport (1977)

Scott and Major (1972); Brown and Newell (1972); Manley (1983)

(1981) Abel (1976); Watling

Inhibition of protein synthesis, ATP content, and uptake of amino acids by various tissues

Viarengo et al. (1980b)

TABLE V1 (CONTINUED)

Stage of organization Heavy where stressor is metal Observed response

Species applied stressor to stressor Reference

M . edulis

C. virginica

M . californianus

Adult

Adult

Excised gill tissue

Copper Most toxic-suppressed growth Maung-Myint and Tyler of young oocytes and vitellogenesis in large oocytes

development and severe lysis of gametes

Cadmium Least toxic-suppressed gametogenesis only in the initial stages

(1982)

Zinc Less toxic-inhibited oocyte

Copper Increase in oxygen consumption by excised gill tissue; ultrastructural changes in terms of mitochondria1 swelling and damage in the ciliated epithelial cells

Inhibition of glycine influx Copper, mercury, iron

Engel and Fowler (1979)

Swinehart and Crowe (1980)

S. p l u m

C . virginica

M . mercennria

C . gigas

M . edulis

M . edulis

M . edulis

Isolated siphonal Copper, Spontaneous contractions of Akberali (1981); Akberali tissue 1-naphthol muscle et a/. (1982a,b)

48-h straight-hinge Copper Inhibition of growth

48-h straight-hinge Copper, zinc Inhibition of growth

larval stage

larval stage

Calabrese et al. (1977)

Calabrese et a/ . (1977)

Larval stage Zinc Inhibition of growth Brereton et nl. (1973)

Unfertilized eggs Copper Stimulation of egg respiration Akberali et al. (1984) Zinc Inhibition of egg respiration

Sperm Copper Inhibition of sperm respiration Akberali et al. (1985) Zinc Inhibition of sperm respiration

and motility

Digestive gland and Copper Biphasic effect-stimulation Akberali et a/. (1984) mantle mitochon- followed by inhibition of dria mitochondrial respiration

Zinc Inhibition of mitochondrial Akberali and Earnshaw respiration (1982a)

M . edulis Digestive gland and Copper Inhibition of mitochondrial Akberali and Earnshaw mantle mitochon- calcium transport (1982~) dria

Zinc Inhibition of mitochondrial calcium transport

I68 H. B. AKBERALI A N D E. R. TRUEMAN

exposures to copper (Viarengo et al., 1980b), and mitochondria1 respiration and calcium transport (Akberali and Earnshaw, 1982a,c; Akberali et al., 1984). It has also been shown that concentrations of 0.05 ppm copper or 0.2 ppm zinc applied continuously to adult M. edulis severely inhibit gametogenesis by suppressing both the growth of young oocytes and vitellogenesis in larger oocytes (Maung-Myint and Tyler, 1982). When the oyster C. virginica was continuously exposed to 50 and 100 ppb copper, significant increases in the rate of oxygen consumption of the excised gill tissue after 14 days of continuous exposure to 100 ppb copper were reported (Engel and Fowler, 1979). It has also been shown that copper, cadmium, lead, and zinc cause reduction of the filtering rates in the bivalve molluscs C. gigas, Crassostrea margaritacea, P . perna, and Choro- mytilus meridionalis, with copper being most toxic to both oysters and mussels (Watling, 1981). Further evidence on the effect of heavy metals on filtration rates has also been reported for M. edulis (Abel, 1976). Mer- cury, copper, and zinc reduced filtration rate by 50% at concentrations of 0.04, 0.15, and 1.6 ppm, respectively. In the gills of M. californianus and Anodonta, mercury has a dramatic effect on amino acid efflux, whereas copper, mercury, and iron inhibit the influx of glycine into Mytilus gills (Swinehart and Crowe, 1980).

C. Effects of Heavy Metals on Released Gametes and Embryonic and Juvenile Stages

Although adult bivalves may be capable of tolerating high body concentration of heavy metals, little attention has been directed toward the effects these metal concentrations may have on gametogenesis, fertilization, and embryology. Laboratory techniques are now available for the rearing of early developmental stages of a variety of marine fishes and invertebrates so that it should be feasible to direct both field and laboratory work to emphasize the impact of heavy metal stress on various stages in the life cycle.

The gametogenic cycle in bivalves, e.g., M. edulis, can be affected by variation in natural environmental parameters, such as temperature and food abundance (Seed, 1976; Bayne et al., 1978; Lowe et al., 1982) but is also subjected to stress imposed by heavy metal input. The application of environmental stress to adult marine bivalve molluscs can result in a reduction in reproductive capacity. For example, prolonged exposure of M. edulis to high temperatures and low ration results in stress to the organism owing to an increase in metabolic rate (Gabbot and Bayne, 1973). Although subsequent oocyte development, vitellogenesis, and fertilization appear to proceed normally, the larvae have a lower growth rate


than larvae from “unstressed” adults. Moreover, the “stressed” adults produce fewer and smaller eggs with less lipid and protein reserves (Bayne et al., 1978) and a larger proportion of abnormal trochophores resulting in a small proportion of normal prodissoconch I larvae (Bayne, 1972; Bayne et al., 1975). It has recently been shown that continuous exposure to sublethal levels of copper and zinc suppresses gametogenesis in M . edulis, with copper being more toxic (Maung-Myint and- Tyler, 1982).

Little information is available in the literature concerning the mechanism of the action of heavy metals either with respect to the inhibition of gametogenesis or in their sublethal effects on the released gametes of bivalve molluscs and on the overall pattern of development. Due to a high surface area : volume ratio and in the absence of behavioural avoidance mechanisms, newly released gametes may well be more susceptible than adults to the direct input of heavy metal into the aquatic environment.

The presence of heavy metals in the environment has been shown to inhibit the gametogenic cycle, embryonic development, and larval growth. For example, Wisely and Blick (1967) exposed M . edulis and C . commercialis larvae with developing dissoconchs to copper and mercury. Fifty percent of the mussel larvae died in 2 h at concentrations of 32.3 ppm copper and 13 ppm mercury, and oyster larvae at 180.5 ppm mercury. They concluded that this comparatively high resistance was a result of the ability of these organisms to withdraw their bodies into their shells, thereby reducing the penetration of the toxic material into the tissues. The lethal concentration values (LCso) obtained would thus be influenced by the length of time that a particular test species could remain closed (Table VII). In the presence of zinc concentrations of 125-500 ppb, growth and development of larvae of the Pacific oyster C . gigas were markedly slower (Brereton et al., 1973). This indicates that exposure of larvae to sublethal concentrations of heavy metals can have subtle but equally deleterious consequences on development as exposure to lethal concentration levels. Calabrese et al. (1973) have reported an LCs0 value for the development of embryos of C . virginica to straight-hinge larval stage of approximately 0.103 ppm copper, 0.0056 ppm mercury, and 0.31 ppm zinc, an indication that the embryos of bivalve molluscs may be more sensitive than the larvae to heavy metal pollution, while the adult animals are fairly resistant (Table VII). Furthermore, a comparison of studies on C. virginica and M. mercenaria larvae (Calabrese et al., 1977) during the development of embryos to the straight-hinge larval stage (Calabrese et al., 1973; Calabrese and Nelson, 1974) indicates that a slightly higher concentration of mercury, silver, and copper was required for 50% mortality of larvae than for the development of embryos. The early develop-

TABLE VII. COMPARISON OF TOXIC EFFECTS OF STRESSORS I N RELATION TO VARIOUS LIFE STAGES^

Stage of Concen- Duration of Observed Species organization Stressor tration exposure response Reference

M. mercenaria Embryos and following Mercury development to Silver straight-hinge larvae Zinc

Nickel Lead

M . edulis Embryos and following Copper development to Mercury straight-hinge larvae Silver

Zinc Lead Cadmium

M . edulis

M . edulis

M . edulis

Unfertilized egg First polar body Two-cell stage 64 cells or greater Ciliated blastula Trochophore Early veliger

Larvae

Adult

Sevin Sevin Sevin Sevin Sevin Sevin Sevin

Sevin

Copper

4.8 pg/l 21 pg/l

166 pg/l 3 10 pg/l 780 pg/l

5.8 pg/l 5.8 pg/l 14 pg/l

175 pg/l 476 pg/l

1200 pg/l

20 mgil 5.3 mg/l

7 mg/l 8.3 mg/l 16 mg/l 19 mg/l 24 mg/l

2.3 mg/l

250 pgll 500 pg/l

42-48 h 42-48 h 42-48 h 42-48 h 42-48 h

48 h 48 h 48 h 48 h 48 h 48 h

I h I h l h I h I h l h I h

2 h

Continuous exposure Continuous exposure

Lc50 Calabrese and Lc50 Nelson (1974) LC50 LC50 LCSO

ECso Martin et al. (1981) ECso EC50 EC50 EC50 EC50

Armstrong and Millemann (1974)

EC50 Stewart et al. ( 1967)

MLT 4-5 days Davenport (1977) MLT 2 days

M . edulis planulatus

C . virginica

C . gigas

C . commercialis

0. edulis

M . arenaria

S . plana

Larvae with develop- Mercury ing dissoconch Copper

Embryos and following Mercury development to Silver straight-hinge larvae Copper

Embryos and following Copper development to Mercury straight-hinge larvae Silver

Zinc

Zinc Cadmium Lead

Larvae with develop- Mercury ing dissoconch

Larvae Adult

Adult

Zinc Zinc

Copper Zinc

Adult Copper

Zinc

1-Naphthol

13 mg/l 32.2 mg/l

5.6 pg/l

103 pg/l 310 pg/l

5.8 pgll

5.3 pgll

22 pg/l 6.7 pgll

119 pg/l 611 pg/l 758 pg/l

180.5 mg/l

1 mgll 100 mg/l

5 mg/l 52 mg/l

500 pg/l

10 mg/l

20 mg/l 10 mg/l

2 h 2 h

42-48 h 42-48 h 42-48 h 42-48 h

48 h 48 h 48 h 48 h 48 h 48 h

2 h

96 h 96 h

48 h 48 h

Continuous exposure

Continuous exposure

Continuous exposure Continuous exposure

LCSO LCSO

LCSO

LCSO LCSO

EGO

ECso

EGO E G O

LCSO

LCJO LCJO

LCSO

MLT 5-7 days

MLT 6 days

MLT 5 days MLT 9 days

Lc50

EC50

EC50

Lc50

Wisely and Blick

Calabrese et al.

(1967)

(1 973)

Martin et a / . (1981)

Wisely and Blick

Walne (1964);

Eisler (1977)

( 1967)

Portman (1970)

Akberali and Black

Akberali et a / . (1980)

(1981)

Akberali et a / . (1982b)

Abbreviations: MLT, median lethal time for 50% mortality; LCso, lethal concentration for 50% mortality; ECso, concentrations that caused anomalous development of 50% test animals.

I72 H . B . AKBERALI A N D E. R. TRUEMAN

mental stages in M . edulis was most sensitive to the insecticide Sevin during the first hour after fertilization. The toxicity of Sevin decreased with increase in age of the larvae (Armstrong and Milleman, 1974). For example, the l-h EC50 values for the first polar body stage and for the veliger were 5.3 and 24.0 mg/l, respectively (Table VII).

