The Regulation of the Internal Sodium Concentration of Asellus Aquaticus in the Absence of Sodium Chloride in the Medium

Many small freshwater animals survive in distilled water for a considerable time. The factors concerned in the maintenance of the ion concentrations in the haemolymph at levels compatible with life under these circumstances have not been fully examined.

Beadle & Cragg (1940), measuring the haemolymph concentration of Gammarus pulex which had been in distilled water for various periods, showed that there is initially a fairly rapid fall in the haemolymph chloride concentration followed by a long period during which the concentration falls very slowly. They have therefore suggested that retention of ions may play an important part in the maintenance of the ion concentrations in the haemolymph.

In the present paper details are given of the effect of treatment with distilled water on the haemolymph concentration of the isopod Asellus aquaticus. In addition, a study has been made of the actual rate of loss of sodium from the body of this animal to demonstrate that the maintenance of an almost constant haemolymph concentration does not necessarily imply retention of ions.

The Asellus used were in general large males, though a few unberried females were also included. Specimens were from a drainage ditch on Coe Fen, Cambridge. The animals were not fed during the course of the experiments. It has been shown that a starvation period of 8 days does not affect the ionic or total concentration of the haemolymph (Lockwood, 1959).

Osmotic pressure was estimated by the method of Ramsay & Brown (1955), sodium by flame photometry and chloride by the first method of Ramsay, Brown & Croghan (1955). For details of procedure and method of haemolymph collection see Lockwood (1959).

In the earlier experiments on the effect of ion lack on the haemolymph concentration the animals were kept in a 700 ml. chamber through which a slow current of distilled water was passed. The purity of the water in the chamber was tested at intervals and the sodium concentration was not found to rise above 4 μequiv./l. In later experiments smaller chambers of 3–10 ml. were used and the incoming stream of water (5-25 ml./min.) was first passed through a column of Amberlite M.B. 3 ion-exchange resin. The de-ionized water prepared in this way did not contain more than 1 μequiv./l. sodium.

Animals used in tracer experiments were also kept in the 3–10 ml. chambers which were mounted on a tray designed to fit into an E.R.D. lead castle. The activity of tracer in the animals could thus be readily determined. The water was de-ionized with resin and lifted by an air pump to a Polythene storage tank. Continuous aeration and circulation were thus achieved. All the tubing used was Polythene.

The fact that the animal has freedom of movement in the chamber entails the disadvantage that the thickness of water between the animal and the counter is liable to vary from time to time. This results in a variable degree of absorption of β-particles by the water. Errors arising from this source were reduced by interposing a brass screen (990 mg./cm.²) between the chamber and counter. This screen absorbs all the soft β-particles but allows a high and constant proportion of the γ-rays to pass. Absorption of γ-rays is not appreciably altered by the small changes in water thickness.

A standard was counted before and after the animal to correct the count rate and decay, and in addition the usual corrections for background and dead time were applied.

All experiments were carried out at room temperature (15-22° C.). In the first series of tracer experiments animals were loaded in a solution containing ²²Na until their tracer count reached a steady state. This took about 3 weeks and food was provided during the loading period. In a later series of experiments animals were loaded for only a few hours before being washed out with de-ionized water.

Most animals survived in distilled water for 6-8 days, the longest observed period of survival being 16 days. Osmotic pressure (0·P·1) falls rapidly during the first day, but after this time only a very slow decrease in the concentration occurs (Fig. 1). This appears to suggest that the rate of salt loss has been much diminished as the ratio Na/o.p. remains constant (Lockwood, 1959). Less haemolymph can be obtained from animals which have been washed out for a few hours than from normal controls of a similar size.

The initial rate of fall of Na₁ is about 1·6% Na₁/hr. This is probably close to the rate of loss by diffusion and in the urine in fresh water, since the diffusion gradient is little altered by transferring the animals to distilled water. As Na₁ decreases so does the concentration gradient between haemolymph and medium. This would be expected to result in a gradual rather than a rapid decrease in the loss of salt from the body by diffusion. However, after the first day in distilled water Na₁ falls only very slowly.

