Osmotic Relations in Molluscan Contractile Tissues: I. Isolated Ventricle-Strip Preparations From Lamelli-branchs (Mytilus Edulis L., Ostrea Edulis L., Anodonta Cygnea L.)

A study of the osmotic pressure relations of rhythmic muscles from three polychaete species was published by Wells & Ledingham (1940). They drew attention to the physiological importance of rate of change of salinity, as distinct from its absolute value. To distinguish between these factors they made experiments of two types, the ‘constant exposure’, in which the osmotic pressure is suddenly changed to a new value at which it is then held constant, and the ‘drift’, in which it changes gradually at a controllable rate. The distinction between the actions of osmotic pressure per se and of osmotic pressure change was further emphasized during the course of the present work.

The genus Mytilus is very widely distributed while M. eduEs is found on both sides of the North Atlantic. The species occurs over a wide salinity range from full sea water down to below 15 % ^† (Haas, 1926; Dodgson, 1928; Percival, 1929; White, 1937; Conklin & Krogh, 1938).

The suggestion has been made that the individuals collected from the lowest salinities, in the Baltic, constitute a physiologically distinct race (Wells, Ledingham & Gregory, 1940). Experimentally, the animals have been acclimatized to external environments of down to 2% sea water (Dodgson, 1930; Topping & Fuller, 1942) and even to fresh water according to Beudant (1816). However, workers with M. galloprovincialis (which is sometimes regarded as a subspecies of M. edulis) and M. callfornianus have recorded somewhat less tolerance of dilution (Bouxin, 1931; Ricci, 1939; Fox, 1941). Several authorities have compared the osmotic pressure of the body fluids with that of the surrounding water; Monti (1914) found a slight degree of hypertonicity in sea water, but according to Schlieper (1929), Maloeuf (1937), Conklin & Krogh (1938) and Krogh (1938) there is no active regulation in external concentrations down to 29 % (Conklin & Krogh 1938) and 20% (Schlieper, 1929). There may be some degree of osmotic regulation at the very lowest salinities at which the mussels occur and to which they can be acclimatized; nevertheless, the demonstrated isotonicity over a wide range of external salinity suggests a considerable resistance of the tissues to osmotic variations.

The cardiac physiology of this species has been little studied. The rate of beat of the heart in situ varies from 10 to over 50 beats per minute, and is slowed by closing of the shell (Carlson, 1906; Field, 1922; Woortman, 1926; Diederichs, 1935). The heart of small specimens can be seen through the transparent shell, and Schlieper (1929) found by this means that transfer from an external concentration of 22% to one of 49% reduced the heart frequency to 62–84% °f previous value. As these results were got with whole animals, they may have been due to factors other than the direct action of salinity on the heart tissue.

M. edulis was obtained weekly from Plymouth, and kept in cool aerated aquarium sea water. The animal was removed entire from the shell, pinned ventral-side down in a dish of sea water and the roof of the pericardium removed. A thread was tied about each end of the ventricle and the auricles cut away, on one side the cut being extended right along the ventricle giving access to the rectum which was then removed. The ventricle was freed beyond the threads and transferred to the apparatus. The preparation consisted, therefore, of roughly the whole ventricle open along one side so that all parts were easily accessible to the bathing fluids. It was attached to an isotonic lever weighted to a tension of 100–150 mg. for specimens of 8–10 cm. shell length. After an hour or two of irregular activity, a regular rhythm was obtained in about 80% of the animals. Such preparations have been maintained active up to 3 days after mounting without aeration or addition of any nutrient; the medium was replaced by new, well-aerated sea water daily.

The preparations were mounted in the apparatus (usually in pairs) by attaching one thread to a fixed glass rod and the other to the kymograph lever. They were immersed in 250 ml. of medium in a beaker resting on a block of wood ; by removing the wood and replacing the beaker with one containing a fresh medium a very rapid and complete change was effected; the new bathing medium thus retained its composition and quickly reached all parts of the preparation. This will be referred to as the ‘Constant Exposure’ method following the terminology of Wells & Ledingham (1940). Dilution of sea water was made with aerated M/400-NaHCO₃ to maintain the pH between 7·8 and 8·2.