The mechanisms involved in the disruption by heavy metal of the gametogenic cycle (Maung-Myint and Tyler, 1982) and embryonic development (Calabrese and Nelson, 1974) are unknown, but it is possible that these heavy metals inhibit metabolic processes (Table VI). Zinc is known to inhibit the respiration of mitochondria isolated from the mantle or digestive gland of M . edulis (Akberali and Earnshaw, 1982a), fish liver (Zaba and Harris, 1978), and rat liver (Kleiner and von Jagow, 1972; Kleiner, 1974; Akberali and Earnshaw, 1982a). In contrast, copper initially stimulates and then inhibits respiration of mitochondria isolated from fish liver (Zaba and Harris, 1978), rat liver (Zaba and Harris, 1976), and mantle tissue of M . edulis (Akberali et al., 1984).

Recent studies have examined the action of copper and zinc on both the sperm and the unfertilized eggs of M . edulis, with emphasis placed on their effects on respiration (Akberali et al., 1984) and sperm motility, and on uptake and intracellular localization of heavy metals (Akberali, unpublished). This approach establishes the mode of action of copper and zinc on the released gametes at a cellular level, in order to avoid complexities involved when working at organismal and tissue levels. Such an approach would serve as a convenient starting point in seeking to understand the observed heavy metal toxicity associated with the gametogenic cycle and development. Many metal accumulation studies with adult bivalve molluscs have been undertaken with little thought as to their reproductive condition at the onset of the exposure period or the effect that spawning during the exposure period may have on metal elimination via the released gametes and on gamete viability itself. For example, in M . edulis, the toxicity of copper varies during the reproductive period (Delhaye and Cornet, 1975). In the months preceeding the spawning (January-Febru- ary) the TL, is 9-10 days for a l-ppm copper concentration; this de- creases to 6 days and 2-3 days during the spawning period (March) and postspawning period (April-May), respectively. It was concluded that this increase in sensitivity during the spawning season, which is characterized by a period of high respiration (de Vooys, 1976), was more a result of increased metabolic rate, resulting in a faster absorption of copper, than an increase in sensitivity. The decrease in TL , to 2-3 days observed during postspawning period may well be due to the metabolic condition of the adults. It is well known that during the period following spawning the metabolic reserves of the adults are at a minimum (Bayne, 1976); and


hence the presence of copper or other heavy metals may well provide an added burden on the adult metabolic state.

It is also reported by Cunningham and Trip (1975) that when C. uirginica were continuously exposed to sublethal levels of mercury, a 60% decline in the accumulated mercury levels in the tissues was observed during the spawning season. These authors have suggested that if mercury was accumulated in the gonadal tissue then an appreciable loss may occur during spawning, with the implication that the metal-gamete interaction during the spawning season may result in a decrease in reproductive potential, Indeed, Maung-Myint and Tyler (1982) have reported that continuous exposures to both copper and zinc suppress gametogenesis in adult M . edulis, with copper being more toxic (Table VI). Further- more, transfer of heavy metals, e.g., cadmium or copper, from adult female oysters to their eggs has also been reported (Greig et al., 1975).

Direct addition of 0.1-0.5 mM (6.3-31.5 ppm) copper to unfertilized eggs of M . edulis resulted in an immediate stimulation of respiration, but a similar direct addition of zinc (6.5-32.5 ppm) had no such effect (Fig. 34). By contrast, a similar direct addition of copper to sperm has no effect, but the respiration is inhibited by zinc (Fig. 34). Additional experiments, in which the gametes were preincubated with the heavy metal at 0°C for 20 min before measurement of respiration, showed that egg respiration was inhibited by zinc and sperm respiration by copper (Akberali et al., 1985). Presumably the time-dependent increase in the cytoplasmic concentration effects of copper and zinc is variable and appears to be relevant in its mode of action on sperm and egg respiration. Furthermore, the effects of direct application (Fig. 35) or preincubation (Akberali et al., 1985) with

9

2 min -

cf

FIG. 34. Oxygen electrode traces showing the effect of direct additions (4) of copper or zinc on egg and sperm respiration in filtered sea water at 10°C. Heavy metal ions were present at a final concentration of 0.5 mM. Numerals refer to respiration as nanomoles oxygen/minute/milligram protein (from Akberali ef a / . , 1984).


0 ' I I r I

1 2 3 4

cu2+ ( m M )

FIG. 35. Egg respiration as afunction of direct additions of various copper concentrations. The experiment was carried out as in Fig. 34 (from Akberali et a / . , 1984).

copper shows that a concentration of ca. 0.5 mM copper causes a maximal stimulation in Mytilus of egg respiration, and that higher copper concentrations result in a progressively smaller stimulation of respiration. The uptake of copper and zinc by sperm and eggs of Mytilus, expressed on the basis of cell volume in order to take account of markedly different volumes, indicates that the uptake of both metal ions by the gametes increases with increasing metal concentration. The metal ion uptake in the sperm is approximately three times that in the eggs, with more zinc than copper uptake in both the egg and sperm (Akberali ct ul., 1985).

Akberali et al. (1984) have further reported that respiration in unfertilized M. edulis eggs is only partially released, since the addition of uncoupler carbonyl cyanide rn-chlorophenylhydrazone (CCCP) causes a several-fold increase in egg respiration. Presumably the low rate of basal respiration in the unfertilized egg ensures that the substrate reserves are not depleted prior to fertilization. These authors have suggested that respiration in the unfertilized egg is inhibited by a high ATP/ADP ratio in the cytosol. Respiration can, therefore, be stimulated by either the addition of H+-translocating uncoupler (CCCP) or by copper which may act by stimu- lating mitochondria1 K+ influx (see Section V1,D). It is tempting to suggest that the observed effects of copper and zinc in suppressing gameto-


genesis in adult M . edrnlis (Maung-Myint and Tyler, 1982) may be partly caused by their disruptive effects on normal cellular respiration of gametes (Table VI). Adult bivalve tissues, including gonads, accumulate high levels of heavy metals (Cunningham and Trip, 1975; Bryan and Gibbs, 1983). It is likely that low levels of heavy metal incorporated during gamete development from adult tissue (Greig et al . , 1975) could well affect subsequent gamete viability and postfertilization development. Un- der natural conditions, the long-term effects of heavy metals on gametogenesis may well be of more importance to a species than, for example, their action in reducing the performance of gametes and larvae.

The presence of a heavy metal scavenging system in M . edulis or other bivalve gametes has not yet been established. Recently, zinc-binding protein has been demonstrated in the unfertilized egg of the sea urchin Antho- cidaris (Ohtake et al . , 1983). It has been shown that zinc concentrations which were lethal to “naive” fathead minnow (Pimpehales promelas) cell cultures were not lethal to cells which were slowly adapted to higher levels of zinc (Adragna and Privitera, 1978). This suggests that the cells were able to tolerate a gradual increase in the metal load. Such an exciting approach, which has been adopted for vertebrate cell cultures, could well yield interesting results in heavy metal stressor studies of bivalves.

D. Effects of Heavy Metals on Cellular Organelles

The ability of bivalve molluscs to concentrate heavy metals in different tissues (Bryan and Gibbs, 1983) and apparently to survive and reproduce indicates that they have evolved control or tolerance mechanisms at the cellular level. The accumulation of heavy metals in various tissues in the estuarine bivalve molluscs, e.g., S . plana and M . edulis, has been shown to vary in different localities depending on the environmental concentration (Phillips, 1977; Bryan and Gibbs, 1983). Although studies on uptake and respiratory effects of heavy metals on cellular organelles, e.g., fish and rat liver mitochondria (Kleiner and von Jagow, 1972; Kleiner, 1974; Zaba and Harris, 1976, 1978) have been reported, there is little published work with cellular organelles from bivalve tissues. Emphasis should be placed especially with reference to tolerance and sensitivity, which may exist at the organelle levels in various tissues in relation to the observed differences in the amount of heavy metal accumulated by the tissues. In fish and mammals, the liver is known to be of considerable importance in the storage, uptake, and detoxification of heavy metals by metallothionein synthesis (O’Dell and Campbell, 1971 ; Zaba and Harris, 1976, 1978). Fur- thermore, studies on mammalian liver mitochondria indicate that these

I76 H . B . AKBERALI A N D E. R. TRUEMAN

organelles are capable of active uptake of divalent cations (Vainio el al., 1970; Bygrave, 1978). In M . edulis, digestive gland and mantle mitochondria have been shown to possess a calcium transporter comparable to vertebrate mitochondria (Akberali and Earnshaw, 1982b). The addition of calcium to M . edulis mitochondria results in a transient stimulation of respiration which is less pronounced than in rat liver mitochondria. This stimulatory response by mussel mitochondria to calcium ion addition is strongly indicative of an energy-linked uptake of calcium which is similar to that reported for vertebrate preparations (Lehninger, 1970; Bygrave, 1978; Zaba and Harris, 1978).

Mitochondria are known to act as regulators of intracellular calcium levels (Bygrave, 1977; Carafoli and Crompton, 1978a; Nicholls and Crompton, 1980), and may play a role in both nerve conduction (Alnaes and Rahamimoff, 1975; Carafoli and Crompton, 1978b) and muscle contraction (Huddart and Price, 1976; Carafoli and Crompton, 1978a). Since bivalve molluscs accumulate excessive levels of heavy metals in their tissues, it is likely that mitochondria1 calcium transport may at some stage be affected prior to immob,ilization or excretion of heavy metals. Akberali and Earnshaw (1982~) examined the effects of copper and zinc on the respiration of digestive gland mitochondria in M . edufis and their calcium transport in a sucrose reaction medium. Copper has little effect on the initial rate of basal respiration, but dramatically inhibits the initial rate of calcium transport, with 50% inhibition occurring at 50 p M copper (Fig. 36A). Calcium transport is also inhibited by copper in rat liver mitochondria (Zaba and Harris, 1976), probably in a fashion similar to lead, which at low concentrations is a competitive inhibitor of calcium transport and at high concentrations inhibits the production or use of respiratory energy (Parr and Harris, 1976). On the other hand, zinc produces a similar degree of inhibition in the initial rates of both basal respiration and calcium transport, with 50% inhibition occurring at approximately 60-80 p M zinc (Fig. 36B). Akberali and Earnshaw (1982~) suggested that the inhibition of calcium transport in M . edulis mitochondria caused by zinc is largely a respiratory inhibition.

The effects of copper and zinc have also been examined on M . edulis digestive gland mitochondria which have been preloaded with calcium (Akberali and Earnshaw, 1982~). The addition of 100-200 p M copper results in an immediate efflux of calcium, whereas the accumulated mitochondrial calcium was stable on addition of 100 p M zinc (Fig. 37). A delayed and much slower rate of calcium efflux occurred at 200 p M zinc. On the basis of these findings, Akberali and Earnshaw (19824 put forward a hypothesis for the copper-induced contraction observed in the isolated bivalve mollusc siphon (Akberali, 1981 ; Akberali et al., 1982a). They


I\ - 1 0

-*-•

0- 100 200 300 400

cuso4 OJM)

0 ' 100 ZOO 300 400

Z ~ S O ~ ( p M )

FIG. 36. M. edulis digestive gland mitochondria: The effect of copper (A) and zinc (B) on the initial rate of basal respiration in the absence of calcium (0) and on the initial rate of calcium transport (0) in the sucrose reaction medium. Control rates (100%) for respiration were 12.3 ng atoms oxygen/min/mg mitochondria1 protein (A) and 12.7 ng atoms oxygen/ min/mg mitochondria1 protein (B). Control rates (100%) for calcium transport were 8.0 ng ions calcium/lO s/mg mitochondria1 protein (A) and 7.3 ng ions calcium/lO s/mg mitochondrial protein (B) (from Akberali and Earnshaw, 1982~).

