The Excretion of Magnesium by Carcinus Maenas

Clasical studies on the ionic composition of the blood of Crustacea have shown that the magnesium concentration in the blood of most decapods is markedly lower than that in sea water (Robertson, 1939, 1949, 1953; Webb, 1940).

Webb (1940) and Robertson (1939) demonstrated that the lowering of the magnesium concentration in the blood could be accounted for by the fact that magnesium is eliminated in the urine at a high concentration.

It is only comparatively recently that the mechanisms by which the magnesium concentration in the blood is regulated by the excretory system have been studied directly (Gross & Marshall, 1960; Prosser, Green & Chow, 1955 ; Riegel & Lockwood, 1961; Gross & Capen, 1966) though Robertson (1953) suggested that it was more likely that magnesium was secreted into the urine than that the withdrawal of water accounted for the raised magnesium concentration in the urine.

Using ¹⁴C-inulin as a marker to determine the amount of water withdrawn from the urine in the excretory system, Riegel & Lockwood (1961) demonstrated that the raised magnesium concentration found in the urine of Carcinus is indeed due primarily to the secretion of magnesium into the urine, though withdrawal of water does make some contribution. Secretion is also responsible for raising the urine concentration of magnesium in Pachygrapsus crassipes and in this animal it has been shown that the site of magnesium secretion is the bladder wall and not the excretory organ itself (Gross & Capen, 1966).

When the concentration of magnesium in the urine of crabs is raised the sodium concentration is depressed (Webb 1940; Prosser et al. 1955;Gross, 1957; Gross, 1959; Gross & Marshall, 1960; Riegel & Lockwood, 1961; Gross, 1964; Gross & Capen, 1966). There is, however, no absolute relationship between the drop in sodium concentration and the rise in magnesium concentration, which suggests that the secretion of magnesium into the bladder does not involve an obligatory 1:1 exchange with sodium (Riegel & Lockwood, 1961 ; Gross & Capen, 1966). The latter authors have produced further evidence in support of this conclusion by showing that some magnesium secretion can still occur even when the active transport of sodium has been poisoned with ouabain.

As would be expected, the rate of secretion of magnesium is under facultative control and is influenced by the concentration of this ion in the blood (Gross & Capen, 1966). Despite this control the rate at which magnesium is lost from Pachy-grapsus in the urine is largely determined by the rate at which water passes through the excretory system (Gross & Marshall, 1960) so that in magnesium-free sea water the animal rapidly becomes depleted of magnesium. Survival of Pachygrapsus is poor in magnesium-free sea water (Gross & Marshall, 1960).

By contrast, Carcinus survives well in magnesium-free sea water, which implies that its magnesium metabolism may differ from that of Pachygrapsus.

In the present paper a simultaneous study has been made of the clearance of inulin from the blood and of the levels of various ions in the blood and urine in an attempt to evaluate the rôle of the excretory system of Carcinus in regulating magnesium excretion.

For the early experiments in the series Carcinus maenas were obtained from the Plymouth Marine Laboratory and maintained prior to use in a circulating sea-water system. Later experiments were carried out at the Plymouth Marine Laboratory using crabs maintained in the circulatory system of that laboratory. The majority of the crabs used exceeded 40 g. in weight. They were not fed during the course of experiments.

Three separate sets of experiments were undertaken. (1) The concentrations of sodium, calcium, magnesium and inulin were determined in the blood and urine of crabs at 24 hr. intervals after transfer to 50% or 150% sea water. (2) The concentrations of magnesium, sodium and inulin in blood and urine were followed after injection of magnesium into the blood. (3) The concentration of magnesium was measured in blood and urine following transfer of the animals to magnesium-free sea water or magnesium-free 50% sea water.

When measurements of inulin concentration were to be made the ¹⁴C-inulin was injected some days before the start of the experiment to ensure that it had equilibrated throughout the excretory system and that any diuretic effect following injection had passed off before analyses were started.

Urine was obtained by lifting the nephropore flap with a steel needle and sucking the emerging fluid into a glass pipette. Wads of Kleenex paper were inserted below the third maxillipeds prior to collecting urine to ensure that there was no possibility of contamination by fluid from the branchial chamber or mouthparts.

