ADAPTATION TO CHANGES OF SALINITY IN THE POLYCHAETES

Transcription

1 ADAPTATION TO CHANGES OF SALINITY IN THE POLYCHAETES I. CONTROL OF BODY VOLUME AND OF BODY FLUID CONCENTRATION IN NEREIS DIVERSICOLOR BY L. C. BEADLE University of Durham College of Medicine; Newcastle-on-Tyne {Received 18 July 1936) (With Five Text-figures) INTRODUCTION THERE are several possible means by which marine invertebrates could survive the passage into brackish water. They may be summarized as follows: A. The animal may be able to maintain its body fluids hypertonic to the external medium, thus avoiding undue internal dilution and distension of the body resulting from osmotic inflow of water. To this end the following factors might contribute: (a) Excretion of an hypotonic urine. This has been demonstrated only for the freshwater crabs Potomobius astacus and P. leptodactylus (Herrmann, 1931; Scholles, 1933). On the other hand the concentration of the blood of these crabs is little altered by blockage of the excretory organs (Herrmann, 1931). The urine is isotonic with the blood in the brackish water crab Carcinus moenas and in the fresh-water forms Telphusa Jluviatile and Eriocheir sinensis (Schlieper & Herrmann, 1930; Scholles, 1933). (b) Low permeability of the body surface to water and salts. Bateman (1933) argues from the blood dilution curve of Carcinus moenas that there is probably a decrease in passive permeability to salts in dilute sea water. Bethe (1934) and Nagel (1934) produce evidence that brackish water forms are less permeable to salts than marine forms. Experiments on Gunda (Procerodes) ulvae suggest a decrease in permeability to water as a result of treatment with dilute sea water (Beadle, 1934). Reduction in permeability, unless it were reduced to zero, could not of course by itself maintain hypertonic body fluids. (c) Active resistance to inflow of water and/or outflow of salts by all or part of the body surface involving the expenditure of energy. The fact that there is an increase in the rate of respiration on transference to dilute sea water of forms such as Nereis diversicolor, Gunda ulvae and Carcinus moenas which can survive these changes has been adduced as evidence of this activity (Schlieper, 1929; Beadle, 1931). Further support is given by the action of cyanide, which causes an increased accumulation

2 Adaptation to Changes of Salinity in the Polychaetes 57 of water in the body in dilute sea water (Beadle, 1931). But this increase in rate of respiration is not observed in some resistant forms (e.g. Eriocheir sinensis, Schlieper, 1935), anc^ th e increase is not always maintained (e.g. Nereis diversicolor, Schlieper, 1929; Beadle, 1931). Pieh (Schlieper, 1935), from measurements of the water content of various organs, concluded that the increase of respiration is due to hydration of the tissues and is not directly connected with osmo-regulation. (d) Active uptake of salts from the external medium. Nagel (1934) showed that if Carcinus moenas after treatment with dilute sea water is transferred to water of a higher concentration but still lower than that of the blood, the concentration of the latter is raised. In view of the isotonicity of the urine he concluded that this must be brought about by active uptake of salts from the water against a concentration gradient. (e) The maintenance of a high internal hydrostatic pressure to balance the osmotic pressure difference. The possibility of this playing a significant part has apparently not been systematically investigated though Bethe (1934) has considered it as a possibility. B. There may be no mechanism for the maintenance of hypertonic body fluids. But the latter might be diluted to a considerable extent without harm, provided that the body is not unduly distended. This would entail either a high permeability to salts or a rapid removal of bodyfluidsby the excretory organs. The degree of dilution of the external medium which the animal could withstand would in this case obviously be limited. It will thus be seen that maintenance of body volume and of a favourable internal concentration are two problems confronting brackish-water invertebrates which are not necessarily completely interdependent. That in the polychaetes they are less connected than might have been expected will emerge from these and other results to be published later. The object of the following work on Nereis diversicolor, which can survive large changes of salinity, was to make parallel determinations of weight and of internal concentration changes resulting from treatment with dilute sea water, to investigate the effects upon the body fluid concentration of cyanide and calcium-deficiency, both of which influence the weight curve (Beadle, 1931; Ellis, 1933), and to decide the relationship, if any, between the rate of respiration and the maintenance of hypertonic body fluids. Direct measurements of hydrostatic pressure and of the amount and concentration of the fluid excreted by the nephridia were not possible, but certain deductions are made concerning these from the results of the experiments. METHODS The animals were all obtained from the same position in the River Blythe estuary. Northumberland. Weight. After placing on dry filter paper for a few moments to remove excess of water they were weighed in water to the nearest o-oi g. Calcium-free sea water and cyanide. Calcium-free sea water and sea water containing M/1000 NaCN were prepared as previously described (Beadle, 1934).