I78 H . B. AKBERALI A N D E. R. TRUEMAN

10

I

C m ._

- 8 2 a

7 3 Z 6

+' n

.- m C I

+ L

0

5 4

.- s m I

U - 3

2

0

A

1 2 3 4 5 6 7

B

1 2 3 4 5 6 7

Time (rnln)

FIG. 37. The effect of copper (A) and zinc (B) on accumulated mitochondria1 calcium-45: Mitochondria were isolated from M . edulis digestive gland. Mitochondria1 calcium transport was determined at 5°C using calcium-45. Copper was added at 2 min (J) at concentrations of 100 p M (0) and 200 pM (W), and (0) represents calcium transport in the control (from Akberali and Earnshaw, 1982~).

suggested that, in the presence of copper, release of calcium ions takes place from mitochondria or possibly another cellular site, and that this increase in free intracellular calcium then induces excitation-contraction coupling in muscle cells. Mitochondria are thought to play a role in calcium movements during excitation-contraction coupling in muscle cells; this may be particularly important in molluscan smooth muscle in the absence of an organized sarcoplasmic reticulum (Huddart et al., 1977). Akberali and Earnshaw (1982~) have alternatively suggested that the induced calcium efflux by copper from mitochondria and possibly smooth endoplasmic reticulum in the presynaptic nerve terminal may result in an increase in intracellular calcium activity, which possibly triggers transmitter release (Katz and Miledi, 1965, 1967; Shapiro et al., 1980).

It has been shown that fish liver mitochondria are capable of binding certain cations to a considerable degree (Zaba and Harris, 1978); thus calcium, copper, and manganese are taken up much more rapidly than

EFFECTS O F STRESS O N MARINE BIVALVE MOLLUSCS I 79

zinc. Furthermore, copper possesses a biphasic reaction on state 4 oxidation of mitochondria isolated from rat liver (Zaba and Harris, 1976), fish liver (Zaba and Harris, 1978), and the mantle tissue of M. edulis (Akberali et al., 1984). Zaba and Harris (1976, 1978) have explained the biphasic mode of action of copper on vertebrate mitochondrial respiration. They have shown that the initial stimulation of mitochondrial respiration is a result of enhanced K + influx, which is accompanied by mitochondrial swelling. The ensuing progressive inhibition of mitochondrial respiration appears to be linked to K+ loss, which seems to take place at higher copper concentrations, where copper may have a general inhibitory effect upon respiratory enzymes. It seems reasonable to assume that the biphasic mode of action of copper on M. edulis mitochondrial respiration in KCI reaction medium (Fig. 38) is comparable, particularly as the addition of copper to M. edulis mitochondria in a reaction medium containing sucrose does not produce a respiratory stimulation (Fig. 36A). Further- more, it is also reasonable to suppose that in the presence of copper, the stimulation of M. edulis unfertilized egg respiration (Figs. 34 and 35) and the increase in oxygen consumption rates of excised oyster (C. virginica) gill tissue (Engel and Fowler, 1979) is due to a similar uncoupling of mitochondrial respiration by copper, as previously described (Akberali er al., 1984). In fact, Engel and Fowler (1979) have demonstrated ultrastruc-

FIG. 38. Oxygen electrode trace showing the action of copper on the respiration of mitochondria isolated from M. edulis mantle tissue. The substrate addition (J S) was made 1 min after addition of the mitochondria to the potassium chloride reaction medium and consisted of 6 mM succinate plus 6 mM glutamate. Copper (4) was added at a final concentration of 0.4 mM. Numerals refer to respiration as nanomoles oxygen/minute/milligram mitochondrial protein (from Akberali et al . , 1984).

180 H . B. AKBERALI AND E. R . TRUEMAN

tural changes in the ciliated epithelial cells of gill tissue in relation to cellular swelling and mitochondrial damage. These authors have suggested that the observed increase in gill-tissue respiration may be related to increased cellular or mitochondrial membrane permeability.

Akberali et al. (1984) have further reported a lack of inhibition of respiration in the eggs of M . edulis in the presence of copper as would be expected from the biphasic mode of action on mitochondrial respiration (Fig. 38). This could possibly be caused by low intracellular copper concentrations, since it is noticeable that the degree of stimulation from direct application (Fig. 35) or pretreatment (Akberali et al., 1985) with copper concentrations greater than 0.5 mM is reduced. This may be an important consideration in the case of the egg where the vitelline membrane, which surrounds the plasma membrane, could be responsible for binding a large proportion of heavy metal taken up from the medium, thus resulting in low intracellular metal concentrations.

On the other hand, Akberali et al. (1984) reported that direct addition of copper has no effect on the respiration of the sperm (Fig. 34) of M . edulis, whereas pretreatment of sperm with copper inhibits respiration (Akberali et al., 1985). This different effect of copper on sperm and egg respiration results from greater copper uptake by the sperm (Akberali et d., 1985), which inhibits mitochondrial respiration; it may also be the result of different physiological states of the mitochondria in the two gametes. The latter authors have suggested that sperm mitochondria are presumably carrying out state 3 oxidation in which the rate of respiration is not limited by ADP, and therefore possess little potential for copper-induced uncoupling. Egg mitochondria, however, show a considerable potential for uncoupling by a H+-translocating uncoupler (CCCP), or by copper, and it has been suggested that their respiration is inhibited by a high cytoplasmic ATPIADP ratio.

Unlike copper, zinc inhibits respiration of mitochondria isolated from the mantle or digestive gland of M . edulis (Akberali and Earnshaw, 1982a), fish liver (Zaba and Harris, 1978), and rat liver (Kleiner and von Jagow, 1972; Kleiner, 1974; Akberali and Earnshaw, 1982a). Approxi- mate zinc concentrations giving a 50% inhibition of respiration are 70, 135, and 280 p M for rat liver, mantle, and digestive gland mitochondria, respectively (Akberali and Earnshaw, 1982a). The authors have suggested that the increased resistance of digestive gland and mantle mitochondria to zinc in M . edulis could be advantageous in an organism that accumu- lates excessive amounts of zinc (Fig. 39). The greater resistance of the digestive gland as compared to the mantle mitochondria could be an adaptation in a tissue that has been shown to accumulate greater zinc concentrations (Pentreath, 1973; George and Pirie, 1980; Bryan and Gibbs, 1983).

EFFECTS OF STRESS O N MARINE BIVALVE MOLLUSCS 181

1 , 1- ) 100 200 300 400 1000

Zinc added ( V M )

FIG. 39. The effect of zinc on the oxidation of succinate plus glutamate by M. edulis digestive gland, M. edulis mantle, and rat liver mitochondria. The control rates (100%) were 10.22 -+ 1.40 (SE) nmol oxygenfmidmg mitochondrial protein for M. edulis digestive gland (-.-); I1 5 0 * 0.40 for M. edulis mantle (- - - -), and 53.31 ? 4.88 for rat liver (-). The points represent means of five to seven separate mitochondrial isolations from different groups of animals. Vertical bars represent SE (from Akberali and Earnshaw, 1982a).

In fish, Zaba and Harris (1978) have shown that zinc is taken up by the mitochondria to a very limited extent, for example, raising the external zinc concentration from 10 to 60 ng ions/mg protein resulted in an increase in uptake from 0.4 to only 1.2 ng ions/mg protein. They found, however, that zinc was the most potent inhibitor of mitochondrial respiration, 75% inhibition occurring at a mitochondrial uptake level of only 2 ng ions/mg protein. They also reported that for mitochondria to accumulate this amount of zinc, the external concentration must be raised to 65 p M . This concentration is lower than the 50% inhibition concentration of 135 and 280 p M for M . edulis mantle and digestive gland mitochondria, respectively. It seems that mitochondria isolated from bivalve tissues are more resistant than vertebrate mitochondria. Future work involving bivalve mollusc populations with differing tissue zinc and other metal con-


centrations is certainly required in order to elucidate this difference in resistance between tissues and species at cellular organelle level.

The acute effects of heavy metals in a number of bivalve molluscs have been extensively investigated, and it is now perhaps the right time for sublethal effects of heavy metals to be examined in detail at tissue and cellular level. The work reviewed here indicates that sublethal effects can have far more serious long-term consequences on various processes which can ultimately affect the survival and propagation of species. Stud- ies on the effects of heavy metals on genetic material of cells are still unclear. There is some evidence for metal resistance (Bryan, 1976; Adragna and Privitera, 1978) whereby tolerance can be increased to the toxic effects of heavy metals by previous exposures to sublethal concentrations, but these studies are few and limited to fish, brown fouling algae, and seaweeds.

VII. Conclusions

The development of electronic and other analytical techniques has led to significant advances in the understanding of the behavioural adaptations in bivalve molluscs to adverse environmental changes. It is evident that bivalves can distinguish between favourable and unfavourable environmental conditions and make an appropriate behavioural response either to minimize the harmful effects of unfavourable conditions or to exploit favourable conditions. Many bivalves studied so far utilize valve closure as a protective mechanism to counteract transient or recurrent short-term adverse environmental changes, and they are capable of sensing immediate changes in environmental conditions. Sensory receptors are present on the marginal lobes of the mantle and the siphons, and changes in the medium are detected even when the valves are apparently closed. During periods of valve closure, bivalve molluscs are capable of sustaining basal metabolism using anaerobic respiration. The mobilization of bound calcium from the shell is also of great significance to an organism in order to buffer the end products of anaerobic respiration and hence minimize the toxic effects of metabolites. Although valve closure allows the organism to overcome short-term adverse changes in environmental conditions, it is of no survival value to long-term or persisting environmental changes.

Acknowledgments

Our gratitude is expressed to the Natural Environment Research Council (1978-1982), the Leverhulme Trust (1983-1985), and The Nuffield Foundation (April-June, 1983) for support

EFFECTS O F STRESS ON MARINE BIVALVE MOLLUSCS I83

and to Professors E . G. Cutter and D. M. Guthrie for the generous hospitality of the Botany and Zoology Departments, University of Manchester. Sincere gratitude is expressed by H.B.A. to Dr. M. J. Earnshaw, a friend and colleague, for his constant encouragement, advice, and moral support. We would like to thank Mrs. S. E. Hardman for patiently typing the manuscript.

References

Abel, P. D. (1976). Effects of some pollutants on the filtration rate of Mytilus. Marine Pollution Bulletin 7, 228-23 1 .

Adragna, P. J., and Privitera, C. A. (1978). Zinc effects on fathead minnow cell cultures. Comparative Biochemistry and Physiology 60C, 159-163.

Ahmad, T. A, , and Chaplin, A. E. (1977). The intermediary metabolism of Mytilus edulis (L.) and Cerastoderma edule (L.) during exposure to the atmosphere. Biochemical Soci- ety Transactions 5 , 1320-1323.

Ahsanullah, M. (1976). Acute toxicity of cadmium and zinc to seven invertebrate species from Western Port, Victoria. Australian Journal of Marine and Freshwater Research 27, 187-196.

Akberali, H. B. (1978). Behaviour of Scrobicularia plana (da Costa) in water of varying salinities. Journal of Experimental Marine Biology and Ecology 33, 227-249.