Blood was sampled by means of a hypodermic syringe inserted through the arthrodial membrane at the base of a walking leg.

Blood and urine samples were subdivided immediately after collection for analysis of sodium, calcium, magnesium-plus-calcium, and inulin.

Sodium and calcium measurements were made on an EEL flame photometer, appropriate corrections being made for interference by other ions in the case of the calcium determinations. Magnesium-plus-calcium was determined by EDTA titration and magnesium was estimated from this by subtraction of the calcium. In later experiments magnesium was determined directly by atomic absorption flame spectrophotometry using a Tektron Model AA4.

Estimation of ¹⁴C-inulin was made by means of a conventional gas-flow Geiger counter. The details of the analytical methods used are as described by Riegel & Lockwood (1961). An appropriate correction was made to inulin data to take account of the fact that there is a greater self-absorption of β-particles in blood samples than in urine samples.

The experiments were conducted at room temperature which fluctuated in the range 16–18° C.

Seventy-two hours prior to the start of the experiment 12 crabs were each injected with 0·05 ml. of 0·447% ¹⁴C-inulin. These animals were then replaced in sea water to allow the inulin in blood and urine to reach steady state. At the start of the experiment blood and urine samples were taken from each animal for inulin determination and ion analyses. Half the crabs were then transferred to 50 % sea water and the other six to sea water concentrated by evaporation to 150% sea water. Blood samples of 50 μl. and urine samples of 20 μl. were taken for inulin counts after the animals had spent 24, 48 and 72 hr. in these media at 18° C.

The ratio of the concentration of inulin in the urine to the concentration of inulin in the blood (U/B for inulin) is given in Table 1. In this particular experiment there is some difference between the U/B ratios after 72 hr. in the two media but, because of one exceptionally high value among the animals from 50 % sea water and one low value among the animals from 150% sea water, the means are not significantly different. A combination of the data from three similar experiments in which the inulin U/B for inulin was measured after exposure of animals for 72 to 96 hr. to the media gave a more positive indication of differences in U/B ratio. The following values were obtained: in 100% seawater, 1· 51 ± 0·10 s.e. N = 10; in 50% seawater, 1· 12 ± 0· 05 s.e. N = 13; in 150% sea water, 1·61 ± 0· 12 s.e. N = 10. There is a significant difference at the 5% level of confidence between the means of values for animals in 50% and 100% sea water but not between those of animals in 100% and 150% sea water.

Data derived from a number of similar experiments where animals were maintained in 100% sea water or transferred to 50% or 150% sea water some days after the injection of ¹⁴C-inulin indicate that there are differences in the rate of clearance of inulin from the blood in different media. The clearance rates 72 to 168 hr. after transfer to the media were respectively: in 100% sea water, 0· 38 ± 0· 12 ml./100 ml. blood/hr. (N = 10); in 50% sea water, 2· 14 ± 0· 61 ml./100 ml. blood/hr. (N = 16); and in 150% sea water, 0· 61 ± 0· 23 ml./100 ml. blood/hr. (N = 9). An intriguing feature of these results is that the clearance rate, and hence presumably the rate of primary urine formation, is faster in 150% sea water than in 100% sea water.

Samples of blood and urine were taken from each animal before transfer to 50 or 150 % sea water and again at 24, 48 and 72 hr. after transfer. The concentrations of sodium, calcium and magnesium were measured in each sample.

Figure 1, which illustrates the results of the sodium analyses, demonstrates three points of note: (1) the sodium concentration in the urine of animals taken directly from sea water is markedly lower than that in the blood (mean U/B = 0· 767) ; (2) the sodium concentration in the urine remains lower than in the blood in animals transferred to 150% sea water (mean U/B = 0· 764 after 72 hr.), Fig. 1b; (3) the sodium concentration in the urine comes to exceed that in the blood after animals have been in 50% sea water for 48 hr. (mean U/B = 1· 052 at 72 hr.), Fig. 1a.

