Calcium Regulation in the Freshwater Mollusc Limnaea Stagnalis (L.) (Gastropoda: Pulmonata): II. Calcium Movements Between Internal Calcium Compartments

The existence of calcium deposits in the tissues of freshwater molluscs is well established (Numanoi, 1939; Carriker, 1946; Carriker & Bilstad, 1946; Little, 1965 a; Kapur & Gibson, 1968a, b). Greenaway (1970) gave values for the Ca content of the fresh tissues of Limnaea stagnalis. In a histological investigation of the alimentary canal of L. stagnalis appressaCarriker & Bilstad (1946) described cells containing calcium granules in the digestive gland. Although appreciable amounts of calcium may be present in the fresh tissues, the shell of freshwater molluscs contains a very much larger amount and may be regarded as the major calcium-containing compartment. The blood forms a third calcium compartment, containing a relatively small amount of calcium the majority of which is in diffusible form and the remainder bound to blood proteins (Schoffeniels, 1951c; van der Borght & van Puymbroeck, 1964; Little, 1965a, b). There is evidence to suggest that movement of calcium occurs between the three calcium compartments described above (blood, fresh tissue deposits and shell). Calcium absorbed from the medium appears in the blood, fresh tissues and shells of certain freshwater molluscs (Schoffeniels, 1951a; Horiguchi et al. 1954; van der Borght, 1963; van der Borght & van Puymbroeck, 1967). Furthermore, Schoffeniels (1951b) found that radiocalcium injected into the blood of Anodonta appeared in the shell and fresh tissues, suggesting the movement of calcium from the blood to the other two calcium compartments. In support of this Horiguchi (1958) claimed that calcium absorbed by the freshwater bivalve Hyriopsis schlegeli was transferred from the blood to the shell and fresh tissues. The reverse movement of calcium from the shell to the blood has been observed in several marine bivalves (Dugal, 1939; Collip, 1921) and in Anodonta (Dotterweich & Elssner, 1935), calcium from the shell acting to buffer the blood against pH changes caused by carbon dioxide build-up. Greenaway (1971) has provided evidence indicating movement of calcium in both directions between blood and shell in L. stagnalis. Wagge (1952) obtained evidence for the transfer of calcium from shell to tissues in the terrestrial pulmonate Helix aspersa. Calcium movements between internal calcium compartments also occur in aquatic Crustacea. Dall (1965) demonstrated that calcium absorbed from sea water by the shrimp Metapenaeus became distributed throughout the fresh tissues but was mainly concentrated in the exoskeleton. The reverse movement from exoskeleton to blood occurred when Metapenaeus was placed in calcium-free sea water. Similarly, in Astacus pallipes a movement of calcium from exoskeleton to blood occurred during depletion in calcium-free water (Chaisemartin, 1965).

In this investigation measurements of the calcium content of the blood, of certain other fresh tissues and of the shell of L. stagnalis have been made to ascertain the relative magnitude of these calcium compartments. In addition ⁴⁵Ca has been used to examine the fate of calcium absorbed from the medium by Limnaea and to investigate the movements of calcium between the internal calcium compartments described above.

Materials and methods used in this investigation, apart from those detailed below, were as described previously (Greenaway, 1970, 1971). All snails used in this series of experiments were of 1–2 g total weight.

In experiments involving measurements of specific activity the following procedure was used. In experiments on uptake of ⁴⁵Ca snails were removed at intervals from their experimental media containing radiocalcium and washed in distilled water. Two 5 μl blood samples were removed from each snail and one diluted for calcium determination. The other sample was diluted appropriately on a planchet for radioactive assay. Each snail was then removed from its shell and the mantle and digestive gland were dissected off ; each part was placed in a silica crucible and dried at 100 °C for 24 h. The remaining fresh tissues plus washings from the shell interior and from dissecting instruments were treated similarly. The dry tissues were then ashed, dissolved in HC1 and diluted to suitable volumes for determinations of calcium and radioactivity. Shells were dissolved in HC1 and diluted with distilled water for analysis. One-ml volumes of the tissue samples plus a standard amount of 1 M dextrose, as a spreading medium, were dried on planchets and counted with an I.D.L. low-background counter.

The specific activity of blood calcium was also followed during ⁴⁵Ca efflux, from ⁴⁵Ca-loaded snails, to non-radioactive media. Snails were loaded for 2 weeks in ⁴⁵Ca-labelled artificial tap water containing 0·5 mm-Ca. After loading, the animals were divided into several groups and from one of these groups blood samples were taken for measurements of specific activity. The other groups were placed in unlabelled media for measurements of ⁴⁵Ca efflux. After 2–3 h half the snails from each group were removed and blood samples were taken for measurements of specific activity. At the end of the experiment the specific activity of the blood of the remaining animals was measured.

The term ‘fresh tissues’ in the following experiments refers to the soft parts of the snail exclusive of the blood, whilst the term ‘total fresh tissues’ includes both the soft parts and the blood.