Akberali, H. B. (1980a). Problems of haemolymph sampling in bivalve molluscs. Marine Biology Letters I, 161-166.

Akberali, H. B. (1980b). 45Calcium uptake and dissolution in the shell of Scrobicularia plana (da Costa). Journal of Experimental Marine Biology and Ecology 43, 1-9.

Akberali, H. B. (1981). Effects of copper (Cuz+) on an isolated tissue preparation from the bivalve Scrobiclilaria plana (da Costa). Journal of Experimental Marine Biology and Ecology 52, 1 15-120.

Akberali, H. B., and Black, J . E. (1980). Behavioural responses of the bivalve Scrobicularia plana (da Costa) to short-term copper (Cu2+) concentrations. Marine Enuironmental Re- search 4, 97-107.

Akberali, H. B., and Davenport, J . (1981). The responses of the bivalve Scrobicularia plana (da Costa) to gradual salinity changes. Journal of Experimental Marine Biology and Ecology 53, 251-259.

Akberali, H. B., and Davenport, J. (1982). The detection of salinity changes by the marine bivalve mollusc Scrobicularia plana (da Costa) and Mytilus edulis (L.). Journal of Experi- mental Marine Biology and Ecology 58, 59-79.

Akberali, H. B., and Earnshaw, M. J. (1982a). Studies on the effects of zinc on the respiration of mitochondria from different tissues in the bivalve mollusc Mytilus edulis (L.). Comparative Biochemistry and Physiology 72C, 149-152.

Akberali, H. B., and Earnshaw, M. J. (1982b). The mechanism of calcium transport in mitochondria isolated from the marine mussel, Mytilus edulis (L.). Cell Calcium 3, 55-66.

Akberali, H. B., and Earnshaw, M. J. (1982~). A possible role of mitochondria1 Ca" in the contraction of the mollusc siphon induced by Cuz+. Comparative Biochemistry and Physi-

Akberali, H. B., and Trueman, E . R. (1979). p 0 2 and pC02 changes in the mantle cavity of Scrobicularia plana (Bivalvia) under normal and stress conditions. Estuarine and Coastal Marine Science 9, 499-507.

ology 73C, 395-398.


Akberali, H. B., Maniott, K. R. M., and Trueman, E. R. (1977). Calcium utilization during anaerobiosis induced by osmotic shock in a bivalve mollusc. Nature (London) 266, 852- 853.

Akberali, H. B., Wong, T. M., and Trueman, E. R. (1981). Behavioral and siphonal tissue responses of Scrobicularia plana (Bivalvia) to zinc. Marine Environmental Research 5 , 25 1-264.

Akberali, H. B., Trueman, E. R., and Earnshaw, M. J . (1982a). The influence of external calcium in facilitating copper-induced contraction in the isolated siphon of Scrobicularia plana (Bivalvia). Journal of Molluscan Studies 48, 96-97.

Akberali, H. B., Trueman, E. R., Black, J. E. , and Hewitt, C. (1982b). The responses of the estuarine bivalve mollusc Scrobicularia to the first hydrolytic product of the insecticide SevinR. Estuarine Coastal and Shelf Science 15, 415-421.

Akberali, H. B . , Brear, K., and Currey, J . D. (1983). Mechanical and morphological properties of the shell of Scrobicularia plana (da Costa) under normal and stress conditions. Journal of Molluscan Studies 49, 93-97.

Akberali, H. B., Earnshaw, M. J., and Marriott, K. R. M. (1984). The action of heavy metals on the gametes of the marine mussel, Mytilus edulis (L.). I. Copper-induced uncoupling of respiration in the unfertilized eggs. Comparatiue Biochemistry and Physiology 77C, 289- 294.

Akberali, H. B., Earnshaw, M. J., and Marriott, K. R. M. (1985). The action of heavy metals on the gametes of the marine mussel, Mytilus edulis (L.). 11. Uptake of copper and zinc and their effect on respiration in the sperm and unfertilized egg. Marine Enuironmen- tal Research, in press.

Alnaes, E., and Rahamimoff, R. (1975). On the role of mitochondria in transmitter release from motor nerve terminals. Journal of Physiology (London) 248, 285-306.

Alyakrynskaya, J . 0. (1972). Biochemical adaptations of aquatic molluscs to an air environment. Zoologicheskii Zhurnal51, 1630-1636.

Ansell, A. D. (1967). Leaping movements in two species of Asaphidae (Bivalvia). Proceed- ings of Malacological Society 37, 395-398.

Ansell, A. D., and Trueman, E. R. (1973). The energy cost of migration of the bivalve Donax on tropical sandy beaches. Marine Behauiour and Physiology 2, 21-22.

Armstrong, D. A,, and Millemann, R. E. (1974). Effects of the insecticide Sevin and its first hydrolytic product, 1-naphthol, on some early development stages of the bay mussel Mytilus edulis. Marine Biology 28, 11-15.

Badman, D. G. (1974). Changes in activity in freshwater clam in response to oxygen concentration. Comparative Biochemistry and Physiology 47A, 1265-1271.

Badman, D. G., and Chin, S. L. (1973). Metabolic responses of the freshwater bivalve Pleurobema coccineum (Conrad) to anaerobic conditions. Comparative Biochemistry and Physiology 44B, 27-32.

Barber, V. C., and Wright, D. E. (1969). The fine structure of the eye and optic tentacle of the mollusc Cardium edule. Journal of Ultrastructural Research 26, 5 15-528.

Barber, V. C., Evans, E. M., and Land, M. F. (1967). The fine structure of the eye of the mollusc Pecten maximus. Zeitschrft fur Zellforschung 76, 295-3 12.

Barnes, G. E. (1955). The behaviour ofAnodonta cygea L. and its neurophysiological basis. Journal of Experimental Biology 32, 158-174.

Barnes, H., and Barnes, M. (1958). Note on the opening response of Balanus balanoides (L.) in relation to salinity and certain inorganic ions. Ver6flentlichungen des Instituts fur Meeresforschung in Bremerhauen 5 , 160-164.

Barnes, T. C. (1939). Experiments on Ligia in Bermuda. VI. Reactions to common cations. Biological Bulletin, Marine Biological Laboratory, Woods Hole 76, 121 - 126.


Barnes, T. C. (1940). Experiments on Ligia in Bermuda. VII. Further effects of Na+, NH’, and Mgz+. Biological Bulletin, Marine Biological Luhorafory, Woods Hole 78, 35-41.

Bayne, B. L. (1971). Ventilation, the heart beat, and oxygen uptake by Mytilus edulis (L.) in declining oxygen tension. Comparative Biochemistry and Physiology 40A, 1065-1085.

Bayne, B. L. (1972). Some effects of stress in the adult on the larval development of Mytilus edulis. Nature (London) 237, 459.

Bayne, B. L. (1975). Aspects of physiological condition in Mytilus edulis L., with special reference to the effects of oxygen tension and salinity. Proceedings of the 9th European Marine Biology Symposium (H. Barnes, ed.), pp. 213-238. Aberdeen Univ. Press, Scot- land.

Bayne, B. L., ed. (1976). “Marine Mussels: Their Ecology and Physiology.” International Biological Programme Synthesis, Vol. 10. Cambridge Univ. Press, London and New York.

Bayne, B. L., and Livingstone, D. R. (1977). Responses of Mytilus edulis L. to low oxygen tension: Acclimation of the rate of oxygen consumption. Journal of Comparative Physiol- ogy 114B, 129-142.

Bayne, B. L., Thompson, R. S. , and Widdows, J . (1973). Some effects of temperature and food on the rate of oxygen consumption by Mytilus edulis L. I n “Effects of Temperature of Ectothermic Organisms” (W. Wieser, ed.), p. 21 1 . Springer-Verlag, Berlin and New York.

Bayne, B. L., Gabbot, P. A,, and Widdows, J. (1975). Some effects of stress in the adult on the eggs and larvae of Myrilus edulis (L.). Journal of the Marine Biological Association of the United Kingdom 55, 675-689.

Bayne, B. L., Thompson, R. J . , and Widdows, J. (1976a). Physiology: 1. In “Marine Mussels: Their Ecology and Physiology” (B. L. Bayne, ed.), International Biological Programme Synthesis, Vol. 10, pp, 121-206. Cambridge Univ. Press, London and New York.

Bayne, B. L., Bayne, C. J. , Carefoot, T. C., and Thompson, R. J . (1976b). The physiological ecology of Mytilus ralifornianus Conrad. 2. Adaptations to low oxygen tension and air exposure. Oecologia (Berlin) 22, 229-250.

Bayne, B. L., Holland, D. L., Moore, M. N., Lowe, D. M., and Widdows, J. (1978). Further studies on the effects of stress in the adult on the eggs of Mytilus edulis. Journal of the Marine Biological Association of the United Kingdom 58, 825-842.

Bayne, B. L., Moore, M. N., Widdows, J., Livingstone, D. R . , and Salkeld, P. (1979). Measurement of the responses of individuals to environmental stress and pollution: Stud- ies with bivalve molluscs. Philosophical Transactions of the Royal Society, London, Series B 286, 561-563.

Bayne, B. L., Clarke, K. R., and Moore, M. N. (1981). Some practical considerations in the measurement of pollution effects on bivalve mollusk and some possible ecological consequences. Aquatic Toxicology 1, 159-174.

Beedham, G . E., and Owen, G. (1965). The mantle and shell of Solemya parkinsoni (Proto- branchia: Bivalvia). Proceedings of the Zoological Society of London 145, 405-430.

Bettison, J. C., and Davenport, J. (1976). Salinity preference in Gammarid amphipods with special reference to Marinogammarus marinus (Leach). Journal of the Marine Biological Association of the United Kingdom 56, 135-142.

Black, J. E. (1983). “Behavioural and Sensory Aspects of the Responses of Scrobicularia plana (da Costa) to Stress.” Ph.D. thesis, University of Manchester.

Blandford, P., and Little, C. (1983). Salinity detection by Hydrobia ulvae (Pennant) and Potamopyrgusjenkinsi Smith (Gastropoda, Prosobranchia). Journal of Experimental Ma- rine Biology and Ecology 68, 25-38.


Bone, Q., Pulsford, A,, and Chubb, A. D. (1981). Squid mantle muscle. Journal o f t h e Marine Biological Association of the United Kingdom 61, 327-342.

Booth, C. E., and Mangum, C. P. (1978). Oxygen uptake and transport in the lamellibranch mollusc Modiolus demissus. Physiological Zoology 51, 17-32.

Boyden, C. R. (1972a). The behaviour, survival, and respiration of the cockles Cerasto- derma edule and C . glaucum in air. Journal of the Marine Biological Association of the United Kingdom 52, 661-680.

Boyden, C. R. (1972b). Aerial respiration of the cockle Cerastoderma edule in relation to temperature. Comparative Biochemistry and Physiology 42A, 697-7 12.

Brafield, A. E . (1964). The oxygen content of interstitial water in sandy shores. Journal of -Animal Ecology 33, 97-116.

Brand, A. R. (1976). Heart action of the freshwater bivalve Anodonta anatina during activity. Journal of Experimental Biology 65, 685-698.

Brand, A. R., and Roberts, D. (1973). The cardiac responses of the scallop Pecten maximus (L.) to respiratory stress. Journal of Experimental Marine Biology and Ecology 13,29-43.

Brand, A. R., and Taylor, A. C. (1974). Pumping activity ofArctica islandica (L.) and some other common bivalves. Marine Behauiour and Physiology 3, 1-15.