On transfer of Carcinus from sea water to 50% sea water the U/B for magnesium declines from an average value of 7· 25 to 1· 24 in 72 hr., Fig. 2b. The former figure considerably exceeds the U/B for inulin, thus indicating that magnesium is being secreted into the urinary system when the animal is in sea water. The figure of 1-24 for the U/B for magnesium after 72 hr. in 50% sea water is, however, not significantly different from the U/B for inulin (1-22). This implies that secretion of magnesium is much reduced or has ceased after 72 hr. in 50% sea water. It is probable indeed that secretion has ceased long before this time, since the mean U/B for magnesium is in fact lower than that for inulin (though not significantly so) at 48 hr. This reduction in magnesium secretion contrasts with the situation in Pachygrapsus, where the U/B for magnesium exceeds 4 in 50% sea water (Prosser et al. 1955).

Conversely, when Carcinus is transferred to 150% sea water, there is little tendency for the magnesium U/B for magnesium to increase (Fig. 3b). This does not necessarily mean that the mechanism transporting magnesium into the urine is already operating at its maximum capacity when the animals are in sea water. Indeed, the fact that the U/B shows a small rise, on average, despite the larger inulin clearance which has been noted to occur in 150% sea water implies that the amount of magnesium secreted when the animals are in this medium is greater than that secreted when they are in 100% sea water.

The rate at which magnesium is lost from the body in terms of mM Mg lost/1. blood/hr. has been calculated from the following equation :

Median values for each period of 24 hr. were used in making the calculation. The individual results for animals transferred to 50 and 150% sea water are given in Table 2.

The data indicate that despite the greater rate of urine flow in 50% sea water the output of magnesium is in fact lower in this medium than when the animals are in 150% sea water. This is in contrast to the situation found by Gross & Marshall (1960) for the crab Pachygrapsus.

The U/B for calcium, unlike that for magnesium, remains at a level not significantly different from that for inulin when the animals are put into either 50 or 150% sea water (Figs. 4, 5). As a consequence of this the rate of loss of calcium in the urine is largely determined by the rate of urine flow.

The results described above imply that renal activity with respect to the excretion of magnesium is modified according to the nature of the environment, magnesium being secreted into the urine when the concentration in the blood is high but not secreted when it is low.

In order to ascertain whether the rate of secretion of magnesium is determined by the concentration in the blood or the salinity of the medium the magnesium concentrations in blood and urine of two batches of animals were followed for 10 and 9 days respectively after transfer to magnesium-free sea water. The artificial sea water used was based on the formula given by Hale (1958) but with the MgCl₂ replaced by the equivalent amount of NaCl. The medium was replaced at intervals to prevent a build-up of magnesium as the ion was lost from the animals.

Despite the fact that during the period of the experiment the magnesium concentration in the blood fell to about one quarter of the initial value there was no obvious change in the activity or behaviour of the crabs, all of which remained vigorous and aggressive until the termination of the experiment.

The results (Table 3) indicate that in these conditions there is a proportionally larger drop in magnesium concentration in the urine than in the blood so that the U/B for magnesium declines from 4·34 to 1·90 on average in one experiment and from 5·38 to 2·98 in the other. If it is assumed that the ratio of filtration and water removal from the urinary system remain unaltered during the course of the experiment, this result implies that there is a marked decline in the rate of magnesium secretion into the urine. Gross & Marshall (1960) found no variation in the magnesium concentration in the urine after exposure of Pachygrapsus to magnesium-free sea water for 24 hr., and as the magnesium concentration in the blood fell in magnesium-free sea water the U/B for magnesium in fact rose. Therefore in this respect also Pachygrapsus differs from Carcinus.

In order to investigate the response of the excretory organ to an excess of magnesium in the blood seven animals were acclimatized to 50% sea water and then injected with 0-05 ml. of saturated MgCl₂ (5·68 M/1.). A preliminary experiment had shown that this amount of magnesium approximately doubles the magnesium concentration of the blood. A similar volume of NaCl solution was injected into five control animals which had also been acclimatized to 50% sea water. The concentrations of sodium, magnesium and calcium in blood and urine were determined on both sets of animals prior to injection and again 7, 13 and 25 hr. after injection. Samples were also taken for inulin determinations, all animals having been injected with ¹⁴C-inulin prior to the initial acclimatization to 50% sea water.

