Solute and Water Balance in the Muscle Fibres of the East African Fresh-Water Crab, Potamon Niloticus (M. Edw.)

One of the important factors in the penetration of marine animals into brackish water and eventually into fresh water has been the ability of the cells of the organism to withstand and adapt themselves to a very great reduction in the total concentration of the blood. There are obviously many ways in which this might be accomplished. In the case of the muscle fibres of the marine and estuarine crab, Carcinus maenas, cell adaptation involves the reduction of the osmotic activity of the cell contents by the removal of certain small nitrogenous compounds, such as free amino acids (Shaw, 1958 a).

It is possible that the ability of cells actively to regulate their composition in response to changes in blood concentration, and hence preserve the normal water balance of the cell, is a special characteristic of brackish water animals. These animals normally withstand large changes in the composition of their environment, and it is of great interest to know whether these abilities are still possessed by freshwater animals, which have been derived from marine forms but are now living in a much more stable environment.

In the case of the crab, Eriocheir sinensis, which spends much of its life in fresh water but returns to the sea to breed, the muscles from the marine animals contain greater amounts of free amino acids than those in animals from fresh water (Du-château & Florkin, 1955), although it is not known to what extent these changes are due to changes in water content.

It has been suggested (Ramsay, 1954) that one of the reasons why many freshwater animals are unable to survive in concentrations of sea water much above that of the normal blood concentration, is due to the fact that the muscles are unable to increase their amino acid content.

The East African fresh-water crab, Potamon niloticus, is a very favourable animal on which to pursue this question further. In the first place, it is restricted to fresh water and shows the characteristic inability of fresh-water animals to survive in sea water (Shaw, 1959). In the second place, it is reasonably closely related to Carcinus and therefore the behaviour of its muscle fibres may be compared with that of Carcinus muscle fibres under similar circumstances.

The crabs were collected from the shores of Lake Victoria, at Entebbe and maintained in a laboratory aquarium with circulating lake water. For experiments on the effect of different concentrations of sea water on the animals, specimens were kept, singly, in jars each containing about 500 ml. of the appropriate solution, which was changed at frequent intervals. The crabs remained in the experimental solutions for a week or longer, except in the case of those animals in undiluted sea water. In this solution the animals died within a week and so they were removed from the solution for analysis after only 3 days. It cannot be certain, therefore, that in these animals steady-state conditions were established fully at the time of the analysis. Since natural sea water was not available, all the experimental solutions consisted of artificial sea-water solutions made up from a mixture of standard salt solutions as described below. None of the solutions contained sulphate. In some experiments magnesium-free solutions were used without any noticeably different effects on the animals or on their muscles. The effects described, therefore, cannot be attributed to increased concentrations of magnesium or sulphate ions in the blood.

The procedure for the muscle analysis followed that used for similar analyses on Carcinus muscle (Shaw, 1955a, b 1958a, b). As with Carcinus, the carpopodite extensor and flexor muscles were studied and each muscle was prepared for analysis in the form of a group of washed and separated single fibres. The analyses were thus unaffected by contamination from surrounding blood and so the results could always be directly expressed in terms of intracellular concentrations. Analytical methods were identical with those used previously.

The concentrations of the most important osmotically active substances in the muscle fibres of crabs taken from their normal environment is shown in Table 1. The ionic composition of the muscle shows many close resemblances with that of the muscles of marine Crustacea, like Carcinus (Shaw, 1955 a, b; 1958b) and Nephrops (Robertson, 1957). It is characterized by high concentrations of potassium and organic phosphate compounds but relatively low concentrations of sodium and chloride. As in the marine forms, the balance between the measured cations and anions is satisfactory: in Potamon the cation charge is 155 m-equiv./kg. water compared with the anion charge of 145 m-equiv./kg. water at pH 7·0. Compared with typical muscle analyses of fresh water or terrestrial vertebrates, this freshwater crustacean differs in that the concentrations of sodium and chloride, although low compared with those in the blood, do make a significant contribution to the osmotic activity of the muscle fibre.