3 58 L. C. BEADLE Body fluid concentration measurements. Baldes's modification of the Hill vapourpressure method was used (Baldes, 1934). Three pairs of constantan-maganan thermocouple loops were mounted together so that three determinations could be made simultaneously. The filter paper lining the surrounding moist chamber was soaked with distilled water, and the two loops of each pair were supplied with a drop of distilled water and of the unknown solution respectively. The apparatus was standardized for each experiment with 100 per cent sea water. The galvanometer used was a low resistance Cambridge A and M model with 4 m. between mirror and scale. Reading to the nearest 0.5 mm. on the scale it would theoretically have been possible to obtain results accurate to the equivalent of the nearest 0-5 per cent sea water. In practice the errors occurring from unknown causes made the determination accurate to the nearest 1 -o per cent sea water. All concentrations were expressed as the equivalent of a percentage of sea water. I am much indebted to Prof. A. V. Hill and to Miss B. M. Garrard for advice on the use of this method. Extraction of body fluids. The animal was placed on dry filter paper and the fluid was extracted by means of a fine glass pipette inserted into the body cavity. With the same pipette the fluid was transferred direct to the thermocouple loop. The whole operation was performed in a moist chamber. Respiration measurements. These were made with Barcroft manometers. Increase in rate of oxygen consumption was expressed as percentage of original rate per gram original weight. In all cases the animals were kept in the experimental solutions in a thermostat at 15 0 C. Temperature changes were found to have considerable influence upon the weight curve a subject which will be dealt with in a subsequent paper. WEIGHT AND BODY FLUID CONCENTRATION Experiments on weight changes were made at different times of year with animals collected from the same place in the Blythe estuary. It was found that during the winter months the increase in weight in 25 per cent sea water was greater and more prolonged than during the summer. The curves in Fig. 1 are each typical of four to six obtained at the same time. Five animals were weighed together to obtain each point. The curve 13. ii. 35 is typical of animals which survive the change well with no diminution of activity. The weight rose to a maximum after 5-7 hours and then fell to a relatively steady value at hours. Unfortunately body fluid concentrations were measured only for a few points on the winter swelling curve simultaneously with Fig. 1, curve 9. xii. 35. Body fluid concentrations of single animals expressed as percentage sea water are enclosed in circles in Fig. 1 and will serve as a comparison with those given later. A determination made after 9 days gave 42 per cent and after 5 weeks 32 per cent. Hypertonicity of the body fluids is therefore permanent, but is not very great. It was found that in 100 per cent sea water the body fluids were isotonic ± i-o per cent.