The average magnesium concentration in the blood of the experimental animals rose sharply after injection of magnesium and 7 hr. later was still approximately three times the initial level. The concentration in the blood declines with time (Fig. 6) and by 25 hr. after injection is no longer significantly different from that of the controls. The magnesium concentration in the urine rises in the experimental animals but not in the controls, and the U/B for magnesium is also significantly higher in the experimental than in the control animals at 7 hr. and remains higher till the end of the experiment. There is no systematic variation in U/B for inulin during the course of the experiment and the half-time for clearance of inulin from the blood of the magnesium-injected animals was 17-5 hr., only about half that of the animals exposed to 50% sea water in the experiments mentioned earlier. It is probable therefore that the rise in U/B for magnesium results from an increased rate of magnesium secretion and that it is not brought about by increased residence time of urine in the bladder enabling an increase in urine concentration at a constant rate of secretion.

It is possible that the increased clearance rate, following injection of magnesium, is in part due to a diuresis created by the presence of excess magnesium in the blood. Burger (1957) found a similar diuresis after injection of magnesium sulphate into lobsters. The clearance was also more rapid in the controls injected with NaCl (half-time 25 hr.) than in uninjected animals exposed to 50% sea water, which suggests that the faster rate of clearance is not solely due to magnesium diuresis.

Robertson (1949, 1953) drew attention to the fact that there is an apparent relationship between the activity of certain species of decapod crustaceans and the concentration of magnesium in the blood. Species such as Maia and other spider crabs which are generally rather sluggish have a high concentration of magnesium in the blood but more active forms such as Carcinus, Pachygrapsus and Homarus have low magnesium concentrations. Maintenance of the magnesium concentration in the blood at a level below that in sea water is achieved by the production of urine with a magnesium concentration greater than that of the blood (Webb, 1940; Robertson, 1949; Prosser et al. 1955; Gross & Capen, 1966). The last-named workers have also shown that in Pachygrapsus the raised magnesium concentration in the urine is brought about by secretion of magnesium across the bladder wall from the blood. The rate of secretion is under facultative control and is dependent on the concentration of the ion in the blood. Several of the findings in the present study imply that the secretion of magnesium into the urine of Carcinus is also under facultative control. These are : (1) the calculated loss of magnesium from the body in unit of time when the animals are in 150% sea water is greater than when they are in 50% sea water even though the rate of urine flow is some five times more rapid in the latter than in the former medium ; (2) when the magnesium concentration in the blood of animals acclimatized to 50% sea water is increased by the injection of magnesium chloride the U/B for magnesium 7 hr. later is significantly higher than that of controls injected with sodium chloride, even though the rate of urine production (inulin clearance × U/B for inulin) is faster in the animal injected with magnesium; (3) the U/B for magnesium declines when the animals are transferred from sea water to magnesium-free sea water. The final magnesium concentration of the urine and the total loss in unit time naturally depend on three factors ; (a) the rate of secretion of magnesium into the urine ; (b) the rate of flow of urine ; and (c) the amount of water and other ions withdrawn from the excretory system between the formation of the primary urine and the eventual release of the definitive urine.

It has been suggested that in mammals there may be a common absorptive pathway for both calcium and magnesium in the renal tubule and gut (McIntyre, 1963). The mechanism is assumed to be such that deficit or excess of one of these ions would lead to over-absorption or under-absorption of the other. There is no evidence for the presence of such a common transport system in Carcinus. The U/B for calcium varies little, and in a non-systematic manner, in conditions where magnesium is likely to be secreted rapidly or slowly (Fig. 7).

Magnesium secretion in crabs appears to be associated primarily with an exchange for sodium so that when urine magnesium is high urine sodium is reduced (Webb, 1940; Prosser et al. 1955; Riegel & Lockwood, 1961 ; Gross & Marshall, 1960; Gross & Capen, 1966). Gross and Capen have shown that some magnesium secretion can occur in the bladder even after the poisoning of the active transport of sodium with ouabain, and they suggest therefore that the secretion of magnesium does not involve an obligatory exchange with sodium, though leaving open the possibility that poisoning of the sodium transport system is incomplete because of the complexity of the bladder in brachyurans (Balss, 1944).

