Active Transport and Sodium Fluxes at Moult in the Amphipod, Gammarus Duebeni

The time of ecdysis in Crustacea is a period at which considerable changes occur in both the volume and ionic composition of the blood. Detailed studies made on the concentration and volume of the body fluids during the course of the moult cycle of various crabs (Robertson, 1960; Drach, 1939; Baumberger & Olmsted, 1928; de Leersnyder, 1967) indicate that in general there is a rise in the osmotic pressure of the blood, the blood protein and in the concentration of many of the ionic constituents of the blood during the pre-moult period. The rise in the total concentration of the blood in pre-moult Carcinus is accounted for mainly by increases in the plasma concentrations of sodium, calcium, magnesium and chloride ions (Robertson, 1960). Two factors could contribute to this rise in ion concentration. These are (1) that a rise in cell osmotic pressure produced by an increase in free amino-acids results in absorption of water by the cells from the blood, and (2) that an active uptake of ions across the body surface together with ions reabsorbed from the old cuticle raises the blood concentration. An increase in the amount of free amino-acids in the cells has indeed been observed at the time of moult in Eriocheir (Duchâteau, Florkin & Jeuniaux, 1959) and Robertson (1960) has demonstrated that about one third of the water taken in by Carcinus at moult enters the cells. On the other hand he has shown that the rise in blood osmotic pressure which occurs prior to moult in Carcinus is insufficient by itself to account for the subsequent uptake of water by osmosis. Ion intake accompanies the absorption of fluid into the body. As the net uptake of fluid at moult in Carcinus is equivalent, on average, to 66·3 % of the animal’s pre-moult weight the intake of ions must, in fact, be considerable.

Fluid intake at moult in decapods occurs either largely from the gut as in Carcinus (Robertson, 1960) or across the body surface as in PanuUrus (Travis, 1954). In Maia too there is an isosmotic transfer of fluid across the body surface (Dandrifosse, 1966).

The means by which the fluid and ions taken in are caused to cross the body surface (or gut wall) is not known. Theoretically ions could be taken in by active transport processes and water follow passively or, alternatively, a bulk flow of fluid under the influence of the colloid osmotic pressure of the blood proteins might account for the influx of both water and ions.

Control over the amount of fluid taken in is apparently adequate to permit moulting in a wide range of salinities in the case of a number of species of euryhaline Crustacea.

For example, Haefner & Shuster (1964) have shown that the size increase at moult in the blue crab Callinectes sapidus is not related to the gradient of concentration between blood and medium at the time of moult. Gammarus duebeni itself grows and moults satisfactorily in the salinity range 2%o ^t0 38%o though the rate of growth and moulting frequency is somewhat less at the two extremes studied than at 10%, (Kinne, 1953a, b, 1959). Over this range there are considerable differences in the gradient of concentration maintained between blood and medium (Beadle & Cragg, 1940; Lockwood, 1961) and in media more concentrated than 60% sea water the blood is almost isotonic with the medium. Successful increase of size at moult over the whole salinity range tolerated therefore implies that mechanisms are present to enable isotonic fluid uptake when the animal is isotonic with its medium and to limit the net fluid uptake when it is hypertonic.

In the present experiments the sodium fluxes in post-moult Gammarus duebeni have been examined to determine if there is an increase in the active uptake of sodium sufficient to account for the net water uptake at moult by isosmotic transfer. It is shown that there is a massive increase in the rate of active uptake above the level found in inter-moult animals.

Gammarus duebeni were obtained from salt-marsh pools and ditches at Redbridge on the River Test. They were maintained individually in 20 c.c. crystallizing dishes, the tops of which were covered with muslin or Nylon mesh netting. These vessels were immersed in the flowing sea-water circulation of the Department of Oceanography. During the period over which most of the experiments were carried out (February-May 1968) the temperature in the aquarium water ranged from 15 −19° C. and the salinity was 33 %0-At the time of additional experiments in February 1969 the aquarium temperature was 14 −15° C. The animals were each provided with a piece of Enteromorpha thallus and a few flakes of Bemax as food. Additional food was given as necessary.