The similarity in the ionic composition of the muscle in Potamon and Carcinus extends beyond a mere qualitative likeness in the relative concentrations of the constituent ions : the concentrations of ions in Potamon muscle show a remarkable similarity to those in the muscles of Carcinus, taken from 40 % sea water, and this extends also to the water content of the muscle. In fact, if Potamon has evolved from a marine decapod, similar to Carcinus and with a similar muscle composition, then in the course of its history there has been little change in the muscle composition from that which existed at the time of the initial penetration of brackish water by its ancestors. There has been no return to the original potassium or phosphate concentrations nor a re-establishment of the normal water content.

The inorganic ions and the organic phosphate compounds account for not much more than 50% of the total osmotic activity of the fibre, if this is assumed to be iso-osmotic with the blood. The total contribution from muscle ions may be a little greater if all ions, such as calcium, magnesium, bicarbonate, etc., were to be included, but there is a deficit of, at least, 200 m-osm./kg. water. This is largely accounted for by the measured organic compounds, the amino acids and trimethylamine oxide, which together account for at least 167 mM./kg. water. The remaining deficit may take the form of betaine, which was found in large quantities in lobster muscle (Kermack, Lees & Wood, 1955) or it may be some as yet unidentified substance. The total concentration of the non-protein nitrogenous substances in the muscle is much smaller than in the marine forms. Thus in Carcinus muscle the total concentration of the amino acids with taurine and trimethylamine oxide, but excluding arginine is 524 mM./kg. water (Shaw, 1958 a) compared with the 167 mM./kg. water in Potamon. This lower value is probably characteristic of the fresh-water decapod Crustacea. The total free amino-acid concentration of two other fresh-water crustaceans, Astacus fluviatilis and Eriocheir sinensis, has been estimated at 153 and 239 mM./kg. water, respectively (Camien, Sarlet, Duchâteau & Florkin, 1951) and these are of the same order as found in Potamon.

For the purposes of these experiments animals were maintained in a standard artificial sea water made up in the following manner. About 11. of the solution was made by mixing 801-9 ml. of o·6 M-NaCl, 17·2 ml. of o·6 M-KCI, 27·2 ml. of 0·4 M-CaCl₂ and 139·6 ml. of 0 4 M-MgCl₂. The sodium concentration of the final solution was 490 mM./l. and the potassium concentration, 10·4 mM./l.

The experimental solutions were prepared from this standard solution in the following dilutions: 100, 75, 50 and 25 %. The animals remained in the last three solutions for at least 7 days and in the 100% solution for 3 days. After removal the muscles were analysed for water content, acid-soluble phosphate compounds, potassium, sodium and free amino acids (actually, non-protein a-amino-N compounds). Penetration of salts from the higher sea-water dilutions caused increases in the blood concentration and the effect of these changes on each of the muscle constituents has been studied.

The water content of the muscles was calculated from the difference between the wet and dry weights and thus the weight of water in the muscle was measured. The water contents, expressed as a percentage of the wet weight of muscles of animals from the different external solutions are shown in Table 2.

A simple hypothesis to account for the effects of increased blood concentration on the water balance of the muscle fibre is that the cell functions as a simple osmometer. In this case, the amount of water withdrawn from the cell is related to the increased blood concentration in a definable manner. Thus, if the normal blood concentration = C and if the blood concentration is increased to nC then, assuming that (a) all the muscle water is solvent water, and (b) that there is no loss of dry substance from the muscle, the new water content of the muscle as a percentage of the original water content = 100/n.

The water content of muscles of crabs from the four experimental solutions was calculated in this way from the measured water content and from the mean value of the water content of the normal muscle, taken from Table 2. The results are shown in Fig. 1. The points joined by the continuous line are those calculated on the osmometer hypothesis from the mean value of the water content of the normal muscle and from the mean value of the freezing-point depression of blood of the animals from the four experimental solutions. The open circles are the actual experimental measurements of the muscle water content calculated in terms of the water content of the normal muscle (as above).