Pachygrapsus and Carcinus are crabs which inhabit slightly different ecological niches. Both are forms whose main habitat is the intertidal region of rocky shores, but whereas active Pachygrapsus have been found occurring naturally in a hypersaline lagoon with a salinity of up to 190% sea water (Gross, 1968), Carcinus is intolerant of such raised salinities. Pachygrapsus (Hiatt, 1948) and Carcinus both penetrate into estuaries but Carcinus appears more tolerant of low salinities than Pachygrapsus. Pachygrapsus tends to raise itself out of the medium when placed in 50 % sea water, whereas Carcinus remains immersed.

Comparison of the magnesium excretion of Carcinus and Pachygrapsus reveals certain differences, some of which can be related to the ecological factors.

In Pachygrapsus the control of the rate of magnesium secretion is apparently not adequate to override the effect of variation in urine volume over the range of salinities tolerated by the crab. Consequently when the animals are in 50% sea water there is a more rapid loss of magnesium from the body than when they are acclimatized to 150% sea water even though the magnesium concentration in the urine is six times greater in the latter than in the former medium (Gross & Marshall, 1960). The U/B for magnesium does not fall below 5 in 50% sea water in Gross & Marshall’s experiments or below 4 in those of Prosser et al. (1955).

The present study suggests that Carcinus has a renal secretory system which is somewhat more adaptable than that of Pachygrapsus in regulating magnesium over the same range of salinities. Thus there is a difference of more than 20-fold in magnesium concentration in the urine of crabs after 72 hr. in 50% sea water by comparison with crabs in 150 % sea water for the same period. This variation in concentration more than compensates for the difference in urine volume between the two media (3·0% of body weight/day in 150% sea water and 14·9 %/of body weight/day in 50% sea water). As a result, Carcinus is able to excrete more magnesium when it is in 150% sea water than it does in 50% sea water. On a priori grounds we should expect this to be a physiologically desirable end, since more magnesium would be expected to diffuse into the blood from the more concentrated medium than from the dilute one. The data given in Table 2 suggest that between 48 and 72 hr. after transfer to 50 and 150% sea water Carcinus is eliminating magnesium in the urine more than times as rapidly in the more concentrated medium than in 50 % sea water. The values obtained by Gross & Marshall (1960) suggest that Pachygrapsus excretes magnesium in 150% sea water at the rate of 0·0073 m.equiv./day/g. Calculations based on our results put the equivalent figure for Carcinus at 0·0062 m.equiv./day/g. The implication is that Pachygrapsus excretes magnesium somewhat more rapidly than Carcinus when in 150% sea water. However, in view of the various differences in experimental technique used on the two crabs this conclusion can only be tentative. Comparison of the loss rates in dilute media (50% sea water) reveals larger differences between the two species. Carcinus is calculated to lose magnesium at the rate of 0·0023 m.equiv./day/g. whereas the loss from Pachygrapsus is given as 0·0433 m.equiv./day/g. However, Gross and Marshall comment that this high rate of loss, in what was a very short period of measurement, may not be maintained in the long term.

Another difference between Carcinus and Pachygrapsus is that Webb (1940) found that the magnesium concentration in the urine of Carcinus rises when the animal is placed in sea water with an artificially raised magnesium concentration but this does not happen in Pachygrapsus (Gross & Marshall, 1960). When the converse experiment is tried of placing the animals in magnesium-free sea water Pachygrapsus does not decrease the magnesium concentration of its urine so that as the blood concentration falls the U/B for magnesium rises (Gross & Marshall, 1960). Carcinus in the same medium reduces both the urine concentration and the U/B for magnesium. It should, however, be noted that the reduction in U/B for magnesium becomes more marked after the Carcinus have spent several days in magnesium-free medium (Tables 3, 4), and Gross and Marshall were only able to keep Pachygrapsus in this medium for shorter periods. It appears in fact that Pachygrapsus is ill-adapted for the conservation of magnesium when in media with low Mg concentrations. Gross & Marshall (1960) calculated that the rate of loss of magnesium was four times as large in 50% sea water as in 100% sea water. Calculation of the relative loss rates in these two media for Carcinus based on the inulin clearance and U/B for magnesium suggests that when the animal is fully acclimatized to 50% sea water the rate of loss of magnesium is some-what less than that to 100% sea water. As a reflexion of this difference Carcinus survives in a healthy state for at least 96 hr. (experiment then discontinued) in magnesium-free 50 % sea water, whereas Pachygrapsus lasts only a few hours in this medium.