After settling down in Plymouth aquarium sea water (see Appendix), preparations exhibited a very regular rhythm of simple contractions and relaxations whose amplitude was usually steadily maintained for several hours; some gave an un-accountably fluctuating amplitude, particularly with respect to systolic level. The frequency ranged from 11 to 24 beats/min. between 10 and 16° C., the variation appearing to be due largely to differences between individuals.

When the beaker of sea water was replaced by another with the same fluid there was a sharp contraction of the preparation, followed by relaxation with gradual resumption of the beat to normal in 1–4 min. This effect was probably mechanical, arising from the extra tension on the tissue when the buoyant fluid medium was suddenly withdrawn; it will be termed the ‘mechanical effect of change’ (Fig. 1).

The effect of dilution was to produce a contraction following the mechanical effect of change; in cases of moderate dilution it was short-lived and may or may not have been accompanied by inhibition of the beat. This effect was called a ‘Shock Reaction’ and was followed by accommodation, the beat becoming adjusted in frequency and amplitude to those characteristic of the new medium. By analysing records at intervals during this accommodation it was found that a steady rhythm developed in 1–3 hr. after the change; this was called a ‘Long-term Reaction’. With more dilute media all these effects were correspondingly more pronounced; the contraction and inhibition phases lasted longer and a regular rhythm took up to 6 hr. to appear (Fig. 2).

Fig. 3 shows analyses of frequency for these preparations; the figures have been converted into percentages of those in the initial bathing medium. The lowest concentration of sea water in which even occasional recovery was found was 10%, though a number of other preparations failed to recover below 30%. In the case of the accommodated rhythm, frequency may be greater or less than that in 100% sea water until 40%, beyond which the trend is towards a decrease. There is no clear correlation of amplitude with salinity.

Those preparations which had accommodated to the diluted media were then replaced in 100% sea water. The shock reaction following mechanical effect of change was, in mild treatments, a rapid return to the normal tone level with inhibition of the rhythm; accommodation to regular beat again took 1–3 hr., the contractions often appearing irregularly at first. When the preparation was returned from more dilute media the shock reaction was more pronounced and included a lowering of tone usually below the normal diastole level (Fig. 4). The increase and decrease of frequency and amplitude were about equally common after this treatment; amplitude particularly showed a wide range of response. It was not found possible to accommodate any preparations returned from 10 to 100%. The accommodated beat was commonly less rapid than it had been previously in 100% sea water, especially after great changes in concentration, indicating some degree of damage to the tissue by sudden immersion in very hypotonic media and the return process.

A number of preparations were tested in media consisting of various hypertonic concentrations of artificial sea water (for composition see Appendix). They were first of all transferred from natural to artificial sea water of the same strength ; this supported an almost identical rhythm in most cases and these latter were then subjected to hypertonic sea water. The shock reaction following mechanical effect of change was slight for small increases, but for greater increases in concentration there was a contraction with inhibition or reduction of amplitude ; this was followed by gradual relaxation of tone, the beats increasing in size until they became very large and separated by periods of inactivity ; finally the preparation remained relaxed (Fig. 5). Accommodation led to a resumption of regular rhythm within 6 hr.; there was accommodation to concentrations up to 160% with no significant change in amplitude and only a slight tendency to reduction in frequency. A number of preparations failed to survive in more hypertonic media and 200 % was the limiting concentration; in this, frequency was much reduced in the three successful experiments.

Those which accommodated to the hypertonic media were transferred back to 100 % artificial sea water. The shock reaction was in general similar to that described for hypotonic sea water, i.e. a contraction phase predominated, with inhibition of rhythm for periods which were longer for greater changes in concentration. In these treatments frequency increased progressively with the greater change in concentration, thus reversing the effect due to the initial transfer to hypertonic medium.

It appears from these results that the tissue was normally capable of contracting regularly over the range c. 40–160% sea water; beyond these concentrations frequency, but not amplitude, tended to lessen and the absolute Emits in which any regular activity was present under the experimental conditions were 10–200% sea water.

The English native oyster, though once an off-shore species, appears now to be confined largely to ‘estuarine’ beds ; nevertheless, it tends to live in positions where the salinity of the water remains fairly high (Russell, 1923; Orton, 1924b, 1928; Percival, 1929); further, any individual is unlikely to be subjected to the whole range of salinity variation of an entire area of beds. The species as a whole is commonly exposed to c. 70–100% sea water. In estuarine localities there may be considerable temporary dilution as a result of heavy rains, and in some places (e.g. Essex) evaporation over shallow beds during long dry spells may raise the concentration to 107%.