Brereton, A , , Lord, H . , and Webb, J. S. (1973). Effect of zinc on growth and development of larvae on the Pacific oyster, Crassostrea gigas. Marine Biology 19, 96-10],

Brown, A. C, (1982). The biology of sandy-beach whelks of the genus Bullia (Nassaridae). Oceanography and Marine Biology Annual Review 20, 309-361.

Brown, B. E., and Newell, R. C. (1972). The effect of copper and zinc on the metabolism of the mussel Mytilus edulis. Marine Biology 16, 108-118.

Brown, F. A., Jr. (1954). Resistant activity rhythms in the oyster. American Journal of Physiology 178, 510-514.

Bryan, G. W. (1973). The occurrence and seasonal variation of trace metals in the scallops Pecten maximus (L.) and Chlamys opercularis (L.). Journal of the Marine Biological Association of the United Kingdom 53, 145-166.

Bryan, G. W. (1976). Some aspects of heavy metal tolerance in aquatic organisms. In “Effects of Pollutants on Aquatic Organisms” (A. P. M. Lockwood, ed.), pp. 7-34. Cambridge Univ. Press, London and New York.

Bryan, G. W. (1979). Bioaccumulation of marine pollutants. Philosophical Transactions of the Royal Society, London, Series B 286, 483-505.

Bryan, G. W., and Gibbs, R. (1983). Heavy metals in the Fa1 Estuary, Cornwall: A study of long-term contamination by mining waste and its effects on estuarine organisms. Occa- sional Publication No. 2, p. 1 1 1. Marine Biological Association of the United Kingdom, Plymouth.

Bryan, G. W., and Uysal, H. (1978). Heavy metal in the burrowing bivalve Scrobicularia plana from the Tamar Estuary in relation to environmental levels. Journal of the Marine Biological Association of the United Kingdom 58, 89-108.

Bullock, T. H., and Horridge, G. A., eds. (1965). In “Structure and Function in the Nervous Systems of Invertebrates,” Vol. 1 I . Freeman, San Francisco.

Bygrave, F. L. (1977). Mitochondria1 calcium transport. I n “Current Topics in Bioenerge- tics’’ (D. R. Sanadi, ed.), Vol. 6, pp. 259-318. Academic Press, New York.

Bygrave, F. L. (1978). Mitochondria and the control of intracellular calcium. Biological Review 53, 43-79.

Calabrese, A,, and Nelson, D. A. (1974). Inhibition of embryonic development of the hard clam, Mercenaria mercenaria, by heavy metals. Bulletin of Enuironmental Contamina- tion and Toxicology 11, 92-97.

Calabrese, A., Collier, R. S., Nelson, D. A., and MacInnes, J. R. (1973). The toxicity of


heavy metals to embryos of the American oyster Crassostrea virginica. Marine Biology

Calabrese, A., MacInnes, J. E., Nelson, D. A., and Miller, J. E. (1977). Survival andgrowth of bivalve larvae under heavy metal stress. Marine Biology 41, 179-184.

Calabrese, A., MacInnes, J. R., Nelson, D. A., Greig, R. A., and Yevich, P. P. (1984). Effects of long-term exposure to silver and copper on growth, bioaccumulation, and histopathology in the blue mussel Mytilus edulis. Marine Environmental Research 11,

Carafoli, E., and Crompton, M. (1978a). The regulation of intracellular calcium. Current Topics in Membrane Transport 10, 151-216.

Carafoli, E., and Crompton, M. (1978b). The regulation of intracellular calcium by mitochondria. Annals of New York Academy of Science 307, 269-283.

Carmichael, H. G. (1980). Metals in the molluscan kidney: Uptake and subcellular distribution of ‘ T d , 54Mn and 65Zn by the clam Mercenaria mercenaria. Comparative Biochemis- try and Physiology 65A, 203-206.

Carpene, E., and George, S. G. (1981). Absorption of cadmium by gills of Mytilus edulis (L.). Molecular Physiology 1, 23-34.

Cawthorne, D. F. (1979). “Some effects of Fluctuating Temperature and Salinity upon Cirrepedes.” Ph.D. thesis, University of Wales.

Chapman, G., and Newell, G. E. (1956). The role of the body fluid in the movement of soft-bodied invertebrates. 11. The extension of the siphons of Mya arenaria L. and Scrobi- cularia plana (da Costa). Proceedings of the Royal Society, London, Series B 145, 564- 580.

Chen, C., and Awapara, J. (1969). Intracellular distribution of enzymes catalyzing succinate production from glucose in Rangia mantle. Comparative Biochemistry and Physiology 30,

Chipman, W. A., and Hopkins, J. G. (1954). Water filtration by the bay scallop Pecten irradians, as observed with the use of radioactive plankton. Biological Bulletin, Marine Biological Laboratory, Woods Hole 107, 80-91.

Coleman, N. (1972). “Some Responses of Mytilus edulis L. and Modiolus modiolus L. to Aerial Exposure.” Ph.D. thesis, University of Manchester.

Coleman, N. (1973). The oxygen consumption of Mytilus edulis in air. Comparative Bio- chemistry and Physiology 45A, 393-402.

Coleman, N. (1974). A review of heart rate in the bivalve molluscs. Oceanography and Marine Biology Annual Review 12, 301-313.

Coleman, N. (1976). The aerial respiration of Modiolus modiolus. Comparative Biochemis- try and Physiology 54A, 401-406.

Coleman, N., and Trueman, E. R. (1971). The effect of aerial exposure on the activity of the mussels Mytilus edulis (L.) and Modiolus modiolus (L.). Journal of Experimental Marine Biology and Ecology 7, 295-304.

Collip, J. B. (1920). Studies on molluscan coelomic fluid. Effect of change in environment on the carbon dioxide content of the coelomic fluid. Anaerobic respiration in Mya arenaria. Journal of Biological Chemistry 45, 23-49.

Collip, J. B. (1921). A further study of the respiratory processes in Mya arenaria and other marine molluscs. Journal of Biological Chemistry 49, 297-310.

Coombs, T. L. (1980). Heavy metal pollutants in the aquatic environment. In “Animals and Environmental Fitness” (R. Giles, ed.), pp. 283-302. Pergamon, Oxford.

Comer, E. D. S., and Sparrow, B. W. (1956). The mode of action of a toxic agent. I . Observation on the poisoning of certain crustaceans by copper and mercury. Journal of the Marine Biological Association of the United Kingdom 57, 63-74.

18, 162-166.

253-274.

727-737.

I88 H . B . AKBERALI AND E . R. TRUEMAN

Coughlan, J. (1969). The estimation of filtering rate from the clearance of suspension. Marine Biology 2, 356-358.

Crenshaw, M. A. (1972). Inorganic composition of mulluscan extrapallial fluid. Biological Bulletin, Marine Biological Laboratory, Woods Hole 143, 506-512.

Crenshaw, M. A., and Neff, J . M. (1969). Decalcification at the mantle-shell interface in molluscs. American Zoologist 9, 881-885.

Cunningham, P. A. (1979). The use of bivalve molluscs in heavy metal pollution research. In “Marine Pollution: Functional Responses” (W. B. Vernberg, A. Calabrese, F. P. Thur- berg, and F. J. Vernberg, eds.), pp. 183-221. Academic Press, New York.

Cunningham, P. A., and Tripp, M. R. (1975). Factors affecting accumulation and removal of mercury from tissues of the American oyster, Crassostrea uirginica. Marine Biology 31, 311-319.

Dakin, W. J. (1910). The visceral ganglion of Pecten with some notes on the physiology of the nervous system, and an inquiry into the innervation of the osphradium in the Lamelli- branchia. Mitteilungen aus der Zoologischen Station zu Neapel20, 1-40.

Davenport, J. (1972). Salinity tolerance and preference in the porcelain crabs Porcellana platycheles and Porcellana longicornis. Marine Behaviour and Physiology 1, 123-138.

Davenport, J. (1976). A comparative study of the behaviour of some balanornorph barnacles exposed to fluctuating seawater concentrations. Journal of the Marine Biological Associa- tion of the United Kingdom 56, 889-907.

Davenport, J. (1977). A study of the effects of copper applied continuously and discontinu- ously to specimens of Mytilus edulis (L.) exposed to steady and fluctuating salinity levels. Journal of the Marine Biological Association of the United Kingdom 57, 63-74.

Davenport, J. (1979). The isolation response of mussels (Myrilus edulis L.) exposed to falling seawater concentrations. Journal of the Marine Biological Association of the United Kingdom 59, 123-132.

Davenport, J. (1981). The opening response of mussels (Mytilus edulis L.) exposed to rising seawater concentrations. Joctmal of the Marine Biologic,al Association of the United Kingdom 61, 667-678.

Davenport, J . (1982). Environmental simulation experiments on marine and estuarine animals. Advances in Marine Biology 19, 133-256.

Davenport, J . , and Carrion-Cotrina, M. (1981). Responses of the mussel Mytilus edulis L. to simulated subarctic tide pool conditions. Journal of Thermal Biology 6, 257-265.

Davenport, J., and Manley, A. (1978). The detection of heightened seawater copper concentrations by the mussel Mytilus edulis. Journal of the Marine Biological Association of the United Kingdom 58, 843-850.

Davenport, J., Gruffydd, L. D., and Beaumont, A. R. (1975). An apparatus to supply water of fluctuating salinity and its use in a study of the salinity tolerances of larvae of the scallop Pecten maximus L. Journal of the Marine Biological Association of the United Kingdom 55, 391-409.

Davies, A. G . (1978). Pollution studies with marine plankton. Part 11. Heavy metals. Ad- vances in Marine Biology 15, 382-478.

De Fur, P. L. , and Mangum, C. P. (1979). The effects of environmental variables on the heart rates of invertebrates. Comparative Biochemistry and Physiology 62A, 283-294.

Delhaye, W., and Cornet, D. (1975). Contribution to the study of the effects of copper on Mytilus edulis during the reproductive period. Comparative Biochemistry and Physiology 50A, 511-518.

De Mellon, F . (1965). Nerve pathways and reflex siphon withdrawal in the surf clam. Journal of Experimenral Biology 43, 455-472.

De Vooys, C. G . N. (1976). The influence of temperature and time of year on the oxygen uptake of the sea mussel, Mytilus edulis. Marine Biology 36, 25-30.


De Zwaan, A. (1977). Anaerobic energy metabolism in bivalve molluscs. Oceanography and Marine Biology Annual Review 15, 103-187.

De Zwaan, A,, and Van Marrewijk, W. J. A. (1973). Anaerobic glucose degradation in the sea mussel, Mytilus edulis. Comparative Biochemistry and Physiology 44B, 429-439.

De Zwaan, A., and Wijsman, T. C. M. (1976). Anaerobic metabolism in Bivalvia (Mollusca). Characteristics of anaerobic metabolism. Comparative Biochemistry and Physiology 54B, 313-324.

De Zwaan, A., and Zandee, D. I. (1972). The utilization of glycogen and accumulation of some intermediates during anaerobiosis in Myrilus edulis (L.). Comparative Biochemistry and Physiology 43B, 47-54.

De Zwaan, A,, Van Marrewijk, W. J. A., and Holwerda, D. A. (1973). Anaerobic carbohy- drate metabolism in the sea mussel Mytilus edulis (L.). Netherlands Journal of Zoology 23, 225-228.