The body of L. stagnalis may be regarded as comprising three major calcium compartments : the shell, the blood and the fresh tissues. The calcium contents of the shell, blood, total fresh tissues and certain organs of the fresh tissues are shown in Table 1. The snails used in these experiments, of total weight 1–2 g, contained approximately 1500 μmole of calcium in the shell and 30 μmole calcium in the total fresh tissues of which 3–4 μmole would normally be in the blood. From Table 1 it is apparent that Specific calcium concentration of the mantle and digestive gland tissues is greater than that of the total fresh tissue. However, these two organs, together accounting for 20 % of the total fresh tissue weight, contain only 25–30 % of the total fresh tissue calcium. Appreciable amounts of calcium, therefore, must be stored in the remaining fresh tissues. Carriker (1946) found cells containing calcium deposits lining the outer walls of arteries and capillaries in L. stagnalis appressa suggesting calcium stores may be distributed throughout the fresh tissues.

Batches of snails were placed in 500 ml volumes of artificial tap water containing 0·25 mm-Ca and labelled with ⁴⁵Ca. Snails were removed at intervals and the distribution of absorbed calcium was studied by measuring the specific activity of the calcium in the shell, blood, total fresh tissues, mantle and digestive gland of each animal. In this way the time course of the increase in specific activity was determined for each tissue and the relative specific activities of the tissues at any given time, after the start of the experiment, could be estimated. The increase in the specific activity of the shell calcium with time is shown in Fig. 1. The increase is seen to proceed in a roughly linear manner. Nevertheless, the specific activity of the shell calcium remained low, in comparison with that of the other tissues examined, throughout the experimental period. The time course of the appearance of ⁴⁵Ca in the soft parts is shown in Fig. 2. The slow increase in specific activity of the shell is to be expected in view of the large reservoir of unlabelled calcium present in the shell. Van der Borght (1963) and van der Borght & van Puymbroeck (1967) found that calcium absorbed from the medium by L. stagnalis was mainly located in the shell margin and to a lesser extent in the old shell. In these experiments no attempt was made to examine the shell margin and old shell separately.

Fig. 2 shows that the specific activity of the blood and tissue calcium rose rapidly during the first hours of immersion in the ⁴⁵Ca-labelled media and reached a constant level within 24 h. In the case of the digestive gland calcium, however, the specific activity continued to rise slowly. Half-labelling of the blood took 2–3 h whilst half-labelling of the fresh tissue calcium was less rapid, taking 9–10 h. The final specific activity of the fresh tissue calcium remained much lower than that of the blood, mantle calcium reaching 20 %, digestive gland calcium 40 % and total fresh tissue calcium 30 % of the final specific activity of blood calcium. Inevitably the samples of mantle and digestive gland tissue were contaminated with blood. However, the calcium concentration of the blood is low in comparison with the calcium concentration of these tissues (Table 1) and the amount of blood calcium of high specific activity in these tissued must therefore have been small. The total fresh tissue samples included the bloocr which contributed substantially to the final specific activity.

Table 2 shows values for the total radioactivity of the shell and total fresh tissues at time intervals of up to 122 h after immersion in ⁴⁵Ca-labelled media. The radioactivity of the total fresh tissues rose rapidly at first but between 22 and 47 h reached a fairly constant level. The shell radioactivity rose more slowly but continued to increase-throughout the experiment, and after 47 h exceeded the total fresh tissue activity. At the end of the experiment 41 % of the activity absorbed from the medium was located in the total fresh tissues and 59 % in the shell. Thus there was a continuing transfer of Ca from the medium to the shell which was not related to tissue calcium exchange.

In the experiments described above a marked fall in the specific activity of calcium in the medium occurred making the relationship between the specific activities of calcium in blood and medium rather difficult to interpret. In order to obtain a clearer picture of this relationship a second experiment was carried out under conditions where changes in the specific activity of external calcium would be minimal. A number of snails were placed in a polystyrene vessel, holding 10 1 of ⁴⁵Ca-labelled artificial tap water containing 0·3 mm-Ca and groups of animals were removed at known time intervals for measurement of the specific activity of calcium in the blood. The specific activity of calcium in the medium was measured at the same time. During the experiment the external concentration and specific activity of calcium remained constant. Fig. 3 shows the specific activity of calcium in the blood expressed as a percentage of the specific activity of calcium in the medium. The specific activity of calcium in the blood rose rapidly over the first 20 h and slowly thereafter until a constant level was reached after about 100 h at approximately 70 % of the specific activity of calcium in the medium.