According to Monti (1914) the blood of O. edulis is slightly hypertonic in sea water. Krogh (1938) found identity of internal and external chloride concentrations at sea waters of too and 72%, and he concluded that the blood came into almost complete ionic equilibrium with the more dilute (Limfjord) water. Oysters can completely close the shell and thus avoid any dilution of the blood for days, even when placed in fresh water (Dakin, 1909); in the case of O. circumpicta, which normally lives in 80–100% sea water (Amemiya, 1928), animals placed in 50% sea water resist dilution for some days by this method, but there then follows a gradual dilution of the blood which is ultimately fatal unless the oysters are restored to sea water before a lower Emit of about 55% of sea water is reached (Yazaki, 1932). It appears then that this species has no power of osmoregulation, and that the tissues are not at all resistant to dilution of the body fluids.

In ionic composition, the blood of the Japanese O. circumpicta closely resembles sea water (Kumano, 1929). The same is true for O. edulis ; Robertson (1950, personal communication) finds that the most marked deviation is that of potassium, which is 128–7% that of sea water, but the oyster heart is so tolerant of variations in the ionic ratios (Jullien & Morin, 1931), that this divergence can be disregarded in the present work.

The heart rate of oysters varies greatly, depending on the pH and on the size of the animal among other factors (Pelseneer, 1906; Orton, 1924a; Takatsuki, 1927; Walzl, 1937 ; Otis, 1942). The effect of varying the total concentration of the medium on the heart beat was studied by Jullien & Morin (1931) and by Hamada (1938), but in both cases the effect of the diluted medium was observed very soon after its application to the heart, and, as shown below, there is a shock reaction in Ostrea, just as in Mytilus, during which the behaviour is quite different from that after accommodation; moreover, in both cases a ‘closed’ preparation of the heart appears to have been used, in which the bathing fluids were denied immediate access to the inner surfaces of the heart.

O. edulis was obtained weekly from Whitstable and kept in cool aerated aquarium sea water. The right valve of the shell was removed (it was advantageous to open the shell via the hinge and cut the adductor muscle away from the dorsal direction), the pericardium slit open and a thread tied about both auriculo-ventricular junctions; a second thread was tied about the ventricle as close as possible to the origins of the aortae, or about the aortae themselves if possible. On cutting the auricles and aortae the freed ventricle contracted and the walls bulged outwards—advantage was taken of this to remove parts of these walls so that the preparation consisted of a double longitudinal strip of ventricle (dorsal and ventral walls), both inner and outer surfaces of which were immediately accessible to the bathing medium. Preparations were mounted in sea water with a lever tension of c. 100 mg. ; they settled down in a few hours but for convenience were mostly left overnight (c. 16 hr.). There was no noticeable difference whether the medium was aerated or not during the experiment, but bubbling was maintained even in the constant exposures because it was used as a stirring mechanism in the ‘drift’ experiments. (See p. 305.) In sea water, the frequency on becoming regular varied between individuals from 6 to 18 beats/min. ; the rhythm was similar to that in Mytilus but a little slower.

Both frequency and amplitude were affected by hypotonic sea water; there was a small initial mechanical effect of change followed by a shock reaction similar to those in Mytilus. Inhibition of beat during the contraction phase occurred in 70% and more dilute, and lasted longer the greater the salinity change. Accommodation occurred in dilutions down to 50% sea water, while several preparations recovered in 40% and a few in 30% ; in no case was there any activity in 20% even during 24 hr. exposure. The frequency characteristic of the new medium developed in 1–3 hr. and covered a rather wide range at each level of dilution, but there was a distinct trend in the values; dilutions to 80% produced a slight increase in frequency which then fell, until in 40 and 30% the rhythm was considerably slower. This can perhaps be justifiably extrapolated to zero between 30 and 20%. Amplitude distinctly increased with dilution though there was a wide spread in the actual values at greater dilutions.

