Osmoregulation in Some Freshwater Prawns

The different species of prawns of the family Palaemonidae are to be found in a range of environments from fresh water to marine, and some individual species are able to tolerate changes over almost the whole of this range. Two such species in Britain are Palaemonetes varians (Leach) which may live in very dilute brackish water, in saline marsh pools and in any salinity between these extremes; and Palaemon longirostris (H. Milne-Edwards) which may extend over a similar range. The distribution of these two species in Britain appears to stop just short of a completely freshwater environment (Gurney, 1923; Panikkar, 1941), but P. longirostris is reported from fresh water in Europe (Holthuis, 1950) and P. varians as varieties macrogenitor (Boas, 1898), termajophüus (Garbini, 1881; Seurat, 1922), or lacustris (Sollaud, 1923, 1933) is found in southern Europe. A recent review of the family (Holthuis, 1950) has accorded specific status to this southern European freshwater form as Palaemonetes antennarius (H. Milne-Edwards).

Osmoregulation in Palaemonetes varians has been studied by Panikkar (1941), but very little physiological information is available for Palaemon longirostris or Palaemonetes antennarius. Predictions of the osmoregulatory activity of the former were made by Gurney (1923), Schnakenbeck (1933) and Panikkar (1941), but there is no experimental work; while for the latter species there exists only a single record of the blood freezing-point made by Vialli (1925).

In this paper an account is given of some aspects of osmoregulation in P. antennarius, together with some comparative observations on P. varians and Palaemon longirostris.

Palaemonetes antennarius was collected from two localities near Verona in North Italy: at Caldiero, a thermal spring in the Po valley, and at Peschiera where the River Mincio flows out from the Lake of Garda. The former source has water of some hardness and a temperature of 28°C. (Frederici, 1948) (c. 200 mg./l. CaCO₃, and appreciable amounts of magnesium and sulphate), while the latter has water of a lower mineral content (Marchesoni, 1952) (112 mg./l. CaCO₃).

P. varians in these experiments was collected from brackish farm drainage ditches near Breydon water (Norfolk), where the chlorinity varied from 0·38 to Cl (10·8 to 28·4 mM./l. Cl). Many freshwater invertebrates were present in the same ditches.

Palaemon longirostris was obtained from the River Tamar (Devon) just downstream from the Calstock Bridge. The chlorinity at low water was 0·67 ‰ Cl (18·8 H1M./1. Cl).

Measurements of the freezing-point depression were made with the apparatus described by Ramsay (1949) and Ramsay & Brown (1955). The freezing-point depression may be converted into terms of salt concentration by means of the relationship Δ/° C. ≡285 mM./l. NaCl (Ramsay, 1949). Chloride was estimated in the blood and urine of P. longirostris by the method of Sendroy (Sendroy, 1937; van Slyke & Hiller, 1947), in which the chloride in solution is replaced by iodate and determined iodometrically. The remainder of the chloride measurements were made by a microdiffusion technique (Conway, 1951). Copenhagen sea water was used throughout as a standard.

The measurements of the rate of urine flow were made by a dye excretion method described in a previous paper (Parry, 1955).

The freezing-point measurements of the blood and urine of the three species in fresh or slightly brackish water show that these two fluids are always approximately isosmotic, and are maintained at a level of concentration very much higher than that of their respective media (Table 1). The figures for Palaemonetes varians confirm the level of the mean for the blood of animals in a similar environment given by Panikkar (1941), who used a Hill-Baldes thermo-electric method. The measurements of blood and urine in Palaemon longirostris confirm that this species maintains the concentration of its body fluids at about the same level as Palaemonetes varians.

On the other hand the freezing-point depression of the blood of P. antermarius is of the same order as that of other freshwater decapods, e.g. Cambarus clarkii Δ = 0·64°C. (Lienemann, 1938) and Astacus astacus Δ = 0·8o° C. (Schwabe, 1933), but it is lower than that found for Eriocheir sinensis in fresh water, Δ=1·18° C. (Scholles, 1933), or for Potamon edule in fresh water, Δ=1·17° C. (Duval, 1925), or of Palaemonetes varians or Palaemon longirostris in the lowest level of salinity they will tolerate. The urine of Palaemonetes antennarius is approximately isosmotic with the blood as it is in the brackish water prawns and in contrast to the dilute urine produced by the freshwater crayfishes.

The chloride analyses (Table 2) show that in each of the three species the chloride concentration of the urine is of the same order as that of the blood, and they confirm the high salt content which was to be expected from the freezing-point measurements. However, the proportion of the total osmotic pressure contributed by chlorides varies between the different species. In P. antennarius the figures for chloride are low compared with those of the other two species, even when the lower value of the freezing-point depression is taken into account. If the concentrations of blood and urine are calculated solely in terms of sodium chloride, and the concentration of chloride calculated from this, it will be found that in P. varians and Palaemon longirostris the ratio Cl estimated : Cl measured is near to unity (0·85–1·20), while the ratio for Palaemonetes antennarius is only 0·56 for blood and 0·66 for urine. This suggests that chlorides are relatively less important than other constituents in maintaining the high osmotic level of the body fluids in this freshwater species.