Carcinus is a species which normally does not inhabit waters with a salinity in excess of 100% sea water and it is therefore not surprising that neither the highest individual U/B for magnesium nor the average U/B for magnesium after 72 hr. in 150% sea water is significantly larger than the corresponding values in 100% sea water. However, the observation that the clearance of inulin increases in this medium whilst the U/B for magnesium does not fall even though the concentration of magnesium in the blood rises leads to the conclusion that the animal is able to eliminate more magnesium per unit time when in 150% sea water than when it is in 100%. It thus seems likely that Carcinus is able to increase the rate of secretion of magnesium if the rate of primary urine formation is raised though it is not able to raise the U/B for magnesium significantly above the level which is found when the animal is in sea water. Gross & Marshall (1960) similarly observed that there is no increase in the U/B for magnesium when Pachygrapsus is transferred to 150% sea water from 100%. In both media this remained at about 15 as compared with the average of 8 in Carcinus. Although the maximum U/B for magnesium of Pachygrapsus in 150% sea water exceeds that for Carcinus by a considerable margin, the maximum concentrations of magnesium in the urine and the gradient between urine and blood are much more closely similar, though the advantage still lies with Pachygrapsus (Table 5). Care must be taken in interpreting these differences, since they may be due in part to differences in the techniques used to obtain the results. The values given by Gross and his collaborators for 50, 100 and 150% sea water refer to acclimatization periods of 24 hr. or 48 hr. in the case of the desiccation experiments, whereas Riegel & Lockwood (1961) and the present study have taken values only after 3–5 days acclimatization. When Gross (1961) examined Pachygrapsus taken from a hypsersaline lagoon which had at that time a salinity of 175% sea water the values obtained were close to the Carcinus values in 150% sea water. Burger (1957) noted that the U/B for magnesium may drop to 0·8 when Homarus is in dilute sea water (60–70 %) but there is no evidence to suggest that either Pachygrapsus or Carcinus can reabsorb magnesium from the excretory system if the blood becomes depleted of this ion. Robertson (1953) found no evidence for the binding of magnesium by blood proteins. If the primary urine is formed as an ultrafiltrate of the blood then the difference in magnesium concentration of the blood and primary urine should be no greater than can be accounted for by the Donnan effect. Since the U/B for magnesium in the final urine of Carcinus has not been observed to decline to a value significantly less than the U/B for inulin (even when there is a net loss of magnesium from the body) it is unlikely that there is any mechanism for the withdrawal of magnesium from the urine. This may be unimportant in normal conditions to Carcinus, as the present experiments suggest that even a reduction of the magnesium concentration in the blood to 25 % of the normal value found in animals maintained in sea water has no apparent adverse effect over a period of a few days. Possibly this may be related to the observation of Shaw (1955) that there is a marked cellular retention of magnesium when the blood concentration falls.

The fact that Carcinus is better able to conserve magnesium than is Pachygrapsus and also that it tolerates low magnesium concentrations in the blood can perhaps be related to the ecological and behavioural differences between the two animals. If given a choice Pachygrapsus tends to raise itself out of dilute media, whereas Carcinus remains submerged. Similarly where Pachygrapsus penetrates along estuaries it tends to occur at and above the high-tide line (Hiatt, 1948), whereas Carcinus may be found submerged in diluted sea water.

We would like to express our appreciation to the Director and staff of the Plymouth Marine Laboratory for the facilities made available to us during our visit in August 1968 and especially to Dr G. W. Bryan for the use of the Tektron absorption spectrophotometer. We would also like to thank Miss M. A. Riegel and Mr M. H. Davis for technical assistance.