De Zwaan, A., De Bont, A. M. T., and Klutymans, J. H. F. M. (1975). Metabolic adaptations on the aerobic-anaerobic transition in the sea mussel, Mytilus edulis L. In “Pro- ceedings of the Ninth European Marine Biological Symposium” (H. Barnes, ed.), pp. 121-138. Aberdeen University Press, Scotland.

Dicks, B. M. (1972). “The Occurrence of Rhythm in the Activity of the Marine Bivalve Mya arenaria (L.) and Some Aspects of its Importance and Control.” Ph.D. thesis, University of Reading.

Dietz, T. H., and Tomkins, R. U. (1980). The effect of temperature on heart rate of the freshwater mussel, Ligumia subrostrata. Comparative Biochemistry and Physiology 67A, 269-27 I .

Dixon, M., and Webb, E. C. (1967). “Enzymes.” Longmans, London. Dotterweich, H., and Elssner, E. (1935). Die Mobilisierung des Schalenkalkes fur die Reak-

tions-Regulation der Muscheln (Anodonfa cygnea). Biologisches Zentralblatt 55, 138- 163.

D’Silva, C., and Kureishy, T. W. (1978). Experimental studies on the accumulation of copper and zinc in the green mussel. Marine Pollution Bulletin 9, 187-190.

Dugal, L. P. (1939). The use of calcareous shell to buffer the product of anaerobic glycolysis in Venus (Mercenaria) mercenaria. Journal of Cellular Comparative Physiology 13,235- 251.

Dykens, J. A., and Mangum, C. P. (1979). The design of cardiac muscle and the mode of metabolism in molluscs. Comparative Biochemistry and Physiology 62A, 549-554.

Earl], R. (1975a). “Some Aspects of Heart Rate and Activity in Bivalve Molluscs in the Laboratory and the Natural Environment.” Ph.D. thesis, University of Manchester.

Earll, R. (3975b). Temporal variation in the heart activity of Scrobicularia plana (da Costa) in constant and tidal conditions. Journal of Experimental Marine Biology and Ecology 15,

Earl], R. C., and Evans, D. T. (1974). Selector systems in recording physiological and behavioural activity in sedentary aquatic animals. Journal of Experimental Marine Biol- ogy and Ecology 15, 35-41.

Edwards, R. R. C., and Steele, J. H. (1968). The ecology of 0-group plaice and common dabs at Loch Eve. I. Populations and Food. Journal of Experimental Marine Biology and Ecology 2, 215-238.

Eisler, G. (1977). Acute toxicities of selected heavy metals to the soft shell clam, Mya arenaria. Bulletin of Environmental Contamination and Toxicology 17, 137-145.

Eisler, R. (1979). Behavioural responses of marine poikilotherms to pollutants. Philosophi- cal Transactions of the Royal Society, London, Series B 286, 507-521.

Engel, D. W., and Fowler, B. A. (1979). Copper- and cadmium-induced changes in the metabolism and structure of molluscan gill tissue. In “Marine Pollution: Function Re-

35-41,


sponses” (W. B. Vernberg, A. Calabrese, F. P. Thurnberg, and F. J . Vernberg, eds.), pp. 239-256. Academic Press, New York.

Foster-Smith, R. L. (1976). Some mechanisms for the control of pumping activity in bivalves. Marine Behaviour and Physiology 4, 41-60.

Freeman, R. F. H., and Rigler, F. H. (1956). The responses of Scrobicularia plana to osmotic pressure changes. Journal of the Marine Biological Association of the United Kingdom 36, 553-567.

Frenkiel, L. (1980). Les organs sensoriels chez les lamellibranches. Annales de Biologie, Paris 19, 147-162.

Frenkiel, L., and Moueza, M. (1980). Ciliated receptors in the cruciform muscle sense organ of Scrobicularia plana (da Costa). Zeitschrift fur mikroskopisch-anatomische Forschung

Gabbot, P. A., and Bayne, B. L. (1973). Biochemical effects of temperature and nutritive stress on Mytilus edulis (L.). Journal of the Marine Biological Association of the United Kingdom 53, 269-286.

Galtsoff, P. S. (1926). New methods to measure the rate of flow produced by the gills of oysters and other molluscs. Science 63, 233-234.

Galtsoff, P. S. (1928). Experimental study of the function of the oyster gill and its bearing on the problems of oyster culture and sanitary control of the oyster industry. Bulletin of the United States Fisheries 44, 1-39.

George, S. G., and Pine, B. J. S. (1980). Metabolism of zinc in the mussel, Mytilus edulis (L.): A combined ultrastructural and biochemical study. Journal ofthe Marine Biological Association of the United Kingdom 53, 269-286.

George, S. G., Carpene, E. , Coombs, T. L., Ovenell, J . , and Youngson, A. Y. (1979). Characterization of cadmium-binding proteins from mussels, Mytilus edulis (L.), exposed to cadmium. Biochimica et Biophysica Acta 580, 225-233.

Gilmour, T. H. J. (1963). A note on the tentacles of Lima hians (Gmelin) (Bivalvia). Pro- ceedings of the Malacological Society of London 35, 82-85.

Graham, A. (1934). The cruciform muscle of lamellibranchs. Proceedings of the Royal Society, Edinburgh, Series B 54, 17-30.

Greig, R. A , , Nelson, B. A., and Nelson, D. A. (1975). Trace metal content in the American oyster. Marine Pollution Bulletin 6,72-73.

Gross, W. J. (1957). A behavioral mechanism for osmotic regulation in a semiterrestrial crab. Biological Bulletin, Marine Biological Laboratory, Woods Hole 113, 268-274.

Gruffydd, L. D., Huxley, R., and Crisp, D. J. (1984). The reduction of growth of Mytilus edulis in fluctuating salinity regimes measured using laser diffraction patterns and the exaggeration of this effect by using tap water as the diluting medium. Journal of the Marine Biological Association of the United Kingdom 64, 401-410.

Hammen, C. S. (1969). Metabolism of the oyster, Crassostrea uirginica. American Zoolo- gist 9, 309-318.

Helm, M. M., and Trueman, E. R. (1967). The effect of exposure on the heart rate of the mussel, Mytilus edulis (L.). Comparative Biochemistry and Physiology 21, 171-177.

Hers, M.-J. (1943). Relation entre respiration et circulation chez Anodonta cygnea L. An- nales de la SociPtP Royale Zoologique de Belgique 74, 45-54.

Heusner, A. A., and Enright, J. T. (1966). Long-term activity recording in small aquatic animals. Science 154, 532-533.

Hildreth, D. I. (1980). Bioseston production by Mytilus edulis and its effect in experimental systems. Marine Biology 55, 309-315.

Hochachka, P. W., and Mustafa, T. (1972). Invertebrate facultative anaerobiosis. Science

94, 881-894.

178, 1056-1060.

EFFECTS O F STRESS ON MARINE BIVALVE MOLLUSCS 191

Hochachka, P. W., Fields, J., and Mustafa, T. (1973). Animal life without oxygen. Basic biochemical mechanisms. American Zoologist 13, 543-555.

Hodgson. A. N. (1982). Some behavioural electrical responses of Scrobic~/i/rrritr plcma (Bi- valvia: Tellinacea) to siphonal wounding. Journal of’ Molluscan Studies 48, 87-94.

Hodgson, A. N., and Trueman, E. R. (1981). The siphons of Scrobicularia plana (Bivalvia: Tellinacea). Observation on movement and extension. Journal of Zoology (London) 194, 445-459.

Hodgson, A. N., Fielden, L., and Cross, R. H. M. (1982). The structure and distribution of peripheral ciliated receptor cells in bivalve molluscs. Proceedings of the Electron Micros- copy Society, Sourh Africa 12, 79-80.

Hoggarth, K. R., and Trueman, E. R. (1967). Techniques for recording the activity of aquatic invertebrates. Nature (London) 213, 1050-1051.

Huddart, H., and Price, N. R. (1976). Review. Calcium movements during excitation- contraction coupling in muscle cells. Comparative Biochemistry and Physiology 54, 375- 386.

Huddart, H., Hunt, S., and Oates, K. (1977). Calcium movements during contraction in molluscan smooth muscle, and the loci of calcium binding and release. Journal ofExperi- mental Biology 68,45-56.

Hughes, R. N. (1969). A study of feeding in Scrobicularia plana. Journal of the Marine Biological Association of the United Kingdom 49, 805-823.

Hughes, R. N. (1970). Population dynamics of the bivalves. Journal of Animal Ecology 39,

Ishida, A. (1935). Oxygen consumption of the oyster. Scientific Reports, Tokyo University (Fourth Series) 10, 619-638.

Janson, B. D. (1962). Salinity resistance and salinity preference of two oligochaetes Aktedri- lus monospermatecus Knoller and Marionina perclitellochaeta from the interstitial fauna of marine study beaches. Oikos 13, 293-305.

Jones, D. S., Williams, D. F., and Arthur, M. A. (1983). Growth history and ecology of the Atlantic surf clam, Spisula solidissima (Dillwyn), as revealed by stable isotopes and annual increments. Journal of Experimental Marine Biology and Ecology 73, 225-242.

Jergensen, B. C. (1966). “Biology of Suspension Feeding.” Pergamon, Oxford. Katz, B., and Miledi, R. (1965). The effect of calcium on acetylcholine release from motor

Katz, B., and Miledi, R. (1967). The timing of calcium action during neuromuscular trans-

Kawaguti, S., and Ikemoto, S. (1962). Electron microscopy on the mantle of a bivalve

Kennedy, D. (1960). Neural photoreception in a lamellibranch mollusc. Journal of General

Kinne, 0. (1971). In “Marine Ecology” (0. Kinne, ed.), Vol. 1, Part 2, pp. 821-995. Wiley (Interscience), New York.

Kleiner, D. (1974). The effect of Znz+ ions on mitochondrial electron transport. Archives of Biochemistry and Biophysics 165, 121-128.

Kleiner, D., and Jagow, G. von (1972). On the inhibition of mitochondria1 electron transport by Zn2+ ions. FEBS Letters 20, 229-232.

Koch, W. (1917). Der Herzschlag von Anodonta under natiirlichen and Kiinstlichen Be- dingungen. Pjugers Archiv f i r die Gesamte Physiologie der Menschen und der Tiere 116,

Kuenzler, E. J. (1961). Structure and energy flow of a mussel population in a Georgia salt

333-356.

nerve terminals. Proceedings of the Royal Society, London, Series B 161, 496-503.

mission. Journal of Physiology (London) 189, 535-544.

Fabulina nitidula. Biological Journal of Okayama University 8, 21-30.

Physiology 44, 277-299.

281-3 17.

marsh. Limnology and Oceanography 6, 191-204.

I 92 H. B . AKBERALI A N D E. R. TRUEMAN

Lagerspetz, K., and Mattila, M. (1961). Salinity reactions of some freshwater and brackish water crustaceans. Biological Bulletin, Marine Biological Laboratory, Woods Hole 120, 44-53.

Land, M. F. (1968). Functional aspects of the optical and retinal organization of the mollusc eye. Symposium of the Zoological Society of London 23, 75-96.

Laverack, M. S. (1968). On the receptors of marine invertebrates. Oceanography and Ma- rine Biology Annual Review 6, 249-324.