The return of preparations from 90 and 80% to sea water was uneventful, but from greater dilutions the shock reaction was prominent; it comprised a contraction phase, with inhibition as a rule, then relaxation with a renewed beat vigorous at first but slowing to give irregular isolated contractions and finally relaxation at a low tone level; this picture closely resembled the reaction of the Mytilus preparation to hypertonic media (Fig. 5). On accommodation the beat underwent changes clearly reversing its reaction after dilution, i.e. frequency was little altered (average value) until the step 70–100% was reached when concentrating the medium led to an increase.

The preparations beat as well in artificial as in natural sea water, and from the former they were transferred to hypertonic concentrations up to 200%. The shock reaction was slight, without loss of rhythm in the case of 110 and 120%, these being the only concentrations in which accommodation occurred; in 130% and stronger sea water the reaction followed the pattern described for increasing the concentration above (and for Mytilus), but there was no accommodation in these cases, though they were quite capable of resuming beat when returned to 100% later. In no and 120%, accommodation led to a small decrease of frequency and increase of amplitude ; reversing the process led to the opposite effects.

On the whole, then, the reactions of the oyster heart to salinity change resemble those of the blue mussel heart, but, as anticipated in reviewing the ecological data, the oyster heart is the more sensitive, and beats over the more restricted range.

A comparison of the shock reactions following the above treatments was brought out by analysing the kymograph records with reference to whether the heart was beating or, if inactive, whether it was relaxed or contracted. The change from 100 to 90 or 80% sea water entailed no loss of rhythm, but beyond here a period of contracture was usually present and its duration increased with the degree of hypotonicity; it is noteworthy that in a few cases of dilutions to 70, 60 and 50% complete contracture did not occur—these were only apparent exceptions because marked tone rises were present at the corresponding times, but rhythmic activity was not completely suppressed. The change from hypotonic to 100% sea water did not inhibit the beat until the 60–100% step; in this and lower groups relaxation became very pronounced and was preceded by a short contracture phase (or a tone rise only, in apparent exceptions).

It was found that similar cycles of events constituted the shock reaction in the hypertonicity experiments, and that it was the direction of the change in concentration that governs this reaction. The shock reaction, then, is a well-patterned set of events related to the sudden change in osmotic pressure of the bathing medium or, almost certainly, to the change in ion-concentration-gradient between tissue and medium. It does not seem to be related to the accommodated rhythm, e.g. in sea water hypertonic beyond 120% the shock reaction was typical but no accommodation occurred.

This technique, elaborated by Wells & Ledingham (1940), was used for two reasons: first, to eliminate the mechanical effect of change and, more especially with low concentrations, to eliminate the shock reaction effects occurring with the constant exposure technique; the curve of rate of change of concentration lies, even in the rapid drifts, in the region well beyond that of any shock reactions of the constant exposure method so that the preparations may be considered as having accommodated to the drift as it proceeded; secondly, such an approach might extend the range of dilution in which the isolated heart would function. The procedure was to allow the preparation to settle down in 100% sea water, then to run in M/4OO-NaHCO₃ at a steady rate from a Mariotte bottle (for details see Wells & Ledingham). The frequency and amplitude were measured at approximately hourly intervals and plotted on a graph against the concentration of the medium as calculated from calibration curves for the apparatus. At the end of each experiment the final concentration was checked by titrating total chlorides with silver nitrate; the results were rejected unless the analysed and calculated concentration values agreed within 4% sea water.

The results of two groups of experiments are shown in Figs. 6 and 7 ; in most the rate of change was such that the concentration reached 50% in hr.; in others hr. The trend of the curves for frequency is similar in both groups, though the slowly drifted ones had a less-marked tendency to increase initially. The rapidly drifted preparations clearly showed an increase in frequency which was maintained as far as 50% sea water or occasionally 40%; there was a rapid falling-off in rate beyond this level, and many preparations passed their maximum frequency well before this. Comparing these results with those of the constant exposure method the range of individual variation was remarkably similar, indicating that the variation was biological rather than experimental, while the trend of the curves ‘frequencyxconcentration’ was the same. Very little extension of the lower end of the curve was obtained by either fast or slow drifts ; a few preparations were feebly active below 30%, but none reached 20% and these low concentrations must be considered well beyond the range of this tissue. The oyster heart resembles the rhythmic muscles of polychaetes studied by Wells & Ledingham (1940), in that the same lower salinity limit is given by the ‘drift ‘and ‘constant exposure ‘methods. The curves ‘amplitude × concentration’ (Fig. 7) were very scattered; fast and slow drifts were not segregated in this respect, and the scatter as a whole resembled that obtained with the constant exposure method.