A significant divergence is apparent in sea-water concentrations greater than 50%. In both 75 and 100% solutions a considerable dehydration of the muscle has occurred, but it is not so extensive as predicted by the simple osmometer hypothesis. In 75 % sea water about half the predicted water loss has occurred, but in full-strength sea water the dehydration has been more complete. The divergence between the measured and calculated water content suggests that the osmotic activity of the muscle fibres has been increased partly by the withdrawal of water, but also partly by the addition of osmotically active solutes. In 75 % sea water, the addition amounts to about 200 m-osm./kg. water, whereas in the undiluted sea water only just under half this amount has been added.

The measurements of muscle phosphate were made on trichloracetic acid extracts of dried muscle. The diluted extract was made normal with respect to hydrochloric acid and hydrolysed on a boiling water bath for 10 min. This treatment hydrolyses arginine phosphate and adenosine triphosphate so that the final solution contained the inorganic phosphate originally present together with phosphate derived from these two sources. These fractions accounted for the large majority of the acidsoluble phosphate compounds in Carcinus muscle (Shaw, 19586). To circumvent the difficulty of the change of water content of the muscles, the total phosphate concentrations were measured in terms of the dry weight and the effect of increasing blood concentrations was studied. The results are shown in Fig. 2, where the total acid-soluble phosphate content is related to the blood concentration, as measured by the freezing-point depression. It is clear that the measured blood concentrations fall into two distinct groups: the group with blood concentrations less than 350mM./I. NaCI equiv, are from crabs in 25 and 50% sea water; the remainder are from crabs in 75 and 100% sea water. There is no indication that the muscle phosphate content has been increased following an increase in the blood osmotic pressure. A statistical test (the ‘t’ test) for a comparison between the two groups showed no significant difference (t= 1· P greater than 0· 05). The behaviour of the phosphate fraction appears, therefore, to be passively determined by changes in the water content of the muscle : there is no evidence of regulation of the concentration of these substances. In this respect the behaviour of the phosphate compounds is similar to that found in Carcinus muscle (Shaw, 1958b).

Fig. 3 illustrates the relation between the potassium content of the muscles, on a dry weight basis, and the blood concentration. As with the phosphate compounds there is little evidence of a change in the potassium content of the muscles over the range of blood concentrations which have been studied. Statistically, the two groups, again, show no significant difference between their means (t = 2·07 ; P greater than 0·05). As in Carcinus muscle, the behaviour of the potassium ions resembles that of the phosphate compounds. There is no evidence of regulation of the potassium concentration and this appears to be determined solely by water movements.

The free amino-acid content was measured on trichloracetic-acid extracts of the dried muscle by the Folin-Danielson method for a-amino nitrogen compounds. The relation between the amino-acid content and the blood concentration is shown in Fig. 4. At the higher blood concentrations there is a small but significant increase in the amino-acid content. The difference between the means of the low and high blood-concentration groups is statistically significant, although not at a high level (t = 2·5; P=0·02). A small quantity of amino compounds has been mobilized and added to the acid soluble fraction in response to an increased blood concentration. Table 3 shows the concentration of these compounds per kg. water content in the experimental muscles, and also the concentration which would have been expected if the change in water content had been the only operative factor.

The expected values have been calculated from the mean concentration in normal muscles (Table 1), the mean water content of these muscles and the mean water content of the muscles from the experimental animals (Table 2). It is noteworthy that the total concentration does not rise much above 300 mM./kg. water and that this concentration may be found in the animals from 75 % sea water. The greater part of this increase is due to the removal of water from the muscle, but a small addition to the osmotically active pool of 50·90 mM./kg. water has been made in the muscles of the animals from 75 and 100% sea water. Since in the animals from undiluted sea water the amount added does not exceed that found in the animals from 75 % sea water where the muscle dehydration is less severe, it seems probable that this represents the maximum amount which can be brought in to boost up the normal level.

Fig. 5 shows the sodium content of the muscles calculated on a dry-weight basis and the relation to the total concentration of the blood. At the higher blood concentrations there is a considerable increase in the sodium content of the muscle.