Lehninger, A. L. (1970). Mitochondria1 and Ca2+ ion transport. Biochemical Journal 119,

Lent, C . M. (1968). Air-gaping by the ribbed mussel, Modiolus demissus (Dillwyn): Effects and adaptive significance. Biological Bulletin, Marine Biological Laboratory, Woods Hole

Lent, C . M. (1969). Adaptations of the ribbed mussel, Modiolus demissus (Dillwyn), to the intertidal habitat. American Zoologist 9, 283-292.

Light, V. E. (1930). Photoreceptors in Mya arenaria with special reference to their distribution, structure, and function. Journal of Morphology and Physiology 49, 1-44.

Loosanoff, V. L. (1939). Effect of temperature upon shell movements of clams Venus mercenaria (L.). Biological Bulletin, Marine Biological Laboratory, Woods Hole 76, 171- 182.

Loosanoff, V. L. (1942). Shell movements of the edible mussel, Mytilus edulis (L.) in relation to temperature. Ecology 23, 23 1-234.

Loosanoff, V. L. , and Engle, J. B. (1947). Effect of different concentrations of microorgan- isms on the feeding of oysters (Ostrea uirginica). Fishery Bulletin of Fisheries Wildlife Service, United Stares 51, 31-57.

Lowe, D. M., Moore, M. N., and Bayne, B. L. (1982). Aspects of gametogenesis in the marine mussel Mytilus edulis. Journal of the Marine Biological Association of the United Kingdom 62, 133-146.

Lowe, G . A. (1974). Effect of temperature change on the heart rate of Crassostrea gigas and Mya arenaria (Bivalvia). Proceedings of the Malocological Society of London 41,29- 36.

Lowe, G . A,, and Trueman, E. R. (1972). The heart and water flow rates of Mya arenaria (Bivalvia: Mollusca) at different metabolic levels. Comparative Biochemistry and Physiol-

McGreer, E. R. (1979). Sublethal effects of heavy metal contaminated sediments on the bivalve Macoma balthica (L.). Marine Pollution Bulletin 10, 259-262.

McLachlan, A., and Young, N. (1982). Effects of low temperature on the burrowing rates of four sandy beach molluscs. Journal of Experimental Marine Biology and Ecology 65,275- 284.

McLusky, D. S. (1970). Salinity preference in Corophium volutator. Joinrnal of the Marine Biological Association of the United Kingdom 50, 747-752.

Manley, A. R. (1980). An apparatus for the preparation of varying concentrations of chemicals for toxicity tests with aquatic organisms. Water Research 14, 1023-1027.

Manley, A. R . (1983). The effects of copper on the behaviour, respiration, filtration, and ventilation activity of Mytilus edulis. Journal of the Marine Biological Association of the United Kingdom 63, 205-222.

Manley, A. R., and Davenport, J. (1979). The behavioural response of some marine bivalves to heightened seawater copper concentrations. Bulletin of Environmental Contamination and Toxicology 22, 739-744.

Manley, R., and George, W. 0. (1977). The occurrence of some heavy metals in populations

129- 138.

134,60-73.

ogy 41A, 487-494.


of the freshwater mussel Anodonla anatina (L.) from the river Thames. Environmental Pollution 14, 139-154.

Manley, A. R., Gruffydd, L. D., and Almada-Villela, P. C. (1984). The effect of copper and zinc on the shell growth of M y t h s edulis measured by a laser diffraction technique. Journal of the Marine Biological Association of the United Kingdom 64, 417-428.

Martin, J. M., Piltz, F. M., and Reish, D. J. (1975). Studies on the Mytilus edulis community in Alamitos Bay, California. V. The effects of heavy metals on byssal thread production. Veliger 18, 183-188.

Martin, M., Osborn, K. E., Billig, P., and Glickstein, N. (1981). Toxicities of 10 metals to Crassostrea gigas and Mytilus edulis embryos and Cancer magister larvae. Marine Pollu- tion Bulletin 12, 305-308.

Martin, M., Ichikawa, G., Goetzl, J., and Reyes de los, M. (1984). Relationship between physiological stress and trace toxic substances in the bay mussel, Mytilus edulis from San Francisco, California. Marine Enuironmental Research 11, 91- 110.

Maung-Myint, U., and Tyler, P. (1982). Effects of temperature, nutritive and metal stressors on the reproductive biology of Mytilus edulis. Marine Biology 67, 209-223.

Moir, A. J. G. (1977). Ultrastructural studies on the ciliated receptors of the long tentacles of the giant scallop Placopecten magellanicus (Gmelin). Cellular Tissue Research 184, 367- 380.

Moon, T. W., and Pritchard, A. W. (1970). Metabolic adaptations in vertically separated populations of Mytilus californianus Conrad. Journal of Experimental Marine Biology and Ecology 5 , 35-46.

Moore, M. N. (1977). Lysosomal responses of environmental chemicals in some marine invertebrates. In “Pollution Effects on Marine Organisms” (C. S. Giam, ed.), pp. 143- 154. Heath, Toronto.

Morton, B. S. (1969). Studies on the biology of Dreissena polymorpha. Part 11. Correlation of the rhythms of adductor activity, feeding, digestion, and excretion. Proceedings of the Malacological Society of London 38, 401-414.

Morton, B. S. (1970). The rhythm of adductor activity and quiescence in Anodonta cygnea L. and Unio pictorium L. and its biological significance. Forma Function 2, 110-120.

Morton, B. S. (1971). The diurnal rhythm and tidal rhythm of feeding and digestion in Ostrea edulis. Journal of Linnean Society 3, 329-342.

Morton, B. S. (1973). A new theory of feeding and digestion in the filter feeding Lamellibran- chia. Malacologia 14, 63-79.

Moueza, M., and Frenkiel, L. (1974). Contribution a I’Ctude des structures palleales des Tellinacea. Morphologie et structure du manteau de Donax trunchulus L. Proceedings of the Malacological Society of London 41, 1-19.

Newell, R. C. (1979). “Biology of Intertidal Animals,” pp. 505-673. Marine Ecological Surveys Ltd., Faversham, Kent.

Nicholls, D. G., and Crompton, M. (1980). Mitochondria1 calcium transport. FEES Letters

O’Dell, B. L., and Campbell, B. J. (1971). Trace elements: Metabolism and metabolic functions. In “Comprehensive Biochemistry” (M. Florkin and E. H. Stotz, eds.), Vol. 21, pp. 179-226. Elsevier, Amsterdam.

Odiete, W. 0. (1976a). The adductor rhythm of Scrobicularia plana (da Costa) and its ganglionic control. Proceedings of the Malacological Society of London 42, 409-430.

Odiete, W. 0. (1976b). Electrophysiological recordings from the adductor nerves of Scrobi- cularia plana (da Costa). Proceedings of the Malacological Society of London 42, 431- 450.

111, 261-268.


Odiete, W. 0. (1978). The cruciform muscle and its associated sense organ in Scrobicularia plana (da Costa). Journal of Molluscan Studies 44, 180-189.

Ohtake, H., Suyemitsu, T., and Koga, M. (1983). Sea urchin (Anthocidaris crassispina) egg zinc-binding protein. Biochemical Journal 211, 109- 118.

Olla, B. L., Bejda, A. J., and Pearson, W. H. (1983). Effects of oiled sediment on the burrowing behaviour of the hard clam, Mercenaria mercenaria. Marine Environmental Research 9, 183-193.

Owen, G., and McCrae, J. M. (1979). Sensory celUgland cell complexes associated with the pallial tentacles of the bivalve Lima hians (Gmelin), with a note of specialized cilia on the pallial curtain. Philosophical Transactions of the Royal Society, London, Series B 287, 46-62.

Pannella, G., and MacClintok, C. (1968). Biological environmental rhythms reflected in molluscan shell growth. Journal of Palaeontology 42, 64-80.

Parker, J. (1978). “Aspects of behavioural and physiological activity in Anodonta cygnea L.” Ph.D. thesis, University of Manchester.

Parr, D. R., and Harris, E. J. (1976). The effect of lead on the calcium-handling capcity of rat heart mitochondria. Biochemical Journal 158, 289-294.

PeCsi, T., and Saltinki, J . (1964). The role of pressure in the periodical changes of cardiac action in the freshwater mussel (Anodonta cygnea L.). Annales Instititti Biologici, Ti- hany, Hungaricae Academiae Scientiarum 31, 65-76.

Pentreath, R. J. (1973). The accumulation from water of h‘Zn, 54Mn, ’*Co and 59Fe by the mussel Mytilus edulis. Journal of the Marine Biological Association of the United King- dom 53, 127-143.

Perkins, E. J. (1979). The need for sublethal studies. Philosophical Transactions of the Royal Society, London, Series B 286, 425-440.

Petit, H., Davis, W. L., and Jones, R. G. (1978). Morphological studies on the mantle of the freshwater mussel Amblema (Unionidae):SEM. Tissue and Cell 10, 619-627.

Phelps, H. L., Hardy, J. T., Pearson, W. H., and Apts, C. W. (1983). Clam burrowing behaviour: Inhibition of copper-enriched sediment. Marine Pollution Bulletin 14, 452- 455.

Phillips, D. J. H. (1976). The common mussel Mytilus edulis as an indicator of zinc, cadium, lead, and copper, I . Effects of environmental variables on uptake of metal. Marine Biol-

Phillips, D. J. H. (1977). The use of biological indicator organisms to monitor metal pollution in marine and estuarine environments-A review. Environmental Pollution 13, 281-317.

Pichon, Y., Moueza, M., and Frenkiel, L. (1980). Mechanoreceptor properties of the sense organ of the cruciform muscle in a Tellinacean lamellibranch, Donax trunculus L.: An electrophysiological approach. Marine Biology Letters 1, 273-284.

Pickens, P. E. (1965). Heart rate of mussels as a function of latitude, intertidal height, and acclimation temperature. Physiological Ziiology 38, 390-405.

Pierce, S. K. (1971). Volume regulation and valve movements by marine mussels. Compara- tive Biochemistry and Physiology 39A, 103-1 17.

Portman, J. E. (1970). A discussion of results of acute toxicity tests with marine organisms using a standard method. F.A.O. Technical Conference on Marine Pollution and its Effects on Living Resources and Fishing. FIR: MPI70iE-31, 1-13.

Prior, D. J. (1972). Electrophysiological analysis of peripheral neurons and their possible role in the local reflexes of a mollusc. Journal of Experimental Biology 57, 133-145.

Pumphrey, R. J. (1938). The double innervation of muscles in the clam Mya arenaria. Journal of Experimental Biology 15, 500-505.

ogy 38, 59-69.


Purchon, R. D. (1968). “The Biology of the Mollusca.” Pergamon, Oxford. Purchon, R. D. (1971). Digestion in filter feeding bivalves-a new concept. Proceedings of

the Malacological Society of London 39, 253-262. Robinson, W. E., Wehling, W. E., and Morse, M. P. (1984). The effect of suspended clay on

feeding and digestive efficiency of the surf clam, Spisula solidissima (Dillwyn). Journal of Experimental Biology and Ecology 74, 1-12.

Roesijadi, G. (1980). Influence of copper on the clam Protothaca staminea: Effects on gills and occurrence of copper-binding proteins. Biological Bulletin, Marine Biological Labo- ratory, Woods Hole 158, 233-247.

Rothstein, A. (1959). Cell membrane as site of action of heavy metals. Federation Proceed- ings, Federation of American Societies for Experimental Biology 18, 1026- 1038.