4 Adaptation to Changes of Salinity in the Polychaetes 59 More exhaustive parallel determinations of weight and body fluid concentration in 25 per cent sea water were made in May 1936 when the weight curve was showing the summer form. The results are shown in Fig. 2 (a and b). The points were obtained by weighing a number of batches of worms (five in each) at the start and at intervals after transfer to 25 per cent sea water and by estimating the body fluid concentration of three of each batch after the second weighing. It can be seen that, though the points are rather scattered in places, the concentration curve (b) follows a logarithmic course towards a value above that of the external medium. On the assumption that no significant loss of salts nor excretion of fluid had occurred during the first half-hour it was possible to construct from the concentration curve a weight curve which would theoretically have been followed if there had been no oc 5 sv 00 a s / / / / / v 18. ix. 34 sluggish / / ^ ^ - ^ is - /^f^^!^^- 49%.44% // / /, i i i i i i i i / 1 Winter Summer 1 i I Time in 25 % sea water (hours) Fig. 1. salt loss nor excretion throughout the whole experiment (Fig. 2 c). The difference between this and the actual curve obtained (a) might be due to continuous loss of salts through the surface and/or to removal of fluid by the excretory organs. The experiment was repeated in the same manner but using 50 per cent sea water (Fig. 3). One of the thermocouple pairs was damaged and only two determinations of body fluid concentration were made at each time. The concentration curve however is obviously of the same form as in the previous experiment and approaches a value closer to the concentration of the external medium (Fig. 3 b). The weight curve (a) shows a marked maximum and subsequent drop, and in general form resembles the curves obtained in the winter using 25 per cent sea water. If this is compared with the theoretical weight curve (Fig. 3 c) calculated as above from the concentration curve it will be seen that the two do not diverge until after one hour. If salts were being lost through the body surface it is reasonable to conclude that

5 6o L.. C. BEADLE they would have left at a maximum rate at the beginning of the experiment when the difference between the internal and external concentrations was greatest. The fact that the curves do not diverge at once suggests that the subsequent divergence was not entirely due to passive salt loss through the surface. This conclusion is further supported by the results of both these experiments (Figs. 2 and 3) if the theoretical amount of salts lost through the surface at intervals Time in 25 % sea water (hours) Fig during the first 5 hours is calculated on the assumption than none occurs during the first half-hour and that salt loss is the sole cause of the lowering of the weight curve. V, the osmotically active weight expressed as percentage of the original weight, can be found from the following equation:

6 Adaptation to Changes of Salinity in the Polychaetes 61 where \ = body fluid concentration at the start (= per cent sea water), A, = concentration of body fluids after time t, and W t = weight increase (per cent) after time t. By substituting this figure for V in the above equation a series of decreasing values can be found for \ corresponding to different moments of time. The figures so obtained represent the theoretical initial concentrations of the body fluids if the weight and internal concentration actually attained at each moment occurred with no loss of salts or water, ioo A,, will therefore give the quantity of salts theoretically lost after a given interval of time expressed as percentage of the original concentration in the body fluids, on the assumption that the difference between the two weight curves (a) and (c) is due entirely to outward diffusion of salts. When these Time in 50 % sea water (hours) Fig figures derived from the two above experiments are plotted against time (Fig. 4) most of the points fall upon a straight line. This result would not be expected on the above assumptions since the rate of salt loss should progressively diminish as the result of the decreasing difference between external and internal concentrations. These considerations combined with the fact that there was apparently no loss of salts during the second half-hour in 50 per cent sea water suggest that the main cause of the divergence of the actual from the theoretical weight curve must be sought elsewhere. The most remarkable feature of the experiment shown in Fig. 3 was the fall in weight after the maximum accompanied by very little reduction of body fluid concentration. If after 6 hours water was still being taken up, the drop in weight must have been due to the removal of fluid hypertonic to the body fluids. If water inflow had somehow been stopped, then the fluid removed must have been isotonic. In

7 62 L. C. BEADLE either case forces other than diffusion or osmotic pressure must have been involved. It seems unlikely that an isotonic or hypertonic fluid could be removed through any other channel than the nephridia. These in the genus Nereis have open nephrostomes and thus afford a clear passage from the coelom to the exterior. The question remains as to how the rate of flow through the nephridia could be sufficiently increased to account for the observed lowering of the weight curve. A possibility which suggests itself is that the rate of flow through the nephridia is partly determined by the hydrostatic pressure in the body cavity. This in turn might be controlled by the tonus of the body wall muscles. The swelling of the body cavity might induce a response in these muscles which would increase the hydrostatic pressure. If this were the main factor controlling the weight, the earlier divergence of the actual from the theoretical weight curve in 25 per cent sea water (Fig. 2 a and c) 1op C o Its los fluid S-o S-S 1 a ore / / 25% seawater/* / /50% seawster Time in hours / than in 50 per cent sea water (Fig. 3 a and c) could be explained. The rate of swelling is greater in 25 per cent sea water and the muscular response might be expected to occur earlier. On this hypothesis it would of course be necessary to postulate that the fall in weight from the maximum (Fig. 3) was due to a relatively violent reaction on the part of the muscles when a certain degree of swelling was reached. The expulsion of fluid as a result of a swollen body cavity could alone explain the result of the experiment summarized in Table I. The concentration of the body fluids of two sets of animals was brought to the equivalent of per cent sea water by different methods of treatment: (a) 4 hours in 25 per cent sea water, and (b) 3 days in 50 per cent sea water. Both sets were then weighed and transferred to isotonic sea water (55 per cent). Subsequent weighings done after 3 and 22 hours showed that the more swollen ones, i.e. those previously treated for a short time with 25 per cent sea water (a), decreased considerably in weight, whereas the weight