The stock of animals was examined each morning and any individuals which had moulted since the previous inspection were removed for experimentation. Moulting was rarely observed to occur during the day so the majority of animals when first studied were within 18 hr. or less of moulting.

To study the effect of moult on the permeability of the body surface and on the rate of active uptake of sodium, animals which had moulted during the preceding night were exposed to the following experimental procedure :

1. Counted in isotonic sucrose (⁠ min.); (2) half an hour in a solution of 32 g. sucrose plus 100 c.c. of 10 MM/L. NaCl labelled with ²²Na; (3) counted in isotonic sucrose (⁠ min.); (4) 1 hr. in sea water; (5) counted in isotonic sucrose (⁠ min.); (6) 1 hr. in de-ionized water; (7) counted in isotonic sucrose (⁠ min.); (8) weighed; (9) returned to sea water circulation for 24 hr.

Most animals were put through this regime daily for the first 6 days after moult and then at intervals of 2 days thereafter. A small proportion died after some days of experimentation but none which survived less than 6 days is included in the data given.

In a later series of experiments ²²Na-labelled sea water was substituted as the loading medium. Measurements of loss of ²²Na to isotonic sucrose and to 10 mM/l. NaCl plus isotonic sucrose were made on separate animals.

All flux measurements were made in a water bath at a temperature of 16 + 0·5° C. The measured pH of the solutions used were: sea water, 7·8; isotonic sucrose, 6·5; labelled 10 mM/1. NaCl plus isotonic sucrose, 6·65. A second stock of the tracer medium had a pH of 6·6.

The same equation has been used to evaluate the gross influx of sodium from sea water but in this case the percentage losses used were those of the outflux to sea water.

Measurements of the potential across the body surface were made by means of calomel electrodes and a Beckmann Research pH meter. Fine-tipped glass cannulae containing 1 M KC1 were attached to the tips of each calomel electrode. The reference electrode was dipped into sea water and this medium was connected to the animal’s container by an agar bridge. The tip of the other electrode was inserted through an intersegmental membrane on the dorsal part of the anterior region of the thorax. The region of the puncture was coated with silicone grease to prevent electrical leakage to the medium and the animal was gripped in a Perspex holder so that the gills were immersed in the medium but the dorsal thorax was in air.

The total body sodium of crabs increases at the time of moult as a result of an expansion of the blood volume and a temporary increase in blood concentration (Robertson, 1960). There is also a change in the ratio sodium content/body wet wt. due to these effects. A similar change may be expected to occur in other crustacea at moult and its extent in Gammarus duebeni has been investigated by comparing the ratio of sodium content/body wet wt. in inter-moult (more than 6 days after moult) and freshly moulted individuals.

The sodium content was determined from measurements of the ²²Na count of animals which had spent a sufficient time in labelled sea water to effect a 99 % exchange of their total body sodium (seven half-times of sodium exchange).

Inter-moult animals in the weight range 52·7; to 88·4 mg. wet wt. had a measured Na content of 0·206 ± 0·009 µg./mg. wet wt. (N = 5). The corresponding value for animals 12-36 hr. after moult was 0·247 ± 0·015µM/mg. wet wt. (weight range 40·8 −88·2 mg. N = 7). The recently moulted animals thus have a sodium content some 20% greater, per unit wet wt., than inter-moult individuals.

The mean weight of the animals used in the subsequent flux experiments was 67·7 mg. (N = 17) and on the basis of the above values it is assumed that their average total sodium content when freshly moulted was 67·7 x 0·247= 16·7 µ^M-This value is presumed to have declined to 14·4 µM by 6 days after moult when allowance has been made for the increase in weight and decrease in relative sodium content which occurs subsequent to moult. Fortuitously, a determination of the total sodium content of five inter-moult animals of average weight 69·4 mg. gave a mean sodium content of exactly 14·4 µM.

These values for total sodium in freshly moulted and inter-moult animals have been used in calculations of the amount of sodium lost from the body in flux experiments.