Fresh-water mussels of the group Unionidae occur in fresh waters throughout the world, the chief variations in their chemical environment being the calcium and magnesium concentrations which may be quite high in regions of hard water. Evidence of their penetration into brackish water is scarce; Haas (1926) records A. cygnea from 8% sea water in Randersfjord (east Denmark), and from 14% near Stockholm, while Välikangas (1933) found some at Helsingfors in 13% but stated that they were ‘. abgestorbene’. Philippson, Hannevart & Thieren (1910) acclimatized A. cygnea to fresh water to which had been added sea-salts raising the concentration to nearly 60% sea water.

The osmotic pressure of the blood has been measured by numerous workers and is known to be exceptionally low (‘.. der niedrigste bis jetzt bekannte Wert’, Koch, 1917). Picken (1937) gives a series of sixteen individual determinations; expressed as % NaCl solution isotonic with the blood they range from 0·05 to 0·14;* these values correspond with c. sea water. His general conclusion is that ‘the blood has a vapour pressure equivalent to that of a solution of c. 0·010 % NaCl’—i.e. c. 3% sea water—and his values are amply confirmed by those of other workers who have studied the blood of the species (Dakin, 1912; Călu-găreanu, 1915; Koch, 1917; Damboviceanu, 1924; Duval, 1925; Jatzenko, 1928; Florkin, 1935). Although the osmotic pressure of the blood is low it is considerably greater than that of the surrounding fresh-water medium, so that in its normal habitats the animal actively maintains Δ_t > Δ_e, i.e. is then homoiosmotic. When the concentration of the external medium is increased, however, the animal soon fails to maintain this hypertonicity and the blood comes to have the same osmotic pressure as that of the outer medium (CălugSreănu, 1915; Duval, 1925; Florkin, 1935; Duchâteau & Florkin, 1950). The animal in these conditions is, therefore, poikilosmotic and its tissues, both internal as well as external, are being bathed by media of increased concentration.

Several workers have studied the heart beat of A. cygnea and of other species, either in the intact animal or in media varying from tap water to frog Ringer diluted to 25%, but there has been no study of the effects of varying the salinity of the bathing medium. The heart beat is always very slow, ranging at room temperatures from about 3 to 10 beats/min., and is much slower when the shell is closed than when it is open (Willem & Minne, 1898; Keber, 1851 ; Noyons, 1908; Koch, 1917; ten Cate, 1923a, b ; Berthe & Petitfrère, 1934; Hers, 1943; Hendrickx, 1945).

A. cygnea were used throughout the investigation; they were obtained from Surrey and kept in running tap water for no longer than 1 month before using. Florkin, Duchâteau & Leclercq (1950) have shown that there is no significant change in the blood ionic composition on starving Anodonta for this length of time.

The animal was removed entire from the shell, the pericardium opened dorsally and the ventricle slit from end to end along the mid-dorsal line; the rectum was removed where it runs through the ventricle and the latter separated into two halves by cutting along the mid-ventral line. Threads were tied at each end of each half-ventricle, the auricles cut away and the ventricle sectioned beyond the threads. In this way two symmetrical halves of the organ were obtained, a situation very convenient for conducting adequately controlled experiments since the two halves proved to be physiologically identical. Each half, termed a ‘ventricle-strip’, was mounted in a beaker separate from its counterpart, but usually together with a strip from another mussel. Under the action of the isotonic lever (tension c. 200 mg. for mussels of 12–13 cm. shell-length) the preparations relaxed slowly and were allowed to settle down for 24 hr. after removal.

Sea water diluted to 4% was chosen as a convenient standard solution, having an osmotic pressure within the range of Anodonta blood, although its individual ionic concentrations are somewhat different (see Appendix).

This proved to be an excellent bathing fluid for the ventricle strip. It supported for several days a regular rhythm comparable to that found in vivo by other workers, even without any added nutrient or aeration. During the period allowed for the strip to settle down, it relaxed considerably and showed at first very irregular beats which were often of large amplitude, and the mechanical activity was interspersed with relaxation periods of varying length. This may be compared with the results of Motley (1934) on American fresh-water mussels where the heart, exposed in situ, passed through four phases, viz. :

• Beats regular and even; lasted only a few minutes after opening shell.

