Palaemonetes varians, variety microgenitor, commonly inhabits only saline water around the British coast. It is, however, abundant in water of extremely low salinity in the Cardiff area, where it occurs in the drainage ditches of farm land adjacent to an area of salt-marsh.
Animals from areas of both high and low salinity were subjected, in the laboratory, to a range of salinities varying from pure tap water to concentrated sea water. The rate of respiration at each salinity was measured.
The population that inhabited the almost fresh water in the ditches differed physiologically from the salt-water form, although morphologically they were identical.
The salt-water population showed a minimum respiratory rate when in a medium of 26‰, NaCI salinity. This was expected, since the animal is isotonic at this salinity and the osmoregulatory work is minimal. The second population respired least when in a medium of 6‰o NaCI. The significance of this is discussed.
Palaemonetes varians has been classified into distinct varieties based on the size of the egg produced. Variety macrogenitor, a fresh-water species, is found in the Mediterranean region but has never been recorded in Britain. The second, variety microgenitor, produces a smaller egg and is always found in water containing a certain amount of salt. It has an extensive distribution around the British coasts, being particularly abundant in areas of salt-marsh. Whilst studying the distribution in one such area (Lofts, 1956), it was found that in addition to being widespread in the highly saline marsh-pools, the animal was also present in a system of drainage ditches traversing cultivated land adjacent to the marsh. Here the water was of very low salinity.
The blood concentration of P. varians is markedly hypotonic to the medium when in normal sea water (Panikkar, 1939), but this condition changes when the medium is brackish or fresh water. Under these conditions the body fluids remain in a state of definite hypertonicity. Isotonicity is attained when the external medium is equivalent to about 25 g. NaCl per 1. (Panikkar, 1941). The average yearly salinity of a marsh-pool is 23·5%, NaCl, thus the prawns are usually in a state of isotonicity with their medium, being slightly hypertonic in the winter when the pool is diluted by rain, and slightly hypotonic during the hottest months. Those inhabiting the drainage ditches, however, must always be hypertonic to their medium, that is, assuming that both populations of prawns are identical.
The purpose of the present study was to ascertain whether there were any physiological differences between these two groups of animals adapting the one to life in a low salinity environment.
The animals used in this investigation inhabited two pools situated within 50 yards of one another and were subjected to the same climatic conditions throughout the year. One population came from a typical salt-marsh pool which lay a little way from high-tide mark. The pool contained highly saline water and was regularly flooded by high tides. The second population inhabited a sluice-pool into which emptied water from drainage ditches traversing the farm land. This water was slightly brackish and of a very low salinity. The sluice-gates were always kept shut and excess water escaped by overflowing the top. It was never flooded by high tides and a high stone embankment sheltered it from sea-spray.
Throughout the year both pools undergo gradual seasonal changes in salinity content and water temperature (Fig. 1), and, in addition, the marsh-pool is also subject to sudden changes brought about by entry of sea water at high tides. Changes in salinity closely follow changes in water temperature; during June and July maximum salinities are reached of 36‰ NaCl in the salt-water pool, but only 2·5 ‰ NaCl in the sluice-pool. The minimum concentration is during January, and figures of 12·8‰ NaCl and NaCl are recorded for the marsh-and sluicepools respectively. The average annual salinity is 23·5‰ NaCl in the marsh-pool and 1·3‰ NaCI in the sluice-pool. These figures show the great difference in the osmotic gradients to which the two populations are subjected.
Both populations are morphologically identical and show no difference in breeding times or in egg size. Egg-bearing females are observed throughout June, and by the end of July both pools swarm with larval prawns. During high tides the larvae in the marsh-pool may become swept out to sea, but the sluice-pool population is not subjected to this dispersal.
MATERIALS AND METHODS
Prawns were transferred to the laboratory and stored in large tanks in a cool room. The two populations were kept in water taken from their respective habitats. No special attempt was made to feed them, but the water was replaced by fresh supplies at monthly intervals, and under these conditions the animals remained in a healthy state for the duration of the investigation.
To study the effects of salinity changes animals were transferred to a medium prepared from clean sea water of the same salinity and pH as the original pond water, and left for a period of 24 hr. to become acclimatized. The rate of respiration was determined and then the salinity of the medium was slightly altered. By a series of dilutions and concentrations effected with tap water and concentrated sea water, the animals were subjected to a range of salinities varying from pure tap water to pure sea water and even concentrated sea water prepared by evaporation and appropriate buffering.
The following uniform procedure was adopted for the measurement of the respiratory rate. Five large bottles were filled to the brim with water of known salinity and immersed in a constant-temperature bath. They were allowed to acquire the temperature of the bath (15° C.). After a 24 hr. acclimatization period in water of the same salinity five prawns of known weight were placed in each of four of the bottles and the fifth was kept as the control without animals. All bottles were then tightly stoppered, care being taken to ensure that no air was trapped inside. Exactly 3 hr. later two samples of water were withdrawn from each bottle and the oxygen content measured.
All salinity estimations were made by titration of a known volume of sample water against standardized silver nitrate solution, with potassium chromate as indicator. This only gave an estimation of the halogens, but since the other salts present were in such minute amounts it was taken as a reading of the total salinity and expressed as grams of NaCI per litre (‰Q NaCI).
