Regulation of Water and Some Ions in Gammarids (Amphipoda): III. Three Euryhaline Species

Gammarus zaddachi Sexton occupies the low-salinity regions of estuaries, including the fresh-water zone influenced by salt water during periods of high spring tides (Sexton, 1942; Spooner, 1947; Kinne, 1954; Hartog, 1964; Dennert et al. 1969). G. locusta (L.) and Marinogammarus finmarchicus (Dahl) are marine-littoral species which also occur in the more saline regions of estuaries (Spooner, 1947; Segerstråle, 1947; Hartog, 1964; Stock, 1967). In addition, G. locusta is found in the Baltic Sea at salinities down to 5·5 ‰ (Segerstråle, 1950, 1951) and M. finmarchicus can tolerate equally low salinities in the laboratory (Werntz, 1963; Sutcliffe, 1968). Both species may therefore be regarded as euryhaline animals with a salinity tolerance range similar to that of the shore crab Carcinus maenas, but less euryhaline than G. zaddachi. These three gammarids provide an interesting comparison with G. duebeni, a highly eury-haline species (Beadle & Cragg, 1940a, b; Shaw & Sutcliffe, 1961; Lockwood, 1961, 1964; Sutcliffe, 1967, 1971a; Sutcliffe & Shaw, 1968).

Several aspects of osmoregulation in G. zaddachi, G. locusta and M. finmarchicus were dealt with by Beadle & Cragg (1940a), Derouet (1952), Werntz (1963) and Sut-cliffe (1968). This paper is concerned with the body water content, and the blood concentrations and total body concentrations of sodium, potassium and chloride in these gammarids. The proportions of body water in the extracellular blood space and in the intracellular space were calculated from the assumption that the distribution of potassium and chloride ions conforms to a Donnan equilibrium between these two spaces (Croghan & Lockwood, 1968; Sutcliffe, 1971a). This assumption is justified by the fact that a Donnan equilibrium exists between the blood and muscle fibres in the euryhaline crabs Carcinus (Shaw, 1955a, b, 1958a, b) and Callinectes (Hays, Lang & Gainer, 1968). It was also shown to be a reasonable assumption in the case of G. duebeni and G. pulex (Sutcliffe, 1971a, b).

G. zaddachi Sexton was obtained during the winter months from underneath stones on the shore where a small stream discharges over the beach near Barrow-in-Furness, Lancashire. G. locusta (L.) and Marinogammarus finmarchicus (Dahl) were obtained from the tidal zone of the shore at Cullercoats Bay, Northumberland. All of the specimens used in this investigation were individually examined and identified under a binocular microscope (Sutcliffe, 1968).

The experiments were carried out at a temperature of 9 ± 1 °C. Animals were fed until about 48 h before removal for analysis. Experiments on G. zaddachi were carried out on three batches of animals (Series A–C). In series A after 3 days acclimatization in approximately 30 % sea water the animals were transferred into higher and lower salinities, including 0·25 mM/l NaCl stirred by a magnetic follower (Sutcliffe, 1971a) for a period of 5 days. In Series B the animals were acclimatized to 40 % sea water for 24 days, followed by acclimatization to 2 % sea water for 2 days and to 0·25 mM/l NaCl for a further 2 days. In Series C the animals were acclimatized to 2 % sea water for various periods of up to 18 days before acclimatization to NaCl-free media for 3–5 days.

The proportion of body water in the extracellular space (blood space) was calculated in the manner described in the first paper of this series (Sutcliffe, 1971 a). That paper also gives details of the analytical methods employed here.

The mean water content varied between 74·7 and 79·7 % body wet weight over the salinity range 100% sea water down to 0-3 mM/l NaCl (Table 1). These values are practically identical with values for the water content in G. duebeni over the same salinity range (Sutcliffe, 1971 a) and in G. zaddachi there is also a tendency towards increased hydration at low salinities.