• Beats irregular and erratic; lasted 2-24 hr.

• Beats regular and uniform; lasted 2-93 hr.

• Beats becoming smaller, slower and finally irregular with the onset of fatigue.

In the present experiments phase (a) was not found, no doubt due to the much more rigorous treatment necessitated by complete isolation of ventricle strips—the other three phases correspond well with those found in the present investigation, though regular uniform beats have been obtained continuously for up to 8 days after removal of the strip, i.e. 7 days after settling down. This is the longest survival and was obtained with four preparations ; 4–5 days was the average survival time. Hendrickx (1945) also found periods of irregularity both before and after the regular rhythm was established in A. cygnea. The rhythm on becoming regular in 4% sea water was uniform though very slow; of approximately 100 hearts (200 preparations) most fell within the range 1·6−5·5 beats/min. at T=8−20° C., with the following exceptions : 6·4(13° C.), 8·1 (14° C.), 9·1 (14° C.), 8·3, 9·0, 12·6, 13·0 (all at 21·5° C.).

The mechanical disturbance produced by the constant exposure technique was very slight and often not noticeable in the case of Anodonta preparations—this was probably due to their larger size, as compared with Mytilus and Ostrea ventricles, so that stretching on removing the buoyant medium was insignificant (Fig. 8).

On increasing the concentration of the medium the preparations showed a loss of tone (Fig. 9), accompanied in the higher concentrations by the absence of any mechanical activity. They remained in this relaxed state for several hours in the cases of great changes then gradually began to beat, irregularly at first, until a regular rhythm was once more produced. The whole process of accommodation was slow, but short recordings taken at intervals showed that it was adequate to leave the preparations for 20–30 hr. after a change of medium. The effect of increasing the concentration of the sea water in simple multiples of 4% showed very clearly an increase in frequency of the beat (though its extent was not proportional to the concentration) and a decrease in amplitude, with some exceptions in the 4–8% group. The highest concentration in which preparations recovered was 40% sea water, representing a tenfold increase in ionic concentration of the medium.

Studies were also made upon the effect of decreasing the concentration of the medium by returning the accommodated preparations to 4% sea water. The immediate reaction was a rise of tone, progressively greater as the change was more severe and often enormous after returning from 20% or stronger sea water. During this tone rise rhythmic activity was inhibited in all cases except some returns from 8%. After further accommodation the beat became characterized by a reduced frequency and usually an increased amplitude.

A number of preparations were treated in the reverse manner, the initial bathing medium being replaced by more dilute sea water, viz. 2 or 1%. Dilution led to a decrease in frequency, while the return to 4% was accompanied by an increase; this is clearly in line with the results of the above experiments and illustrates the relationship between frequency and ionic concentration of the medium. The data for the effect on amplitude do not show the same correspondence; it was more common for dilution to 2% to produce a smaller than a larger beat, while in the case of i % the beat size became irregular. (A number of other preparations became quite inactive in 1 % sea water and resumed beating only after returning to 4%.) It is not considered that these latter results are seriously at variance with the general conclusion that frequency is directly, and amplitude inversely, related to the medium concentration; if it is agreed that 4% sea water is a reasonable blood substitute, at least in respect of its concentration, then 2 % and particularly 1 % are too dilute in one or more ions to support larger contractions. It is always possible, however, to reduce the frequency and if this is dependent on ion concentration(s) it will occur with dilution of the medium.

Tap water failed to support heart beat in the preparations used in this work; the success reported by ten Cate (1923b) may be related to his use of a different type of isolated preparation or to differences in tap waters.

As in Ostrea, preparations submitted to the drift treatment confirmed the results of the constant exposure method, in respect to the dependence of frequency and amplitude on concentration.