Sixteen of the animals used in flux experiments were weighed daily for 6 days after moult and thereafter at intervals of 2 days.

The results (Table 1) suggest that there is a slight increase in wet weight between the day of moult and the third or fourth day after moult. It must, of course, be stressed that this increase is likely to be only a small part of the total increase, since fluid intake will have begun before the first weighing is made.

The corrected effluxes of sodium to sea water, isotonic sucrose solution and deionized water are given in Table 2. All three decrease in value between the day after moult and about the sixth day after moult, thereafter remaining constant.

The average efflux to sea water declines from 87 % of the total body sodium/hr. on the first day after moult to 14% by the tenth day. Fig. 1 illustrates the process in a single individual.

Assuming that the total body sodium is 16·7 µM at moult and 14·4 µM after 6 days the average outflux figures range from 14·9 µM Na/hr. on the first day after moult to 2·0 µM Na/hr. on the tenth day.

The outflux to isotonic sucrose is much smaller than that to sea water, as would be expected, since ion-exchange diffusion is eliminated in this medium and the rate of urine production may also be affected. In the first series of experiments the efflux in this medium declined from 5·48 µM Na/hr. on the first day after moult to 1·84 µM Na/hr. in inter-moult animals (Table 2). In a later series on animals of average wt. 65 mg. a lower average rate of loss was obtained on the first day after moult (3·94 ± 1·45, N = 16). These values suggest that the permeability of the body surface to sodium may be a factor of two to three times higher immediately after moult than in inter-moult animals. The precise change in permeability cannot be obtained directly from these data, however, in the absence of information on the relative urine losses after moult and in inter-moult animals.

For comparison, measurements of the sodium loss to unlabelled 10µM/1. NaCl plus isotonic sucrose were made over the hour preceding or succeeding the latter set of measurements to isotonic sucrose. The loss rate was increased when sodium was present in the medium and in the freshly moulted animals was 4·98 + 2·08. The assumption made here is that the difference between the losses to isotonic sucrose and to 10 µM/L. NaCl plus isotonic sucrose represents the ion-exchange diffusion component of the flux in the latter medium. In fact this is not strictly correct, since differences in the potential across the body wall in the two media will tend to affect the diffusion loss of sodium to isotonic sucrose more than it affects the loss to the unlabelled tracer media (see later). The apparent ion-exchange diffusion component of the flux is thus somewhat exaggerated. Sutcliffe (1967) has shown similarly that the efflux to isotonic sucrose and to 10 mM/l. NaCl differ in inter-moult animals implying the presence of an ion-exchange diffusion component.

The rate of urine production in gammarids is proportional to the gradient between blood and medium (Werntz, 1963) and the loss rate to de-ionized water is therefore large by comparison with that to isotonic sucrose. From Table 2 it can be seen that the outflux to de-ionized water declines nearly threefold in the 6 days following moult. The decline in a single individual is shown in Fig. 1. The difference between the loss in sucrose and that in de-ionized water should approximate to the loss due to the extra urine production in the latter medium. (It will not be exactly the same unless the potential difference across the body surface is the same in the two media.) On the basis of the average values found here the difference between the loss in the two media is about twice as great on the first day after moult as in inter-moult animals. Since the urine will be isotonic with the blood in the conditions of the experiment (Lockwood, 1961) differences in urinary sodium loss must be equated with changes in urine volume. The increase in loss during the first day is therefore probably due to an increase in the permeability of the body surface to water at this time. Any such increase must decline fairly quickly, however, as by the second day following moult the calculated urine loss is comparable to that in inter-moult animals.

The influx of sodium from 10 mM/l. NaCl plus sucrose isotonic with sea water was determined daily from the morning following moult until the sixth day and then at two-daily intervals. On the first day after moult the average influx was 4·48 µM Na/hr./animal. The influx declined markedly with time and by the eighth day after moult had fallen to an average of o·14µM/hr./animal (Table 3). No further decline in sodium influx occurred up to the twentieth day after moult when the experiment was terminated. A single individual followed for a further period showed no additional change in influx (Fig. 2). Determination of the sodium influx on the first day after moult in another, larger batch of animals gave a somewhat lower average value (3·91 + 1·9 µM/hr., N = 16).