8 Adaptation to Changes of Salinity in the Polychaetes 63 of (b) remained relatively constant. Since the body fluids were in both cases at the start isotonic with the external medium, the result must be ascribed to the higher internal hydrostatic pressure in (a) causing the expulsion of fluid. Table I Previous treatment 0 Time in 55% sea water (hours) 22 (a) 3 days 100% 4 hours 25% (6) 3 days 50% Weight Weight i 5 Table II Previous treatment 2"5 Time in 55% sea water (hours) (a) 3 days 100% 4 hours 25% (6) 3 days 50% Body fluid 69 = % sea water Body fluid 61 = % sea water i The experiments illustrated by Figs. 2 and 3 show that there was little connexion between the maintenance of hypertonic bodyfluidsand the regulation of body volume. It was in fact found from experiments done on single animals that, though the weight attained after a given interval varied considerably, the concentration of the body fluids could be predicted fairly accurately. The weight and concentration figures given in Table IV will serve to illustrate this point. The body fluid concentrations correspond fairly closely with those previously found after the same period in 25 per cent sea water (Fig. 2), but there is great variation in the weights. There is therefore no obvious connexion between the degree of swelling and the internal concentration, the latter being determined only by the time elapsed since transfer to dilute sea water. In order to investigate this point further the experiment previously described (Table I) was repeated, but in this case the object was to determine the effect, if any, of isotonic sea water upon the body fluid concentration of swollen and nonswollen animals (Table II). It can be seen that in both sets there was a rise of internal concentration during the first 2-5 hours which was greater in the more swollen set, i.e. those previously subjected to 4 hours 25 per cent sea water (a). The subsequent figures are not very satisfactory but there seems to have been a general tendency for

9 6 4 L. C. BEADLE the internal concentration of the originally more swollen animals (a) to increase still further during the next 20 hours, whereas the concentration of (b) remained more constant. The results of these two sets of experiments (Tables I and II) suggest that the establishment of hypertonic body fluids can be brought about without significant alteration in weight in isotonic sea water (Tables I and II b), but that the process is speeded up when a fall of weight occurs as a result of a high internal hydrostatic pressure (a). This might be explained if the nephridia were excreting an hypotonic urine. The higher internal hydrostatic pressure in (a) would cause a faster flow of fluid through the nephridia, whereas in (b) the lesser degree of concentration might have been due to a balance between the action of the nephridia and the consequent osmotic inflow of water without any extra stimulation of the former as a result of a high internal hydrostatic pressure. Histological examination of animals at intervals after transfer to dilute sea water showed no obvious change other than a swollen body cavity. There were no tissues which appeared swollen such as were found in the case of Gunda vlvae (Beadle, 1934). CALCIUM DEFICIENCY AND CYANIDE It has been shown previously that calcium deficiency and Af/1000 cyanide cause a greater weight increase in dilute sea water than normally occurs, and that this effect is reversible (Ellis, 1933; Beadle, 1931). The following experiments were done Table III Treatment (a) Transferred direct % sea water M/1000 NaCN: (i) 25 hours (ii) 72 hours (iii) 72 hours plus 5 days normal 30% (6) Transferred direct % calcium-free sea water: (i) 48 hours (ii) 72 hours (iii) 72 hours plus 4 days normal 30% (c) Controls. Kept 42 hours normal 30% Body fluid = % sea water 4i Condition Moderately active Swollen and quite motionless but reacting to touch Quite normal Just moving Quite motionless Complete recovery in 22 hours