A few values for the blood sodium concentration over the range 55 % sea water down to 0·25 mM/l NaCl were given by Sutcliffe (1968). Some additional values for blood sodium and chloride are given in Table 2. In animals from 100% sea water the blood sodium and chloride concentrations were identical. The sodium concentration of the blood was about 35 mM/l above that of the medium and the chloride concentration of the blood was about 32 mM/l below that of the medium. Thus the chloride concentration in the blood represented only 94·5 % of the chloride concentration in the sea water. Blood chloride was also slightly below the external chloride in 80% sea water. This maintenance of a blood chloride concentration in the blood below that in the external medium distinguishes G. zaddachi from G. duebeni (Sutcliffe, 1971 a) and Corophium (McLusky, 1968), and in this respect G. zaddachi resembles some marine-estuarine decapods, e.g. Carcinus (Shaw, 1955a; Robertson, 1960), Chionoecetes and Pandalus (Mackay & Prosser, 1970). In G. zaddachi at low salinities the blood chloride concentration was also very similar to the blood sodium concentration. Both fell sharply at salinities below 0·5 mM/l NaCl, even when the medium was stirred (Table 2).

The relationship between the external salinity and the total body sodium and chloride concentrations is similar to that shown by the blood sodium and chloride concentrations. Total body sodium and chloride increased at salinities above approximately 40–50 % sea water, there was a zone of close regulation at salinities between 40 and 2 % sea water, and a sharp fall in body sodium and chloride occurred at salinities below 0·5 mM/l NaCl (Table i, Fig. 2). The values for Na_T and Cl_T resemble the values found in G. duebeni and the similarity between the two species is further shown by the maintenance of a higher sodium content compared with the chloride content in G. zaddachi at low salinities. The ratio Na_T/Cl_T was greater than 1·0 in every individual case, including six animals from 100% sea water, and the mean ratio increased to about 1·85 in animals acclimatized to 0·25 mM/l NaCl (Table 1). This is higher than the mean ratios of 1·48–1·65 found in G. duebeni acclimatized to the same medium (Sutcliffe, 1971a). In fact the ratio Na_T/Cl_T was higher in G. zaddachi when compared with G. duebeni at all external salinities up to 100% sea water.

The total body potassium concentrations showed little change at salinities between 100 and 2 % sea water and there Was only a slight fall in animals acclimatized to NaCl-media for up to 5 days (Table 1, Fig. 2). Thus the regulation of body potassium, and the blood potassium concentration (Table 2), is practically the same as in G. duebeni. However, the detailed features of potassium balance at very low salinities may differ slightly. Some preliminary measurements of the potassium loss rate into 250–1000 ml de-ionized water were made on three groups of G. zaddachi kept in 100% sea water. At 23 °C the initial mean loss rate was 0·25 μM/h/animal. At 9 °C the initial mean loss rate was 0·11 μM/h, identical with the potassium loss rate in G. duebeni from 100% sea water. After a period of 24 h the animals reached a temporary steady state with respect to potassium when the external concentration had increased to 0-027-0-037 HIM/L potassium. In this particular experiment the total potassium loss from the animals represented about 20% of the body potassium. The loss rate was then measured again in 100-400 ml deionized water. The mean potassium loss rates were 0·020–0·023 μ/h at 9 °C, and the ‘balance’ concentrations after a period of 24 h were 0·027–0·035 mM/l. These are higher than the minimum external potassium concentrations of 0·010–0·015 mM/l required for potassium balance in G. duebeni (Sutcliffe, 1971a), but a more detailed study of potassium regulation in G. zaddachi acclimatized to low salinities is necessary for strict comparison between the two species.