Animals acclimatized to sea water would be expected to have a lower active uptake of sodium than those acclimatized to dilute media and it is not surprising therefore that the influx of 0·14 µM. Na/hr./animal shown by inter-moult animals is lower than the average influx of 0·65 µM/hr./animal found by Sutcliffe (1967) for individuals acclimatized to 10 mM/l. NaCl at 10° C. The exceptionally high influx on the day following moult is particularly interesting, however, because it exceeds the influx shown by inter-moult animals even when they have had their active transport system activated by a lowering of the blood concentration. Thus the average influx of four inter-moult animals after 20 hr. in Southampton tap water was only 2·1 µM/hr.

Since the influx is greater in the freshly moulted animals than in stimulated intermoult animals it follows that either the passive influx or the maximum rate of active uptake of sodium is larger shortly after moult than during inter-moult. The further possibility that the increased uptake of tracer after moult is due solely to the animals drinking the medium may be rejected because of the magnitude of the volume of the medium which would have to be imbibed. In order to account for the influx of sodium from 10 mM/l. NaCl on the first day after moult the animals would have to drink 426 µl./hr. if all the sodium in excess of that entering in inter-moult animals is taken in via the gut. Measurement of the drinking rate in freshly moulted animals using the dye Amaranth suggest that less than 1 ·0 µl./hr. is drunk by animals in the first day after moult. This rate compares with 1·06 µl./hr. drunk by inter-moult animals in sea water. As 426 µl. is more than six times the weight of the animal it seems scarcely credible that this volume should be drunk each hour. It is presumed therefore that most of the uptake takes place across the body surface.

Silver staining by exposure of animals to dilute silver nitrate and then a reducing agent has been used as a means of delimiting those areas of the cuticle of crustacea which are sufficiently permeable to permit the ready passage of ions (Croghan, 1958; Ralph, 1965).

Treatment of freshly moulted Gammarus duebeni for 5 min. in N/50 AgNO₃ followed by 3 min. in Johnson’s Reversol diluted 1/3 resulted in the silver staining of the gills alone. Similar treatment of inter-moult animals resulted in the staining of the gills, some intersegmental membranes and a few cuticular bristles. There is thus no direct evidence to suggest that the general body surface contributes to the increased sodium influx in moulted animals and it seems more likely that the bulk of the ion movement occurs at the gills.

The basic dye thionine (mol. wt. 227) is known to decrease the active uptake of sodium both in whole Crustacea and by isolated gills (Koch & Evans, 1956; Koch, Evans & Schicks, 1953)-The inhibition is reversible. In the present experiment this dye has been used as an aid to establishing the proportion of the increase in influx in moulted animals which can be attributed to processes other than diffusion.

For each animal used a comparison has been made of the influx of sodium from ²²Na-labelled 10 MM/L. NaCl plus isotonic sucrose containing 45 mg. thionine per 75 ml. medium, and then later on the influx from the same medium lacking thionine. Between the periods of loading in the two media the animals were washed for 4 or 5 hr. in their acclimatization medium (sea water) to remove any after-effects of the thionine treatment.

The presence of thionine in the medium produces a sharp decline in sodium influx by animals on the first day after moult (Table 4). A smaller proportion of the influx is inhibited by the dye in inter-moult animals.

By contrast with its influence on the influx from 10 mM/1. NaCl thionine has little effect on the sodium efflux from animals in sea water. Five inter-moult animals previously loaded in ²²Na-labelled sea water were unloaded in sea water for 4 hr. and then for a further 3 hr. in sea water to which 45 mg./75 ml. thionine had been added. No change in efflux was seen when transference was made to the medium containing thionine (Fig. 3). It is therefore assumed that thionine has no gross influence on either the diffusion or exchange diffusion components of the flux. If this assumption is correct then the reduction in influx caused by thionine must be due largely to the inhibition of active sodium uptake.