The differences between the figures for chloride concentration in P. antennarius from the two environments are not statistically significant.

Two sets of experiments were designed to measure the rate of loss of salts from P. antennarius when in distilled water. The first, using animals from both Peschiera and Caldiero, entailed measuring the conductivity of 200 ml. of water in which five animals (c. 0·4 g.) were kept. The water was changed hourly, and the quantity of chloride lost in these hourly intervals was calculated. The apparatus was calibrated with dilutions of standard sea water. Examples of these experiments are shown in Table 3. For animals from both environments the rate of loss of salts calculated from these experiments was 34·2μM. NaCl/g./hr. (where the total salts lost are expressed as μM. NaCl).

If the same weight of animals (c. 0·4 g.) is put into 200 ml. of distilled water, and the medium is not changed, the animals lose salts at a decreasing rate. Prawns from Caldiero reach a steady state when the medium has a concentration of 0·99 mM./l. Cl (c. 24 hr.) after which the animals survive for many days. The prawns from Peschiera, on the other hand, show a similar decreasing rate of loss, but when the concentration of the medium was 0·42 mM./l, Cl the animals died (c. 10 hr.).

A second experiment to measure the rate of loss and gain of salts from the blood was made on animals from Peschiera. In this experiment, single animals (c. 0·1 g.) were kept in 100 ml. distilled water changed at 2-hourly intervals. Small samples of blood were taken for measurement of freezing-point. After 6 hr. the osmotic pressure of the blood had been appreciably reduced. On replacing these salt-depleted animals in their natural medium (Peschiera water) the blood concentration gradually regained its normal level in about 5 hr. (Table 4).

In order to calculate from this data the rates of loss and gain of salts it will be assumed that the blood volume in ml. is one-third of the body weight in g. This gives: loss, 2·21μM./g./hr.; gain, 2·56μM./g./hr.

The rate of urine flow was determined by injecting indigo-carmine into the haemocoele and measuring the rate of its elimination. Prawns from Peschiera and Caldiero were used, and a slightly different rate of flow was measured for each, but this difference does not appear to be significant. These rates may be compared with that previously found for P. varians in a dilute medium and the one measurement (obtained by direct cannulation of the gland) for Palaemon longirostris (Table 5). In a previous paper (Parry, 1955) it was estimated that if Palaemonetes varians was to live in completely fresh water its urine flow would be about 2 % body weight/hr. The rates measured for both Peschiera and Caldiero animals are very close to this.

This apparently high rate of urine flow in these prawns is in contrast to the rate reported for other freshwater decapods. In Cambarus and Astacus the rates are only one-tenth of this (Scholles, 1933; Lienemann, 1938). In Eriocheir and Potamon, which like Palaemonetes cannot produce a hypo-osmotic urine, the urine flow is said to be very low (Scholles, 1933; Schlieper & Herrmann, 1940). It is clear that while Palaemonetes may have saved some osmotic energy in the reduction of the blood concentration (Potts, 1955) it is losing salts constantly in the excretion from the antennal glands.

Palaemonetes antennarius has often been described as a variety of P. varians (Garbini, 1881; Boas, 1889; Seurat, 1922; Sollaud, 1923), but more recently has been given the status of a separate species, although Holthuis (1950), in speaking of this and some other species, is uncertain (on morphological and embryological grounds) whether they can be maintained as separate species. Measurements of the freezing-point depression and chloride concentration of blood and urine of P. antennarius indicate that this animal is distinct (on physiological grounds) from P. varians.

Although of limited distribution P. antennarius is not a rare animal and must be regarded as a successful inhabitant of fresh water. Unlike P. varians, it can live in completely fresh and relatively soft water; unlike Palaemon longirostris it does not return to salt waters, for breeding. At the same time, however, it lacks those adaptations to a freshwater environment which have been achieved by the freshwater crayfishes, for while its blood concentration is as low as that of many other freshwater inhabitants, it continues to produce an isosmotic urine in a copious flow. Scholles (1933) failed to find any modification in the size or structure of the antennal glands, compared with those of the brackish Palaemonetes varians, in contrast to the enlarged glands found in the freshwater crayfishes or gammarids.

Experiments designed to show the rate of loss of salts from the blood in distilled water and of gain in a very dilute natural water show that loss and gain are of the same order, and there is little doubt that under natural conditions there is a constant absorption of ions to compensate the osmotic inflow of water and the loss of salts through the antennal glands.

P. antennarius thus appears to be a successful invader of fresh water, but one which is incompletely adapted to this environment, presumably at considerable energetic expense. While distinct from its near relative P. varians, it has changed relatively little physiologically or morphologically. If it is as geographically isolated as it appears, it would be an interesting evolutionary study to await the development of further distinctions. Comparison of the two local populations at Caldiero and at Peschiera gave indications of small differences in osmotic behaviour, but it was not possible to show that these differences were statistically significant.

My grateful thanks are due to the Central Research Fund of the University of London for covering expenses for a visit to Northern Italy; to Prof. V. Tonolli for his kind hospitality on several occasions at the Istituto di Idrobiologia, Pallanza; and to Prof. S. Ruffo, Verona, for assistance in collecting the prawns.