The mean blood space was calculated from the data given in Table 1 and Table 2. The results are shown in Fig. 1. The blood space is equivalent to about 60% body H₂0 in animals from 100% sea water and equivalent to 50% body H₂0 in animals acclimatized to 40% sea water down to 0·5 mM/l NaCl. At lower salinities there was a very marked reduction in the blood space to only 30% body H₂0 in animals from 0·25 mM/l NaCl. In every case the values for the blood space are very close to the maximum calculated on the assumption that all of the body chloride is in the blood space at the concentration found in the blood. This chloride space contains 2·3–4·9% body H₂0 more than the blood space. In contrast the sodium space, calculated on the assumption that all of the body sodium is at the concentration found in the blood, represents approximately 60–70% body H₂O over a wide range of external salinities (Fig. 1). These values for the blood, chloride and sodium spaces are very similar to the values for the same spaces in G. duebeni.

The calculated concentrations of sodium, potassium and chloride in the intracellular water are shown in Fig. 2. The cell sodium concentration was fairly constant, ranging from 65 to 116 mM/kg cell H₂O. In animals from 2% sea water down to 0–25 mM/l NaCl the concentrations of cell potassium-plus-sodium range from 226 to 163 mM/kg cell H₂O, and these are equal to the concentrations of potassium-plus-sodium in t e Wood. In animals from 80 to 100% sea water the concentrations of cell potassium-plus-sodium were 267–296 mM/kg cell H₂O, representing about 50–56% of the blood concentration. The cell potassium concentration in G. zaddachi from 80 to 100% sea water is higher than the cell potassium concentration in G. duebeni, otherwise the concentrations of the three ions are similar to those calculated for G. duebeni and similar to the concentrations found in muscle and some other tissues of marine, brackish and fresh-water decapods.

In animals from 100% sea water the body water content was equivalent to approximately 72% body wet weight in both species (Table 3, Table 4). At lower salinities the water content in M. finmarchicus increased to 76 % body wet weight in animals from 20% sea water. The water content of these two species is slightly lower compared with G. zaddachi and G. duebeni.

Values for the blood sodium concentration in M. finmarchicus at salinities from 100 to 10% sea water were given by Sutcliffe (1968). Some additional analyses of blood sodium and chloride were made on pooled samples (Table 5). These were obtained from 15 to 16 animals with some exceptions noted in the table. Table 5 also includes samples taken from animals exposed to sea water which was then allowed to become slightly concentrated by evaporation. At salinities above 80% sea water the sodium concentration in the blood was maintained at or slightly above the sodium concentration in the medium, but the chloride concentration in the blood was distinctly lower than that in the medium. In this respect M. finmarchicus resembles G. zaddachi, and a low blood chloride concentration is also characteristic of G. locusta. Two large specimens of G. locusta were acclimatized to slightly concentrated sea water with a chloride concentration of 630 mM/l. The blood chloride concentrations in the two animals were 580 and 550 mM/l respectively. Beadle & Cragg (1940a) found a blood chloride concentration of about 420 mM/l in G. locusta from sea water containing 495 mM/l chloride, and the blood chloride level was slightly below that of the external chloride in 75% sea water. Beadle & Cragg also found a lower chloride concentration in the blood of Marinogammarus obtusatus compared with the chloride concentration in undiluted sea water.

In animals from 100% sea water the values for the total body sodium and chloride concentrations in M. finmarchicus (Table 3, Fig. 4) and G. locusta (Table 4) are lower than the values found in G. zaddachi and G. duebeni despite the lower water content of the two marine-littoral species. Another distinguishing feature is the higher value of the ratio Na_T/Cl_T. This ratio was approximately 1 · 2 in the marine-littoral gammarids (Table 3, Table 4) reflecting the lower blood chloride levels maintained in 100 % sea water, although a lower chloride concentration in the cells would also increase the ratio. Furthermore, the ratio Na_T/Cl_T remained constant in M. finmarchicus at salinities down to 40 % sea water, and showed only a small increase in 20 % sea water (Table 3).