10 Adaptation to Changes of Salinity in the Polychaetes 65 to test the effects of these conditions on the body fluid concentration. The results are shown in Table III. There is no doubt that both calcium deficiency and cyanide bring about an osmotic equilibrium between internal and external media and that this effect is reversible. This further confirms the results described in the last section which showed that concentration of body fluids can occur in an isotonic medium. It is remarkable that under both conditions the attainment of equilibrium requires at least two days and that this prolonged treatment has no permanent deleterious effects. Similar results were however obtained in experiments on Gunda ulvae (Beadle, 1934). RATE OF RESPIRATION AND OSMOTIC REGULATION Table IV gives results of experiments done on single animals subjected to 25 per cent sea water for varying periods of time, the increase in weight and in respiratory rate, and the final body fluid concentration being determined in each case. Table IV Time in 25% sea water (hours) % increase in weight % increase in respiration rate Body fluid = % sea water (.") 4-25 (*) 4-25 (c) (J) («) if) It is evident that, though the body fluid concentration approximate to the values previously found at the corresponding times (Fig. 2), both the weights and respiratory rates are extremely erratic. It should however be noted that (d), whose weight had increased by 135 per cent, showed no increase in respiratory rate, though its body fluid was of the same concentration as that of (c) subjected for the same period to 25 per cent sea water, whose weight had increased by only 87 per cent and whose respiratory rate was 42 per cent higher than the original. If, as originally suggested by Schlieper (1929), the increased rate of respiration in dilute sea water is the result of increased osmotic work, it should be of interest to observe the effects upon the respiratory rate of transferring animals from dilute sea water to a solution isotonic with the body fluids. This was tested in the following experiment the results of which are plotted in Fig. 5. Two single animals (Fig. 5 a and b), whose weight and respiratory rate were previously determined, were placed in 25 per cent sea water for 4 hours, when it was assumed that their body fluid concentrations were equivalent to per cent sea water. The weights and respiratory rates were then redetermined and they were transferred to 55 per cent sea water. An immediate remeasurement of the rate of respiration showed that it had risen in both cases by about 30 per cent (Fig. 5). Subsequent determinations from 3 to 45 hours after transfer to 55 per cent showed that the respiratory rate

11 66 L. C. BEADLE had fallen to a steady value approximating to the original in 25 per cent sea water in (a) and about 15 per cent below it in (b), but in both cases this value was well above the original in 100 per cent sea water. The weights of both animals fell during this period practically to the original in 100 per cent sea water (Fig. 5, lower curves). Although it is not possible to draw any positive conclusions from this experiment, the immediate and temporary rise in respiratory rate cannot be ascribed to a temporary intensification of the osmotic regulatory mechanism, since hypertonic body fluids are not only established but also maintained under these conditions (Table II a). It might perhaps have been due to the stoppage of water inflow on Time in 55 % sea water (hours) Fig transfer to isotonic water and the consequent release of internal pressure which was being resisted by the tonus of the body wall muscles. This sudden release might result in the immediate contraction of these muscles, which might be reflected in increased oxygen consumption. It is difficult however to understand why this initial rise should have been of such short duration, since the weight continued to decrease after it was over. Another possible explanation might be that the sudden change in concentration of the external medium stimulated peripheral sensory structures and caused a temporary rise of metabolic rate, which was not connected with muscular or osmotic work. Even though the weight had fallen after 45 hours to the original in 100 per cent sea water an increased rate of respiration was still maintained. This might have been