The influx of sodium from sea water is considerably more rapid than that from 10 mM/l. NaCl in isotonic sucrose (Table 2). The difference can be accounted for in terms of the larger diffusion and ion-exchange diffusion components of the flux in the more saline medium.

The variability of the individual fluxes in the days following moult precludes any detailed analysis of the net changes in total body sodium. However, when the influx and outflux values are compared, the imbalance between the two broadly follows the trend which would be expected from the measurements of the total sodium content of the body. Thus, on the first day after moult, when the weight of the animals is still increasing, the sodium influx exceeds the efflux. From the third to the fifth day the efflux appears to exceed the influx, a result which can perhaps be correlated with the relative decline in total sodium content which occurs between the period shortly after moult and the inter-moult condition.

A number of measurements were made of the potential difference across the body surface 9 min. after immersing the animal in sea water, isotonic sucrose, de-ionized water and 2% sea water. The results, given in Table 5, suggest that the potential difference in any medium is, in general, larger in freshly moulted than in inter-moult animals. The individual scatter in each medium is, however, large.

In the present experiments the sodium fluxes of Gammarus duebeni have been examined for several days following moult in animals maintained in sea water. On the first morning after moult the influx of sodium from 10 mM/l. NaCl plus isotonic sucrose solution was 4·48 µM/hr./animal (3·91 ± 1·9 in a later experiment). This influx is very large by comparison with that from the same medium at a later stage in the moult cycle. Thus the same animals 8 days after moult had an influx of only 0·14 µM/ hr./animal (0·145 in repeat experiment). An increase in influx in fact is already present on the day before moult though the difficulty of detecting pre-moult animals has limited the quantitative study of the flux in this phase. However, it has been observed that individual animals with influxes two to four times that of the average of a randomly selected group frequently moult within 24 hr.

The addition of the basic dye, thionine, to the medium at a concentration of 45 mg./75 ml. solution decreases the influx of sodium by over 90% on the first day after moult. As this substance is known to block the active uptake of sodium (Koch et al. 1953) it is plausible to suppose that a large part of the 4·48 µM/hr./animal influx on the first day represents active uptake of sodium. Both the total influx of sodium and the proportion of the influx which is inhibited by thionine decrease in the days following moult and have returned to the inter-moult level by the sixth to eighth day after moult. Additional support for this conclusion comes from the study of the efflux of sodium to isotonic sucrose and to unlabelled 10 NaCl plus isotonic sucrose. The efflux to these two media differs to the extent of 1 ·03 µM Na/animal/hr., suggesting an ion-exchange diffusion component of the flux of this degree. However, it is almost certain that this value is too large, since the effect of variations in the potential difference across the body surface in the two media has not been taken into account.

The active transport component of the flux should be the total flux less the diffusion influx and exchange diffusion. Even if this exaggerated value for exchange diffusion is deducted from the observed influx together with 0·11 µM Na/animal/hr., which represents the passive flux, the remaining part of the influx is 3·34 µM/animal/hr. (or 2·8 µM/animal/hr. if the other influx value is used). This rate therefore represents the active component of the flux. The magnitude of this active influx considerably exceeds the values for influx of fresh-water and brackish-water races of Gammarus duebeni given by Sutcliffe & Shaw (1968). The maximum influx they found for animals of all environments was of the order of 1·2 −1·4 µM/animal/hr., which is strikingly lower than that found in the present experiments. In part, the difference can be accounted for by the temperature effects, since Sutcliffe & Shaw’s experiments were carried out at 10° C. whilst the influx into the moulted animals was measured at 16° C. However, Sutcliffe & Shaw have shown that a 10° C. rise in temperature only approximately doubles the influx; the 6° C. difference is unlikely to account fully for the difference and it is to be presumed that the rate of active uptake is, for some reason as yet unknown, somewhat more rapid than the maximum influx rate in inter-moult animals. Some confirmation for the assumption that the active influx in the freshly moulted animals exceeds that of stimulated inter-moult animals is provided by the fact that four inter-moult animals which had been acclimatized to Southampton tap water had an average influx of only 2·1 µM/hr. at 16° C.