The blood potassium concentration showed little change in M. finmarchicus from 100 to 20 % sea water (Table 5). In one large specimen of G. locusta exposed to slightly concentrated sea water the blood potassium concentration was 13·5 mM/l (blood chloride = 55° mM/l). In both species the concentrations of total body potassium at 100% sea water were slightly higher than the concentrations found in G. zaddachi and G. duebeni, but the blood potassium concentrations in all of these gammarids were maintained at the same level. This suggests that the intracellular potassium concentration might be maintained at a higher level in the less euryhaline marine-littoral gammarids compared with the more euryhaline species G. zaddachi and G. duebeni.

The calculated blood space (= extracellular water) in M. finmarchicus at salinities between 100 and 20% sea water is shown in Fig. 3. A blood chloride concentration of 372 mM/l (Table 5) was used to calculate the lower value for the blood space (= 36% body H₂O) at 40 % sea water. However, the blood chloride concentration may have been lower than this in the animals used to estimate the concentrations of total body ions. If the blood chloride concentration was 350 mM/l in these animals from 40% sea water, the blood space would then be equivalent to 39·5% body H₂O.

From Fig. 3 it appears that there was remarkably little change in the blood space despite a considerable fall in the blood concentration at salinities between 100% and 20% sea water (Table 5; Sutcliffe (1968), Fig. 6). The values for the blood space in M. finmarchicus are lower than the values for the blood space in G. zaddachi and G. duebeni. This also applies to the values for the chloride and sodium spaces (Fig. 3). These spaces were calculated in the manner described for G. zaddachi. Very similar values were obtained for G. locusta in 100% sea water, assuming blood concentrations of 472 mM/l chloride and 12 mM/l potassium. In this case the blood space is 49·4% body H₂O, the chloride space is 53·5 and the sodium space is 59·6.

The calculated concentrations of sodium, potassium and chloride in the intracellular water of M. finmarchicus are shown in Fig. 4. All three ions appear to be maintained at fairly constant levels. For each ion its intracellular concentration in M. finmarchicus closely resembles its intracellular concentrations in G. zaddachi and G. duebeni. In G. locusta from 100% sea water the calculated intracellular concentrations of sodium, potassium and chloride were practically identical with the concentrations in M finmarchicus at 100% sea water (Table 6). Thus the suggestion made earlier that the higher total body potassium concentration in the marine-littoral gammarids might reflect a higher intracellular potassium concentration is not supported by these results. This is because the undoubtedly higher potassium content in the cells of these gammarids from 100% sea water is offset by the higher water content of the cells reckoned to be equivalent to 50–60% body H₂O. In comparison the water content of the cells in G. zaddachi and G. duebeni from 100% sea water was reckoned to be equivalent to 40–45 % body H₂O.

The distribution of water and ions in the blood space and in the intracellular space was calculated from the assumption that there is a Donnan equilibrium between potassium and chloride ions in these two spaces (Sutcliffe, 1971a). On this basis the blood spaces in Marinogammarus finmarchicus and Gammarus locusta were equivalent to 44 and 50% body H₂O respectively in animals from 100% sea water. Similar values were obtained for the blood spaces in the more euryhaline G. duebeni and G. zaddachi from 2 to 40% sea water, and in G. pulex from fresh water. In the case of G. duebeni and G. pulex it was shown that it is reasonable to assume that a Donnan equilibrium exists between potassium and chloride in the blood and cells (Sutcliffe, 1971a, b). It is suggested that this assumption is also reasonable as an approximation in the case of the euryhaline gammarids considered in this study. Otherwise it is clear that the proportion of body water in the blood space must be lower than that calculated here, and the blood spaces would then contain less than 40% of the body water. In fact, if the extracellular tissue space represents 20% of the tissue water, then the blood situated outside the extracellular tissue space would contain as little as 20% of the body water in animals from low salinities, i.e. approximately 10 mg H₂O in an animal of 65 mg body wet weight. There is no direct evidence in support of blood volumes as low as this. Up to 5 mg blood can be obtained without difficulty from a 65 mg animal through a small puncture made in a dorsal intersegmental membrane, and this technique only removes blood from the part of the haemocoel immediately surrounding the gut.