The difference between the means of the high and low blood-concentrations groups is a highly significant one (t = 5 ; P less than o-oi). Sodium has penetrated into the muscle as a result of the increased blood sodium concentration. In Carcinus muscle it was found that the muscle sodium was probably present in a region of the muscle, where it was freely exchangeable with the sodium of the blood and did not penetrate into the fibre interior (Shaw, 1958b). The concentration of sodium in the muscle was approximately proportional to that of sodium in the blood (Shaw, 19556). In Fig. 6, the relation between the sodium concentrations of muscle and blood in Potamon is shown. The dotted line in the figure represents a linear relation between the two variables and passes through the origin and through the normal mean sodium concentration of the muscle (44 mM./kg. water). It is possible that Potamon muscle behaves like that of Carcinus at blood sodium concentrations not exceeding 450 mM./l. At higher sodium concentrations the penetration is greater than can be explained in terms of a simple proportionality with the blood sodium concentration. It is probable that at these high concentrations penetration of sodium into the fibre interior takes place.

Since potassium, phosphate compounds, nitrogenous substances and sodium contribute largely to the total osmotic activity of the muscle fibre, it is possible to discuss the behaviour of these substances in relation to the problem of the adaptation of the muscle cell to conditions of increased blood concentration. Potassium and phosphate behave passively with respect to water movements, make no contribution towards cell adaptation and are not themselves regulated. Unlike those of Carcinus (Shaw, 1958 a), the muscle fibres of Potamon niloticus do not possess the ability to vary the content of free amino acids and related compounds over a very large range. Since the amount of these substances which can be added does not exceed 50-90 mM./kg. water, little increase in the osmotic activity of the fibres can be produced by this means. It is, however, interesting to find that some increase is possible and this may well be a legacy from the earlier marine ancestry of these animals.

Apart from increasing the amino acid content of the muscles, osmotic equifibration with the blood is achieved by the osmotic withdrawal of water and by the penetration of sodium (probably with chloride) into the fibre. In animals from 75 % sea water the penetration of sodium is probably restricted to those spaces where free exchange of sodium is normally possible and the breakdown of the mechanism excluding sodium from the fibre interior does not occur. The addition to the amino-acid pool is sufficient to prevent excessive dehydration of the fibre and this probably accounts, in part, for the difference between the predicted and measured water contents of these muscles (Fig. 1). However, the added amino compounds (about 80 mM./kg.), together with the extra sodium (about 30 mM./kg.) and probably an equal amount of chloride, falls somewhat short of that required to account for the increased osmotic activity, and a small amount of some unknown osmotically active substance may also be added.

In the muscles of animals from full-strength sea water there is no further addition of free amino acids or sodium and the additional increase in blood concentration causes extensive dehydration of the muscle and the penetration of sodium into its interior. These changes may be highly significant in view of the fact that the animals are unable to survive in 100% sea water, although they can do so in the lower concentrations.

The cause of death in full-strength sea water is at present unknown. It seems unlikely that it is due to general physiological causes : for example, variations in the ionic composition of the sea water have little effect on the animals and, similarly, the termination of urine production (which has often been advanced as an explanation of the death of other fresh-water animals under similar circumstances) can hardly be important in the case of Potamon over a short period, since the normal rate of excretion is extremely slow (Shaw, 1959). It is not unreasonable to suppose that the lethal effect of sea water is due to the action of the raised blood concentration on the cells, although whether all cells are affected in the same way or whether some are more adaptable than others cannot yet be surmised. If the behaviour of the muscle cell is typical of all other cell types, it may well be that the degree of cell dehydration and the maintenance of the normal sodium-excluding mechanism are factors which determine the correct functioning of the cell.

This work was carried out in the Department of Zoology, Makerere College, Uganda, while the author was in receipt of a Royal Society and Nuffield Commonwealth Bursary. It is a great pleasure to acknowledge the hospitality and assistance given by Prof. L. C. Beadle during a most enjoyable stay in his Department.