12 Adaptation to Changes of Salinity in the Polychaetes 67 explained as the result of osmotic work were it not for the experiments previously done (Table IV), which showed that hypertonic body fluids could be maintained with no rise in oxygen consumption. It can at least be concluded from the results described in this section that the majority of the extra oxygen consumption in dilute sea water is not the result of osmotic work. DISCUSSION Two main conclusions can be drawn from the foregoing experiments: (i) that the degree of osmotic regulation of the body fluids is relatively slight, and cannot be considered as being of direct importance in enabling the animals to survive in dilutions of sea water between 100 and 25 per cent, and (ii) that survival is possible mainly because they can prevent excessive swelling of the body due to osmotic inflow of water. Schlieper (1929) made freezing point determinations on the body fluids of Nereis diversicolor. After 3 days in 50 per cent sea water (i7 /oo salinity) he found the body fluids equivalent in concentration to 68 per cent sea water and after 2 days in 25 per cent they were equivalent to 49 per cent sea water. These figures are higher than those found in the above experiments (Figs. 1 and 2), but Schlieper states that in the localities from which he obtained his material he sometimes found specimens living in a salinity as low as 4%o (11-8 per cent sea water). Salinity estimations on the water at Blythe showed that the worms were probably never subjected to sea water of lower concentration than 60 per cent. It is possible therefore that the osmotic regulatory mechanism of the animals with which Schlieper worked was better developed as a result of continual subjection to water of lower salinity. In the case of the animals used in the present experiments it does not appear that their capacity to maintain hypertonic body fluids has any survival value, since it was found that the internal concentration could be reduced to the equivalent of 32 per cent sea water without harmful results even after a period of several weeks. No direct light is thrown upon the nature of the osmotic regulatory mechanism by the present work. But since the concentration of the body fluids is increased when an animal previously treated with dilute sea water is transferred to an isotonic solution (Table II), the mechanism must entail an active process and one which is not merely controlling the inflow of water. There seem to be two remaining possibilities : (i) addition of salts to the body fluids from the tissues or by active uptake from the external medium (as was suggested by Nagel for Carcinus moenas), (ii) excretion of hypotonic fluid by the nephridia. The fact that worms which are more swollen and consequently have a higher internal hydrostatic pressure are able to concentrate their body fluids more rapidly in an isotonic medium than less swollen ones (Table II) might be explained if the nephridia were producing an hypotonic urine. The amount of fluid passing through the nephridia might be greater under the greater hydrostatic pressure. It certainly could not easily be explained if the mechanism entailed the addition of salts to the body fluids, since an increase in hydrostatic pressure is not likely to speed up this process. There appears therefore 5-3

13 68 L. C. BEADLE to be some circumstantial evidence that the nephridia are largely responsible for the maintenance of hypertonic bodyfluidsand that the internal hydrostatic pressure may play an indirect part in determining the amount of fluid dealt with by the nephridia. Bethe (1934) concluded that the departure of the swelling curve of Nereis diversicolor from that theoretically expected if the surface were impermeable to salts is to be attributed to the loss of salts by diffusion through the body surface. Though salt loss may occur in this way, it has been shown above that it cannot be the major cause, since the rate of loss of salts, which would theoretically account for the form of the weight curve, remains constant over a period during which the difference between internal and external concentrations is diminishing (Fig. 4). It would not in any case explain the fall in weight after the maximum. Bethe (1934) has in fact shown that brackish water invertebrates are in general less permeable to salts than marine forms. In that case salt loss cannot be suggested as the cause of the lowering of the weight curve when the curve of typical marine forms such as Nereis cultrifera rises higher and more nearly approaches a perfect osmotic curve (Beadle, 1931). When five animals were weighed together, weight curves of regular form were obtained (Figs. 2 and 3), but it was found that there was considerable individual variation in weight at any given moment (Table IV). In spite of this, the body fluid concentrations determined on single specimens followed a fairly regular curve. Since it cannot be suggested that the internal concentration is independent of the amount of water taken up, the most reasonable conclusion is that the animal behaves as a normal osmotic system as regards uptake of water and change of internal concentration, but that the volume of the body fluids is continually being reduced by removal through the nephridia. The individual variation in weight might then be due to variation in the amount of swelling which has to occur before the muscles will react to cause increased expulsion of fluid. Cyanide and calcium deficiency cause both an increase in weight and an ultimate osmotic equilibrium between internal and external media, both of which are reversible. It cannot be said whether or not these two effects are interdependent, since it was not possible to measure more than once the weight and internal concentration in the same animal, and histological examination revealed no abnormality other than a swollen body cavity. These experiments were therefore not as illuminating as in the case of Gunda ulvae (Beadle, 1934), but they furnished a convenient method of demonstrating the re-establishment of hypertonic body fluids in an isotonic medium. It has been concluded from the results described in the last section that the majority of the extra respiration in dilute sea water is not concerned with osmotic work. Pieh (Schlieper, 1935), from his experiments on Carcinus moenas and Eriocheir stnensis, has suggested that the rate of respiration is dependent upon the water content of the tissues. It does not seem, however, that this could apply to Nereis diversicolor, since there is no obvious relation between the rate of respiration and the concentration of the body fluids. It has previously been shown (Beadle, 1931) that the respiration and weight curves of the same animal in dilute sea water are of