Several workers have used this solution, considerably diluted, as a bathing medium for Anodonta hearts. Experiments were therefore carried out to compare the effect of this medium with that of 4% sea water. Sudden transfer from 4% sea water to 1/15 Frog Ringer (R/15) led to an immediate rise of tone with inhibition of beat—there was no sign of mechanical activity within 24 hr. Similar treatment with undiluted Ringer caused inhibition with relaxation, but in 24 hr. there was in some cases a rapid but minute and irregular beat. Now Frog Ringer has a Δ = 0·45° C. (Clark, 1927, p. 124), so that it is obvious from earlier experiments in this section that R and R/15 are, respectively, too concentrated and too dilute to be suitable for this preparation; indeed their shock reactions show this also. Further experiments were then performed with R/5, which has a Δ = 0·09° C. (calc.) approximating to those of Anodonta blood and 4% sea water; in this case activity was maintained, sometimes with periods of irregularity following the change, but settling down to a steady uniform beat within 24 hr. Nevertheless, survival of the preparations was much shorter (seldom longer than 2 days) than in 4% sea water.

The levels of systole and diastole in R/5 often fluctuated even when the rhythm was otherwise regular; this further suggests that R/5 is not as suitably balanced physiologically for the preparation as is 4% sea water. Similar fluctuations in tone were found by ten Cate (1923 b, figs. 3, 4) who stated that R/4 favoured the production of this effect.

A solution was prepared having an inorganic chemical composition within the range of values given by Florkin et al. (1950) for the analysis of Anodonta blood. On replacing the 4% sea water by this artificial blood there was little or no shock reaction, and the preparations continued beating with no sign of interruption. The rhythms of six different preparations compared before and 21–24 hr. after such a change showed an increased frequency (133–208%, average 177%); while amplitude was in two cases increased (118%), in three others decreased (68–91 %) and in one case remained unchanged (average for the six examples 94%).

Thus an artificial solution made to imitate Anodonta blood in respect to cation concentrations was eminently satisfactory when used on the ventricle strips, supporting a rhythm considerably faster than did 4% sea water, but of amplitude averaging about the same. It is to be noted that this greater frequency is still within the normal range for Anodonta, and also within the range found for isolated preparations in 4% sea water.

Sea water proves to be a suitable medium for the hearts of the two marine species, survival with good activity being possible for 2–3 days. An artificial solution containing the four major cations (as chlorides) in about the same proportions as in sea water is also satisfactory, and for these experiments there appeared no reason to attempt closer imitations of the bloods. Both marine species have a small degree of ionic regulation in that potassium is maintained some 20 % above sea-water level and a few preparations of both Mytilus and Ostrea were tested in sea water with this added potassium ; there was no significant difference in the rhythms. The excellent results with Anodonta in 4% sea water are intriguing in that this solution has more sodium and magnesium, vastly less calcium and about the same potassium as Anodonta blood (see Appendix), though the Δ’s are equivalent on calculation. Furthermore, the preparation beats merely a little faster in an artificial Anodonta blood but does not survive here so long (perhaps having used up its energy source sooner). The increase in frequency may be a reaction to extra calcium, though it is certainly not proportional to the difference in this ion; or more probably to the lower concentration of magnesium, an ion which is known to have a depressant action elsewhere. In 1/5 Ringer also, the beat is faster and survival time shorter; here there is comparatively little calcium and no magnesium, which supports the idea that the relatively large dose of magnesium in 4% sea water keeps the frequency low. In concentrations of sea water greater than 4% the more rapid beat must be determined by another ion(s) acting antagonistically to magnesium.

Motley (1934) showed that on perfusion of fresh-water mussel hearts only a three-fold increase of calcium was tolerated, but more systematic work is needed to determine the regulation by the ions and their antagonisms.

When the results are considered as a whole, the general relationship emerges that the ventricles beat faster or slower as the surrounding medium is in greater or lesser concentration, with a falling-off of frequency at extremes. Within the ranges of sea water permitting beat in the marine species there are narrower limits in which the frequency is maximal (Mytilus 40–160%, Ostrea 70–100%), but the frequency of Anodonta continues to increase up to the maximum capable of supporting beat (40%).

Amplitude shows some tendency to behave in an inverse manner (Ostrea and especially Anodonta), but differences between individuals are large and this property is capricious and does not lend itself to strict quantitative analysis.