Measurements of the specific activity of calcium in the blood have been made during the efflux of ⁴⁵Ca, from loaded snails, to non-radioactive media. In both artificial tap water (1 mm-Ca and calcium-free artificial tap water a fall of specific activity of calcium in the blood was recorded during the experimental period (Table 3). In the calcium-free medium specific activity of calcium in the blood fell by 13 % during the first 3 h of efflux and by 30% over the whole experimental period (7 h). The mean blood concentration of the experimental snails rose slightly during the experiment although a net loss of calcium, equal to the calcium efflux, occurred (Greenaway, 1971). During efflux to artificial tap water, containing 1 mm-Ca the specific activity of calcium in the blood fell by 21 % in the first 2 $12$ h of the experiment and by 35 % over the whole 6 h period. During this experiment there was a net uptake of calcium from the medium at a rate of 0·135 μmole/g/h and a small rise in blood concentration was again observed. The significance of these results is discussed below.

It may be helpful at this point to summarize the calcium movements involved in calcium regulation by L. stagnalis. A diagramatic summary is provided in Fig. 4. Calcium movements between the blood and the medium in normal fed snails may be-separated into several components. First there is a 1:1 exchange, probably on a passive basis, and secondly a loss of calcium from the blood, probably largely in the urine (Greenaway, 1971). Calcium uptake is normally greater than loss, resulting in a net movement of calcium into the blood, calcium gained in this way being deposited in the shell as CaCO₃. A supply of metabolically produced CO₂ is available in the tissues and in Crassostrea is sufficient to render the uptake of HCO₃⁻ or CO₂ from the medium unnecessary. This CO₂ may be readily converted to the carbonate (Wilbur, 1964). It seems likely that the uptake of calcium ions may be in exchange for hydrogen ions or perhaps accompanied by HCO₃⁻. The latter would seem unnecessary as metabolic HCO₃⁻ would then have to be excreted. An exchange of calcium between blood and shell and between blood and tissues has also been demonstrated. During net loss in calcium-free water the deposition of CaCO₃ ceases and a backflow of calcium from shell to blood compensates the loss of blood calcium to the medium.

It has been shown that the blood calcium concentration of L. stagnalis is maintained within fairly narrow limits during conditions of net calcium uptake from the medium and net calcium loss from the blood. Such a precise regulation of the blood calcium concentration under conditions varying from net uptake to net loss would suggest presence of a sensitive mechanism controlling the direction and magnitude of internal calcium movements. The simplest possible hypothesis to account for the situation described above is as follows. Potts (1954) found that the blood of the freshwater bivalve Anodonta was saturated or supersaturated with respect to aragonite. An increase in either pH or calcium concentration in the blood could, therefore, cause precipitation of calcium carbonate, provided other conditions remained unchanged. If it is assumed that the blood of L. stagnalis is also saturated with calcium carbonate, net uptake of calcium into the blood would cause precipitation of calcium carbonate in the shell. Net loss of calcium from the blood, however, would reduce the degree of saturation of the blood, causing a net movement of calcium from shell to blood. Such a mechanism would constantly compensate for changes in the direction of the net calcium flux between the snail and the outside medium and would also permit calcium exchange between the blood and the shell. It must be emphasized that the control of blood calcium concentration is unlikely to be as simple as this and must be co-ordinated with the mobilization of the organic constituents of the shell. Data given by Dall (1965) suggests that the blood of Metapenaeus may be supersaturated with respect to calcium carbonate in sea water of 2O%₀ salinity, calcium deposition thus being favoured and requiring only nucleation to initiate calcification. Dall also suggests that the enzyme carbonic anhydrase, present in the epidermis of Metapenaeus, may catalyse the solution of calcium deposits thus enabling resorption of calcium from the exoskeleton. No attempt has been made to find the site of this enzyme in the present study. Its presence has been demonstrated, however, in the mantles of several molluscs including aquatic species (Freeman & Wilbur, 1948; Tsujii & Machii, 1953;,Kawai, 1954, 1955; Clark, 1957).

This paper forms part of a dissertation for the degree of Ph.D. at the University of Newcastle upon Tyne. The work was carried out under a grant from the Science Research Council. It is a pleasure once again to thank Professor J. Shaw for his helpful advice and criticism. I am also indebted to Professor Shaw for the derivation of the equations presented in the Appendix.

This appendix concerns the calculation of the increase in specific activity of calcium in the blood of animals in an external medium containing radiocalcium and from which there is a net transfer of calcium to the blood. In addition there is a flux between the blood and the shell and a net transfer of calcium to the shell equal to that into the blood.

Consider a three-compartment system (1–3) representing the external solution, the blood and the shell. Let the outer compartment be considered sufficiently large for the total number of calcium ions (a₁) and the number of labelled ions (n₁) to remain constant. The influx from 1 to 2 is m₁ and the efflux is m₂, so that the net transfer is m₁ – m₂. The number of calcium ions in the blood (compartment 2) is a₂ and the number of labelled ions is n₂. The shell compartment (calcium ions, a₃ ; and labelled ions, N₃) is also very large so that n₃ remains at zero. The influx to the shell is m₃ plus the net transfer (m₁ − m₂) and the efflux, m₃. The accompanying diagram illustrates the movement of labelled and unlabelled calcium ions between the compartments (Fig. 5).

Since in the course of a short experiment n₃ = o, the last term diasppears and a₁, a₂ and n₁ can be considered constant.