In comparison with G. duebeni and G. zaddachi the less euryhaline G. locusta and M. finmarchicus maintain higher blood concentrations at salinities between 20% and 100% sea water. They also have a lower body water content, a smaller sodium space, and slightly lower concentrations of sodium and chloride in the body water than animals from 80 to 100 % sea water. When taken together these observations indicate that it is unlikely that the tissue ion content is maintained at a higher level in the two marine-littoral species. Rather, they lead to the conclusion that the mean intracellular concentrations of sodium, potassium and chloride are practically the same in all of these euryhaline gammarids. This is in agreement with direct measurements made on stenohaline and euryhaline arthropods. In Nephrops and Paralithodes (Robertson, 1961; MacKay & Prosser, 1970) the concentrations of sodium, potassium and chloride in the muscles are substantially the same as in Eriocheir, Carcinus, Potamon, Pacifastacus, Callinectes and Limulus (Krogh, 1939; Shaw, 1955a, b, 1959; Kerley & Pritchard, 1967; Hays, Lang & Gainer, 1968; Robertson, 1970).

The above conclusion is also in agreement with the work of Beadle & Cragg (1940 a) who found that the chloride concentrations in the ‘tissues’ of both G. locusta and M. obtusatus from 100% sea water were slightly lower than the corresponding values in G. duebeni. In G. locusta Beadle & Cragg found approximately 120 mM-Cl/kg wet tissue against a blood chloride concentration of 420 mM/l. Derouet (1952) also found about 128 mM-Cl/kg wet tissue against a blood chloride concentration of 440 mM/l in G. locusta from 100% sea water. Now the mean intracellular chloride concentration in this ‘tissue’ can be calculated in the manner described for G. duebeni and G. pulex (Sutcliffe, 1971a, b), assuming that the extracellular tissue space contained 20% of the tissue water with a chloride concentration equal to that of the blood. The extracellular tissue space would then contain 67–70 mM-Cl/160 g H₂O, leaving 53-58 mM-Cl/640 g H₂O in the cells. This gives an intracellular chloride concentration of 83–91 mM/kg cell H₂O compared with the estimate of 37 mM/kg cell H₂O given in Table 6. At least part of the difference would be due to a residual amount of blood in the appendages of the ‘tissue’ preparations (whole animal minus blood), since it is impossible to remove all of the blood (Sutcliffe, 1971a). The entire difference would be accoanted for if only 85% of the initial blood volume was removed during the preparation of the ‘tissue’.

A comparison of the intracellular concentrations of sodium and chloride is shown in Fig. 5, where the cell concentrations are plotted against the blood concentrations in a series of gammarids. In M. finmarchicus, G. zaddachi and G. duebeni the intracellular chloride concentration is approximately proportional to the blood concentration, and this type of regulation is also found in the muscle fibres of Carcinus (Shaw, 1955b). In contrast the freshwater species G. pulex lacks the ability to maintain a low chloride concentration in the cells when the blood concentration is raised. The cell sodium concentration also rises steeply in G. pulex, as in Astacus (Bryan, 1960), and this again stands in contrast to the situation found in the euryhaline gammarids. Here the in-tracellular sodium concentrations rise or fall by less than 50% in the face of much greater changes in the blood sodium concentration. In this respect sodium regulation resembles chloride regulation. In fact, if a major proportion of the intracellular sodium is situated outside the muscles, sodium regulation in the muscles of the euryhaline gammarids could resemble that of Carcinus, where the sodium concentration in the muscles is proportional to the sodium concentration in the blood (Shaw, 1955b). In any case it appears that the regulation of cell sodium in M. finmarchicus differs from sodium regulation (or the lack of it) in the muscles of Limulus (Robertson, 1970). Further speculation on this and related matters, such as the apparently close regulation of the proportions of body water in the blood space and intracellular space of M. finmarchicus, must wait until a more direct estimate of the blood space is available for these gammarids.

I wish to thank Mr T. Carrick for assistance in collecting some of the animals, and my wife for typing the manuscripts.