Oxygen content was estimated by the Winkler (1888) method, which involves the oxidation of manganous hydroxide by the dissolved oxygen in the water to manganic hydroxide. The latter is made to liberate an equivalent of iodine from potassium iodide in an acid medium. The technique used was as follows: (1) to each sample 2 c.c. of Winkler Solution I (33 % MnCl2) were run in with a pipette, the point being kept near the bottom of the sample bottle so that a bottom layer was formed; (2) 2 c.c. of Winkler Solution II (30% NaOH +10% KI) were added in the same way; (3) the bottle was shaken and the resulting precipitate of manganic hydroxide allowed to settle; (4) 2 c.c. of phosphoric acid were added, and the iodine liberated as a consequence was estimated by titration against standard sodium thiosulphate solution, with starch solution as the indicator. From the results obtained the amount of oxygen in each bottle was calculated, and by subtraction from the control volume the quantity of dissolved oxygen used in unit time by a known weight of animals was estimated.
Before the animals were weighed all adherent water was removed with strips of filter-paper, special care being taken to remove the water in the gill chambers and between the cephalothoracic shield and first abdominal segment. They were weighed in a filter-paper-lined tube and the tube was reweighed after their removal. The difference in the two readings was taken as the weight of the animal.
Both populations of P. varians were subjected to salinities ranging from tap water to concentrated sea water of a salinity equivalent to 65 NaCl. Both groups proved to be tolerant of a very wide range of salinities, though the limits in each case were slightly different. The salt-water population failed to five for more than 16 hr. in fresh water, but tolerated a salinity as low as 1·7‰ NaCl. for several days. At the other end of the scale salinities of over 60‰ NaCl were tolerated and some animals remained alive for over a week at a salinity of 66‰ NaCI. Sluice-pool prawns were able to tolerate fresh water, but at salinities above 45‰ NaCI they only survived for a short time.
The estimated respiratory rate in several different salinities is given in Table 1 and plotted against the salinity of the medium in Fig. 2. In both groups the rate decreases as the salinity increases from tap water. It declines to a minimum rate and then increases again as the external medium becomes more and more concentrated. There is, however, a very marked difference in the salinities at which each group shows the minimal respiratory rate. In the case of the marsh population it was recorded at a salinity of 26‰, NaCI, whereas the sluice-pool animals respired least in a medium of 6‰ NaCI, a difference of 20‰ NaCI.
The P. varians taken from the marsh-pool have a respiratory rate which is minimal when the animal is isotonic with its medium. In such a state there is no tendency for water to pass across the body wall and energy expended on osmotic work is at its minimum. When the salinity of the medium is changed the animal becomes hypertonic or hypotonic, depending on whether the salinity is decreased or increased, and the respiratory rate increases. This is to be expected, since a change in the osmotic equilibrium necessitates an increase in the osmoregulatory work done by the animal. The energy requirement for osmotic work forms only a small proportion of the total metabolic energy used by an aquatic invertebrate, for example, in the crab Eriocheir sinensis, Potts (1954) has calculated that the energy used for osmoregulation is only 0·5% of the total metabolic energy when the animal is in fresh water. The respiratory rate of both sluice-and marsh-pool populations, however, increases by as much as 600 % between isotonic conditions and extremes in salinity of the external environment. Thus if what is true of Eriocheir is also true of P. varians, it would seem that these salinity changes not only affect the osmoregulatory mechanism but also some other process which causes a large increase in the total metabolic rate.
The sluice-pool prawns respire least at a salinity of 6‰ NaCl. They are therefore capable of living in low salinity water at a much smaller expenditure of energy than the salt-water population. How this is achieved is a subject for future investigation, but a series of unpublished observations have shown that it is not a lowering of the blood concentration that is responsible. The animals are not isotonic with the medium at 6‰ NaCl. Potts (1954) has demonstrated that a production of urine hypotonic to the blood, even if it is still many times more concentrated than the external medium, can greatly reduce the osmotic work of an animal living in fresh water. Normally Palaemonetes produces a urine which is isotonic to the blood, and it would be of great interest to investigate whether this condition differs at all in the sluice-pool prawns. P. varians collected from salt-marshes in Kent and Essex have a rate of urine production which is minimal when the external medium is approximately isotonic with the blood, but which increases progressively with increasing dilution of the external medium and also, to a lesser extent, with increasing concentration (Parry, 1955).
This population of prawns inhabiting an almost fresh-water environment probably represents a stage in the physiological adaptation of this species to fresh water, a process which has already been completed in Mediterranean regions. Environmental conditions are far more favourable to the production of a local race in the sluice-pool than in the adjacent marsh-pools. Dispersal of larval prawns by high tides and their intermingling with other populations washed out to sea will minimize the chances of a local race being evolved in the salt-marsh areas, but in the sluice-pool, protected as it is from tidal dispersal of the larvae, offspring that differ from the parents and perhaps more suited to low salinity conditions will be fostered by the more stable conditions, and the evolution of a different race is more likely to occur.
This work was carried out in the Department of Zoology, University College, Cardiff, and thanks are due to Prof. James Brough, for providing me with the facilities which enabled me to undertake this investigation and for his help and criticism. I would also like to thank Mr W. A. L. Evans who assisted me in many ways.