14 Adaptation to Changes of Salinity in the Polychaetes 69 similar form and that both have a roughly simultaneous maximum and subsequent fall. This suggests a relationship between the degree of swelling and the respiratory rate. It is therefore possible that the increase in the latter is the result of work done by the body wall muscles in resisting an increase of volume. If this were true, animals which swell to an unusual extent might be expected to show little increase in the rate of oxygen consumption (e.g. Table IV d). From former experiments it was concluded (Beadle, 1931) that the effect of cyanide in causing an increase of weight in dilute sea water was evidence that the extra oxygen consumption is connected with osmotic regulation. In view of the lack of connexion between the weight and internal concentration this conclusion can no longer be upheld. The osmotic regulatory mechanism may entail oxygen consumption, since it is inhibited by cyanide, but it is possible that the effect of cyanide upon the weight is a separate phenomenon due to inhibition of the body wall muscles. SUMMARY 1. Nereis diversicolor collected from the same locality at different times showed smaller weight increases in dilute sea water (25 per cent) during the winter than during the summer months. 2. In spite of great variations in the weight curve, the body fluid concentration curve was very constant. 3. The maintenance of hypertonic body fluids and the regulation of body volume are largely unconnected. 4. The lowering of the weight curve below that theoretically expected from the concentration curve cannot be attributed to passive salt loss through the body surface. It is suggested that this is due to the removal of fluid through the nephridia under the hydrostatic pressure produced by the contraction of the body wall muscles. 5. Animals previously subjected to dilute sea water, when placed in water isotonic with the body fluids, will increase the concentration of the latter. This result is more marked when the internal hydrostatic pressure is high. 6. The results suggest that the osmotic regulatory mechanism involves the removal by the nephridia of fluid hypotonic to the body fluids. But no direct evidence for this is available. 7. Calcium deficiency and cyanide in dilute sea water cause an increase of weight and ultimately inhibit the maintenance of hypertonic body fluids. Both these effects are reversible. 8. The mechanism by which body fluids are maintained hypertonic to the external medium is not sufficiently developed to be of survival value in the locality in which the animals were found. 9. The control of body volume is probably of greater importance. 10. The majority of the extra oxygen consumption in dilute sea water is not the result of osmotic work. It is suggested that it may be due to work done by the body wall muscles in resisting swelling.

15 70 L. C. BEADLE The cost of the apparatus used in these experiments was born by a grant from the Beaverbrook Fund of the University of Durham College of Medicine. REFERENCES BALDES, E. J. (1934). J. td. Inttrum. 11, 223. BATKMAN, J. B. (1933). J. exp. Biol. 10, 355. BEADLE, L. C. (1931). J. exp. Biol. 8, 211. (1934). J. exp. Biol. 11, 382. BETHE, A. (1934). PflUg. Arch.ges. Pkytiol. 234, 629. ELLIS, W. G. (1933). Nature, Lond., 132, 748. HERRMANN, F. (1931). Z. vergl. Pkytiol. 21, 214. NAGEL, H. (1934). Z. vergl. Physiol. 21, 468. SCHLIEPER, C. (1929). Z. vergl. Phytiol. 9, 427. (1935)- Biol. Rev. 10, 334. SCHLIEPER, C. & HERRMANN, F. (1930). Zool.Jb., Abt. 2., 62, 624. SCHOLLES, W. (1933). Z. vergl. Pkytiol. 19, 522.