Salinki, J. (1966a). Comparative studies on the regulation of the periodic activity in marine lamellibranch. Comparative Biochemistry and Physiology 18, 829-843.

Salanki, J. (196613). Daily activity rhythm of two Mediterranean lamellibranchia (Pecten jacobaens and Lithophaga ~ithophugu~ regulated by light-dark period. Annales Instituti Biologici, Tihany, Hurigaricae Academiae Scientiarum 33, 135- 142.

Salanki, J., Hiripi, L., and Nerncsok, J . (1974). Seasonal variations of activity level in the freshwater mussel, Anodonta cygnea L. Zoologische Jahrbiicher Abreilung Allgemeine Zoologie und Physiologie der Tiere 78, 369-377.

Schlieper, C. (1955). Die Regulation des Herzschlages der Miesmuschel Mytilus edulis L. bie geoffneten und bei geschlossen Schalen. Kieler Meeresforschung 11, 139- 148.

Schlieper, C. (1957). Comparative study of Asteras rubens and Mytilus edulis from the North Sea (3o%Os) and the western Baltic sea (15%0S). AnnPe Biologique, Paris 33, 117- 127.

Scott, D. M., and Major, C. W. (1972). The effect of copper (11) on survival, respiration, and heart rate in the common blue mussel Mytilus edulis. Biological Bulletin, Marine Biologi- cal Laboratory, Woods Hole 143, 679-688.

Seed, R. (1976). Ecology. In “Marine Mussels” (B. L. Bayne, ed.), pp. 13-65. International Biological Programme 10. Cambridge Univ. Press, London and New York.

Segal, E. (1956). Microgeographic variations and thermal acclimation in intertidal molluscs. Biological Bulletin, Marine Biological Laboratory, Woods Hole 111, 129- 152.

Segal, E. (1962). Initial response of the heart rate ofagastropod, Acmaea limatula, to abrupt changes in temperature. Nature (London) 195, 674-675.

Shapiro, E., Castellucci, V. F., and Kendel, E. R. (1980). Presynaptic inhibition in Aplysia involves a decrease in the Ca2+ current of the presynaptic neuron. Proceedings of the National Academy of Science, U.S.A. 77, 1185-1 189.

Shumway, S. E. (1977). Effect of salinity fluctuation on the osmotic pressure and Na’, Caz+, and M&+ ion concentrations in the haemolymph of bivalve molluscs. Marine Biology 41, 153- 177.

Simkiss, K. (1983). Lipid solubility of heavy metals in saline solution. Journal ofthe Marine Biological Association of the United Kingdom 63, 1-7.

Simkiss, K., Taylor, M., and Mason, A. Z. (1982). Review metal detoxification and bioaccumulation in molluscs. Marine Biology Letters 3, 187-201.

Stephens, P. J. (1978a). Mechanical and chemical sensitivity at the mantle edge of the file clam Lima scabra (Born). Marine Behaviour and Physiology 5, 79-90.

Stephens, P. J. (1978b). The sensitivity and control of the scallop mantle edge. Journal of Experimental Biology 75, 203-221.

Stephens, P. J., and Boyle, P. R. (1978). Escape response of the queen scallop Chlamys opercularis (L.) (Mollusca: Bivalvia). Marine Behauiour and Physiology 5, 103-1 13.


Stephenson, R. R., and Taylor, D. (1975). The influence of EDTA on the mortality and burrowing activity of the clam (Venerupis decussata) exposed to sublethal concentrations of copper. Bulletin of Environmental Contamination and Toxicology 14, 304-308.

Stewart, N. E., Milleman, R. E., and Breese, W. P. (1967). Acute toxicity of the insecticide SevinR and its hydrolytic product 1-naphthol to some marine organisms. Transactions of the American Fisheries Society 96, 25-30.

Stickel, W. B., and Ahokas, R. (1975). The effects of tidal fluctuation of salinity on the haemolymph composition of several molluscs. Comparative Biochemistry and Physiology

Stokes, T. M., and Awapara, J. (1968). Alanine and succinate as end products of glucose degradation in the clam Rangia cuneata. Comparative Biochemistry and Physiology 25, 883-892.

Stone, N. M. (1980). “Growth Line Patterns in Bivalve Molluscs: The Mechanism and Periodicity of Line Formation.” Ph.D. thesis, University of Manchester.

Swinehart, J . H., and Crowe, J. H. (1980). Effects of heavy metals on amino acid and divalent cation transport in gills of bivalve molluscs. American Zoologist 20, 73.5.

Taylor, A. C. (1976a). Burrowing behaviour and anaerobiosis in the bivalve Arctica islundica (L.). Journal of the Marine Biological Association of the United Kingdom 59, 95- 109.

Taylor, A. C. (1976b). The cardiac responses to shell opening and closure in the bivalve Arctica islundica (L.). Journal of Experimental Biology 64, 751-759.

Thomas, G. E., and Gruyffdd, L. D. (1971). The types of escape reactions elicited in the scallop Pecten maximus by selected sea star species. Marine Biology 10, 87-93.

Thomas, N. J. , Lasiak, T. A., and Naylor, E. (1981). Salinity preference behaviour in Carcinus. Marine Behaviour and Physiology 7 , 277-283.

Thompson, R. J., and Bayne, B. L. (1972). Active metabolism associated with feeding in the mussel Mytilus edulis L. Journal of Experimental Marine Biology and Ecology 9, 1 11- 124.

Trueman, E. R. (1966a). The dynamics of burrowing in Ensis (Bivalvia). Proceedings of the Royal Society, London, Series B 166,459-476.

Trueman, E. R. (1966b). The fluid dynamics of the bivalve molluscs Mya and Margaritifera. Journal of Experimental Biology 45,369-382.

Trueman, E. R. (1967). Activity and heart rate of bivalve molluscs in their natural habitat. Nature (London) 214, 832-833.

Trueman, E. R. (1971). The control of burrowing and the migratory behaviour of Donax denriculatus (Bivalvia: Tellinacea). Journal of Zoology (London) 165, 453-469.

Trueman, E. R. (1983a). Observations of the responses of the tropical surf clam Donax denticulatus to changes of temperature and salinity. Journal of Molluscan Studies 49, 242-243.

Trueman, E. R. (1983b). Burrowing and physiological responses of a burrowing bivalve to stress. In “Sandy Beaches as Ecosystems” (A. McLachlan and T. Erasmus, eds.), pp. 669-673. Dr. W. Junk, The Hague.

Trueman, E. R., and Akberali, H. B. (1981). Responses of an estuarine bivalve, Scrobicula- ria plana (Tellinacea) to stress. Malacologia 21, 15-21.

Trueman, E. R., and Lowe, G. A. (1971). The effect of temperature and littoral exposure on the heart rate of a bivalve mollusc, Zsognomon alatus, in tropical conditions. Comparative Biochemistry and Physiology 38A, 555-564.

Trueman, E. R., Blatchford, J. G., and Jones, H. D. (1973). Recordings of heart rate and activity of molluscs in their natural habitat. Malacologia 14, 377-383.

50A, 291-296.


Vainio, H., Mela, L., and Chance, B. (1970). Energy-dependent bivalent cation transloca- tion in rat liver mitochondria. European Journal of Biochemistry 12, 387-391.

Van Dam, L. (1935). On the utilization of oxygen by Mya arenaria. Journal of Experimental Biology 12, 86-94.

Vero, M., and Salanki, J. (1969). Inductive attenuator for continuous registration of rhyth- mic and periodic activity of mussels in their natural environment. Medical and Biological Engineering 7, 235-237.

Viarengo, A., Pertica, M., Mancinelli, G., Zanicchi, G., and Oruneso, M. (1980a). Rapid induction of copper binding proteins in the gills of exposed mussels. Comparative Bio- chemistry and Physiology 67C, 215-218.

Viarengo, A., Pertica, M., Mancinelli, G., Capelli, R., and Orunesu, M. (1980b). Effects of copper on the uptake of amino acids, on protein synthesis, and on ATP content in different tissues of Mytilus galloprovincialis Lam. Marine Environmental Research 4,

Viarengo, A., Zanicchi, G., Moore, M. N., and Orunesu, M. (1981). Accumulation and detoxification of copper by the mussel Mytilus galloprovincialis. A study of the subcellular distribution in the digestive gland cells. Aquatic Toxicology 1, 147-158.

Von Brand, T. (1946). Anaerobiosis in invertebrates. Biodynamica Monographs 4, pp. 328. Wald, G. (1964). The origins of life. Proceedings of the National Academy of Science,

U.S .A. 52, 595-611. Waldichuk, M. (1979). Review of the problems. Philosophical Transactions of the Royal

Society, London, Series B 286, 399-424. Walne, P. R. (1964). The culture of marine bivalve larvae. In “Physiology of Mollusca” (K.

M. Wilbur and C. M. Yonge, eds.), Vol. 1, pp. 197-210. Academic Press, New York. Walne, P. R. (1972). The influence of current speed, body size, and water temperature on the

filtration rate of five species of bivalves. Journal of the Marine Biological Association of the United Kingdom 52, 345-374.

Watling, H. (1981). The effects of metals on mollusc filtering rates. Transactions of the Royal Society of South Africa 44, 44 1-45 1 .

Widdows, J. (1973). Effect of temperature and food on the heart beat, ventilation, and oxygen uptake of Mytilus edulis. Marine Biology 20, 269-276.

Widdows, J. (1978). Physiological indexes of stress in Mytilus edulis. Journal of the Marine Biological Association of the United Kingdom 58, 125-142.

Widdows, J., Bayne, B. L., Livingstone, D. R., Newell, R. I. E., and Donkin, P. (1979). Physiological and biochemical responses of bivalve molluscs to exposure to air. Compara- tive Biochemistry and Physiology 62A, 301-308.

Wijsman, T. C. M. (1975). pH fluctuations in Mytilus edulis L. in relation to shell movements under aerobic and anaerobic conditions. In “Proceedings of the Ninth European Marine Biological Symposium” (H. Barnes, ed.), pp. 139-149. Aberdeen Univ. Press, Scotland.

Wilson, D. F., and Nystrom, R. A. (1968). Innervation and activity of nonstriated muscle in a pelecypod mantle. Comparative Biochemistry and Physiology 26, 663-673.

Wisely, B., and Blick, R. A. P. (1967). Mortality of marine invertebrate larvae in mercury, copper, and zinc solutions. Australian Journal of Marine and Freshwater Research 18, 63-72.

Yonge, C. M. (1948). Formation of siphons in Lamellibranchia. Nature (London) 161, 198. Yonge, C. M. (1949). On the structure and adaptations of the Tellinacea, deposit-feeding

Eulamellibranchia. Philosophical Transactions of the Royal Society, London, Series B

145-152.

234,29-76.


Yonge, C . M. (1957). Mantle fusion in the Lamellibranchia. Stazione Zoologica, Napoli XXIX, 151-171.

Zaba, B. N., and Hams , E. J. (1976). Uptake and effects of copper in rat liver mitochondria. Biochemical Journal 160, 707-7 14.

Zaba, B. N. , and Harris, E. J. (1978). Accumulation and effects of trace metal ions in fish liver mitochondria. Comparutiue Biochemistry and Physiology 61C, 89-93.

Zylstra, V. (1972). Distribution and ultrastructure of epidermal sensory cells in the freshwater snails Lymnea stagnalis and Biomphalaria pfeiffeeri. Netherlands Journal of Zool- O ~ Y 22, 283-298.

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