The possibility that a true measure of the rate of salt loss from the body is not obtained by measurements of the haemolymph concentration was therefore tested by measuring the rate of loss of ²²Na from loaded animals. After being loaded to a steady state the animals were placed in small plastic chambers and washed with de-ionized water. The count rate was recorded at intervals. The curve of the loss of ²²Na from the body differed in shape from that of the Na₁. The initial rapid fall and levelling out were now replaced by a slower fall, and only a comparatively slight decrease in the rate of loss with time (Fig. 2). Four repetitions of the experiment, though differing quantitatively, confirmed this difference.

Loss of ²²Na is proportional to rate of sodium loss from the body. It seems, therefore, that sodium is lost at a rate which only declines with time to about a quarter or half the initial value. Clearly sodium is not being retained in the body to the extent that a study of the concentration of ions in the haemolymph might appear to indicate.

In a number of cases animals were loaded in ²²Na for only a few hours before being washed out. The curve of falling ²²Na obtained from these animals differed in a number of respects from that of the long-term loaded animals. The ²²Na falls rapidly to about 40-50% of its initial value, and then only a very slow loss occurs for a long period (Fig. 3). In some cases the loss may again increase slightly before the death of the animal. Determinations of the 0.P.1 and Na₁ of animals which had lost 50-60% of their initial ²²Na showed that the haemolymph concentration was still 60-65% of normal.

If all the sodium in the body of Asellus were in the haemolymph, then the length of the initial loading period in tracer could not result in any qualitative difference in the curves of loss of ²²Na on washing with de-ionized water. These results are therefore incompatible with the assumption that all the sodium in the body of Asellus is in the haemolymph.

Beadle & Cragg (1940) have postulated that retention of ions is an important feature of the survival of such animals as Gammarus pulex in diluted media. Like Gammarus, Asellus has been found to maintain a remarkably constant haemolymph concentration (after an initial rapid fall) for a long period in running distilled water. However, the fact that the rate of loss of ²²Na from long-term loaded animals in distilled water is only decreased slightly with time indicates that the constancy of the Na₁ does not give a true picture of the actual loss of sodium from the animal. In Asellus, at least, a decreased rate of loss of ions is not primarily responsible for the maintenance of the Na₁ when the animal is in distilled water.

Two possible theories could explain the apparent anomaly between loss of sodium and maintenance of Na₁. Either (a) sodium from the tissues replaces that lost from the haemolymph and thus maintains Na₁, or (b) the volume of the haemolymph is decreased as the concentration falls so that the concentration is maintained at a higher level than would be expected from the amount of sodium lost from the body.

As the short-term and long-term loaded animals give curves of different shapes on washing out, it appears that a considerable proportion of the total sodium in the body is not rapidly exchangeable and hence presumably is not in the haemolymph. There is as yet no evidence to suggest that this extra-haemolymph sodium may be released to assist in the maintenance of the haemolymph concentration.

Attempts to determine changes in the haemolymph volume directly have not been successful as the total amount of haemolymph in Asellus is small. The observation, however, that it becomes difficult to obtain normal quantities of haemolymph from washed-out animals, though qualitative, may indicate that there is some reduction of the haemolymph volume as the concentration falls.

Camien, Sarlet, Duchâteau & Florkin (1951) have shown that a considerable part of the osmotically active substances in the cells of Crustacea are organic in nature. Any reduction in the haemolymph concentration would therefore be expected to result in a movement of water into the cells. Shaw (1955) has shown that the muscle cells of Carcinus swell as the result of just such a water shift when the haemolymph concentration is lowered. It is thus not unreasonable to suppose that a similar water shift to the cells occurs in the case of Asellus. If the cells are regarded as being always isotonic with the haemolymph, the decrease of haemolymph volume would be sufficient to account for the maintenance of haemolymphion concentration.

Active processes may possibly play some part in the slight decrease in the rate of loss of sodium from the body, but unfortunately no details are available as to the effect of a reduction in the haemolymph concentration on the output and concentration of the urine.

The presence of a considerable quantity of sodium in the tissues is an unusual feature of Asellus. This subject will be dealt with in detail in a later paper and will not be considered further here.

I would like to thank Dr J. A. Ramsay, F.R.S., for all his interest and advice in this work, and Dr P. C. Croghan for many helpful discussions. I am indebted to the Department of Scientific and Industrial Research for a maintenance grant and the Earl of Moray Endowment for the Promotion of Original Research for a grant for instruments.