In some Crustacea much of the fluid uptake which occurs at around the time of moulting can be attributed to medium which has been drunk and subsequently reabsorbed from the gut into the blood (Robertson, 1960). The drinking rate of freshly moulted Gammarus is totally inadequate to explain the amount of sodium taken up. A drinking rate of over 400 µl./hr. would be required to account for the thionine-inhibited portion of the sodium influx. This is nearly six times the total volume of the animal. The observed drinking rate, as measured with Amaranth, is less than 1 µl./hr. It is probable therefore that most of the sodium influx occurs across the body surface. Dandrifosse (1966) has also concluded that much of the fluid which enters the crab, Maia, at moult does so across the body surface. The rapid active uptake of sodium in freshly moulted animals is clearly related to the uptake of water which occurs at moult.

Diamond (1965) has discussed various ways (local osmosis, the double membrane effect and co-diffusion) by which an active transport of inorganic ions could result in a passive movement of water in the same direction. A large active uptake of sodium, such as that observed in Gammarus, could thus, theoretically, enable an animal to increase its fluid content even when the blood is isotonic with the medium if the active transport of inorganic ion initiates a passive movement of water by one of these methods. The present experiments provide no proof that an isosmotic entry of water is achieved in this way, but the fact that Dandrifosse (1966) found that transport of fluid across the surface of Maia ceased when a sucrose solution was substituted for sea water suggests that the water movement is dependent in some manner on the transport of ions.

The area of the body surface which stains on treatment with silver nitrate varies at different seasons of the year in the mysid, Neomysis integer (Ralph, 1965), perhaps reflecting variations in the area of body surface capable of transporting ions. There is, however, no increase in the silver staining area in Gammarus at moult and this makes it doubtful that areas other than the gills are responsible for the increased uptake of sodium.

The observation that there is a large increase in active uptake of sodium at moult raises a number of questions for future study. For instance, it would be interesting to know if any marine forms obtain water for urine production by isosmotic transfer at the body surface and also how many other Crustacea behave like Gammarus in having a high rate of sodium uptake across the body surface at the time of moult. Perhaps the most obvious question of all, however, concerns the nature of the stimulus which is responsible for the initiation of the increased sodium uptake. Various factors have been reported in the literature to result in increased ion uptake. These include a decrease in blood concentration (Shaw, 1959, 1961 ; Bryan, 1960), an imbalance between the sodium and chloride levels of the blood (Shaw, 1964) and a reduction in blood volume (Lockwood, 1968). It seems unlikely that either of the first two factors could be responsible for increased uptake of sodium in an animal moulting when isosmotic with its medium. A system monitoring body volume might well be stimulated at moult, however, particularly if it involved stretch receptors located on the cuticle. These would perhaps not be extended at moult until the new cuticle was fully expanded. The suggestion that such a system may be operating is, of course, entirely speculative and it might equally be true that the raised rate of ion uptake is due to some other process which is solely an adjunct of moult.

The relatively small size of the increases in permeability of the surface to water and sodium at moult which has been found in Gammarus also pose additional problems. Gammarus duebeni is a species which inhabits a wide range of environments from sites on the sea shore where it is reached by undiluted sea water (Hynes, 1954) to fresh water in the case of a physiological race living in Ireland (Reid, 1939; Sutcliffe, 1967, 1968). It also tolerates salinities in excess of sea water and experimentally has been found to survive evaporation of its medium from 21 ‰, to 85 over a period of 11 days (Forsman, in Segerstråle, 1946). In a species which lives and moults over a wide range of salinities it is not unlikely that selection pressure during the course of its physiological evolution has resulted in some degree of limitation of change in surface permeability at moult. In the marine environment the need to reduce permeability at moult would perhaps be less critical in an animal isotonic with the medium. Comparison of the changes in the sodium and water permeabilities of a stenohaline, but otherwise similar, marine species at moult would, therefore, also merit study.

We are indebted to the Science Research Council for a grant to support this research and also to Mr M. H. Davis for technical assistance.