It is interesting to compare the properties of the heart preparations with other characteristics of the three animals (Table 1). The comparison suggests that there is a direct correlation between blood concentration and the whole metabolism of the animal; that the dilute internal medium of Anodonta calls the tune for its low heart rate and general activity; for example, subjecting the isolated ventricle to concentrated media causes it to develop a rapid beat, while dilution slows it down. With Ostrea and Mytilus the same relationship holds for dilutions below 100% sea water. It would be interesting to know what the activity of whole Anodonta was in experiments where they were acclimatized to dilute sea water.

The results of the experimental work with respect to salinity tolerance of the tissues and the data for ecological ranges from the literature are summarized in Table 2. In Mytilus there is no experimental evidence of osmoregulation but this probably occurs in very dilute sea water, so that in all other conditions the heart is bathed from both sides by the same medium (in terms of inorganic ions); it is shown capable of approximately normal activity over a range greater than that found in the ecology of English specimens. There is a particularly striking adaptability of the tissue to hypertonic media, and even much beyond the range supporting normal activity it is not killed by the treatments. These facts suggest that slow decreases and increases of salinity could probably be withstood experimentally by the species to as far as Fox found with M. califormanus.

Ostrea edulis does not appear normally to live in sea water below 70%, so that although osmotic regulation does not operate, except perhaps in much more dilute conditions, the heart may never in an intact animal be subjected to a body fluid more dilute than this. It is significant that the frequency of the heart beat in these experiments is maximal in those concentrations which the heart may experience normally, while in more dilute media the beat becomes very slow. Similarly, the amplitude, while not showing such a clear trend, becomes very irregular at low concentrations.

Anodonta is the only species constantly to maintain Δ_i> Δ_e. Were it not to do so as successfully, it seems that its heart beat would become so extremely slow as to be almost ineffective in circulating the blood. The finding that the beat is not maintained beyond 40% seawater, whereas Philippson et al. (1910) accommodated the animal to c. 60% is reasonably explained by the slow acclimatization possible with whole animals; this is precluded in experiments on isolated tissues, but obviously the heart must have been beating in Philippson’s experiment and thus functioning in a very hypertonic medium.

The overall picture is that the tissues studied are capable of functioning well in fluids of both greater and lesser concentration than those normally bathing them, though extremes of concentration support reduced levels of activity. These capabilities may be considered as safety margins, allowing latitude in the case of a temporary breakdown or overshoot in some regulating mechanism.

There is evidence, then, that adult animals of the three species can survive quite wide fluctuations of salinity of their environmental media; restriction of their ecological distribution in respect to salinity, therefore, seems to be related to some other factor(s), e.g. less tolerant stages in the life histories, as suggested by Needham (1930).

1. An investigation was made into the effects of variations of osmotic pressure of the bathing fluid on isolated ventricle preparations from three species of lamellibranchs.

2. Suitable media were found in sea water for Mytilus and Ostrea and 4% sea water for Anodonta. Under the conditions described, a regular beat resembling the natural beat is maintained for days, although the media used differ somewhat in ionic composition from the natural body fluids.

3. Sudden changes in concentration of the bathing media lead to temporary ‘Shock reactions’ followed by accommodation. The shock effects can be avoided by gradual changes in concentration, the final effects being essentially the same whether the change is gradual or abrupt.

4. In all species, the shock reaction included reduction in amplitude or even inhibition of the rhythm, and also the following effects, which depend on the direction of change of concentration. Change from a normal to a hypotonic medium, or return from a hypertonic to a normal medium, caused tone rise or systolic contracture ; change from a normal to a hypertonic medium, or return from a hypotonic to a normal medium, caused tone lowering or diastolic relaxation.

5. The beat after the shock reactions had passed off was in general faster in hypertonic and slower in hypotonic media. In the marine species, however, there was also slowing in the most concentrated media capable of supporting a beat.

6. A comparison is drawn between features of the activity and metabolism of the three species and the concentration of their bloods. It is suggested that the very dilute blood of the fresh-water mussel restricts its heart rate, general activity and metabolism.

7. The tissues investigated are shown to tolerate ranges in the concentration of media wider than those encountered under natural conditions, and it is suggested that salinity of the environment is not a critical factor in restricting the ecological distribution of adults of the species. The tolerance of the tissues may have a safe-guarding effect in the event of temporary inefficiency in the osmoregulatory mechanism.

Ionic composition of the body fluids and of the media used. AU in gramsllitre