1. Three major calcium compartments have been identified in L. stagnalis: the shell, the blood and the fresh tissues.

  2. The distribution of 45Ca, absorbed from the medium, in the tissues of L. stagnalis has been studied. Absorbed calcium appears first in the blood and then in the shell and other tissues.

  3. 30% of the total fresh tissue calcium and about 20% of mantle calcium exchanges with blood calcium. A continual exchange between shell and blood calcium occurs.

  4. During net calcium loss from L. stagnalis a net movement of calcium from shell to blood occurs at a rate similar to the rate of net loss. During net calcium uptake, the reverse movement from blood to shell at a rate similar to the rate of net uptake occurs.

  5. A simple mechanism which might account for the control of blood calcium concentration has been proposed.

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.

Calcium compartments in Limnaea stagnalis

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.

Table 1.

Tissue calcium contents in Limnaea stagnalis

Tissue calcium contents in Limnaea stagnalis
Tissue calcium contents in Limnaea stagnalis

Distribution of 45Ca absorbed from the medium

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. 1.

The increase in specific activity of shell calcium of snails kept in labelled artificial tap water containing 0·25 mm-Ca Each point represents the mean for six animals.

Fig. 1.

The increase in specific activity of shell calcium of snails kept in labelled artificial tap water containing 0·25 mm-Ca Each point represents the mean for six animals.

Fig. 2.

The increase in specific activity of calcium in fresh tissue of snails kept in labelled artificial tap water containing 0·25 mm-Ca. ●, Mean values for blood; ○, mean values for total fresh tissue ; ▄, mean values for mantle ; □, mean values for digestive gland. Each point represents the mean for 4–6 animals.

Fig. 2.

The increase in specific activity of calcium in fresh tissue of snails kept in labelled artificial tap water containing 0·25 mm-Ca. ●, Mean values for blood; ○, mean values for total fresh tissue ; ▄, mean values for mantle ; □, mean values for digestive gland. Each point represents the mean for 4–6 animals.

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.

Table 2.

The radioactivity of the shell and total wet tissues during absorption of 45Ca

The radioactivity of the shell and total wet tissues during absorption of 45Ca
The radioactivity of the shell and total wet tissues during absorption of 45Ca

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.

Fig. 3.

Specific activity of calcium in the blood, as a percentage of the specific activity of calcium in the medium, plotted against time. Each point is the mean for 5–6 animals. Vertical lines represent standard errors.

Fig. 3.

Specific activity of calcium in the blood, as a percentage of the specific activity of calcium in the medium, plotted against time. Each point is the mean for 5–6 animals. Vertical lines represent standard errors.

Loss of radioactivity from loaded snails

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.

Table 3.

Changes in specific activity of blood calcium during 45Ca efflux to unlabelled media

Changes in specific activity of blood calcium during 45Ca efflux to unlabelled media
Changes in specific activity of blood calcium during 45Ca efflux to unlabelled media
The data presented on the distribution of 45Ca in the tissues, following absorption from the medium, provides a considerable amount of information concerning calcium movement between internal compartments. 45Ca absorbed from the medium was distributed throughout the snail within 1 h of immersion in radioactive medium. Clearly the calcium absorbed from the medium must have been transported rapidly around the body, presumably in the blood. Certainly the blood became labelled more rapidly and reached a higher specific activity than the other tissues. Horiguchi (1958) demonstrated that 45Ca absorbed from the medium by the freshwater bivalve Hyriopsis schlegeli, was transported by the blood. In the marine shrimp Metapenaeus calcium absorbed from sea water by the gills passed rapidly into the blood (Dall, 1965). As the total 45Ca found in the mantle and digestive gland of L. stagnalis was greater than could be accounted for by contamination from labelled blood contained in the tissue samples it follows that an exchange of blood calcium and tissue calcium had occurred. The specific activity of the total fresh tissues reached only 30% and the mantle calcium only 20% of the final specific activity of calcium in the blood, these levels being attained within 30 h. The half-time of labelling was short (9 h) so the exchangeable fraction of tissue calcium must turn over rapidly. Exchange of the digestive gland calcium appeared to comprise a fast and a slow component. It is possible that the slow component may not represent exchange between blood calcium and digestive gland calcium but deposition of further calcium in the cells of this tissue. The shell became labelled at a fairly constant rate throughout the period of measurement indicating a continual gain of 45Ca from the blood. Again this could have been due to a deposition of labelled calcium in the shell or to exchange with blood calcium. It has been shown that the specific activity of calcium in the blood reached a constant level 30 % below that in the external medium. Two factors could account for this difference between the specific activities. In the first case, if a large proportion of blood calcium was bound, and also non-exchangeable, the specific activity of calcium in the blood would remain lower than that in the external medium by a percentage similar to that of bound calcium in the blood. In fact only a small amount of blood calcium is non-dialysable (0–6 %) and the remainder is freely diffusible (van der Borght & van Puymbroeck, 1964). Bound calcium, therefore, can account for only a small part of the discrepancy between the specific activities of calcium in blood and in medium. Thus the second possibility, that the specific activity of calcium in the blood is kept at a lower level than that in the medium by exchange with a large reservoir of calcium of low specific activity (the shell) must account for the major part of the difference between final specific activities of calcium in the blood and in the medium. Using the equation
(for derivation see Appendix), the calcium influx (m1) into the blood, calculated from data given in Fig. 3, was 0–20 μmole-Ca/g/h which agrees fairly closely with the calcium influx at the same external concentration found previously (Greenaway, 1971). From the same equation the rate of calcium exchange between the shell and the blood was 0·09 μmole-Ca/g/h. Information regarding calcium movements between blood and shell can also be obtained from the data on changes in the specific activity and concentration of calcium in the blood during 45Ca-efflux from loaded snails to calcium-free artificial tap water. Efflux to calcium-free media was accompanied by a fall in the specific activity and a slight rise in concentration of calcium in the blood. Efllux to calcium-free media was due to a net loss of calcium to the medium (Greenaway, 1971) so a transfer of calcium of low specific activity from the shell to the blood must have occurred at a rate similar to the rate of net loss to the medium. Calcium exchange between the shell and the blood has been calculated to occur at a rate of 0·09 μmole-Ca/g/h, a value similar to the rate of net loss to the medium by snails in calcium-free artificial tap water. It appears likely therefore that the normal rate of calcium movement from shell to blood may account for the maintenance of blood calcium concentration during net loss. During 45Ca efflux to artificial tap water (1 m-Ca) a fall in specific activity of calcium in the blood was also measured. However, since a mean net uptake rate of 0·135 μ mole-Ca/g/h was recorded during these experiments, a fall in the specific activity of calcium in the blood was not surprising.

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.

Fig. 4.

Diagram illustrating the calcium movements between the internal calcium compartments and the external medium.

Fig. 4.

Diagram illustrating the calcium movements between the internal calcium compartments and the external 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.

Van Der Borght
,
O.
(
1963
).
In and outfluxes of calcium ions in freshwater gasteropods
.
Archs int. Physiol. Biochim
.
71
,
46
50
.
Van Der Borght
,
O.
&
Van Puymbroeck
,
S.
(
1964
).
Active transport of alkaline earth ions as physiological base of the accumulation of some radionuclides in freshwater molluscs
.
Nature, Lond
.
204
,
533
4
.
Van Der Borght
,
O.
&
Van Puymbroeck
,
S.
(
1967
).
Ionic regulation and radiocontamination: ‘active transport’ of alkaline earth ions in freshwater gasteropods (Mollusca). Radioecological concentration processes
.
Proceedings of an international symposium
,
925
930
.
Stockholm 1966
:
Pergamon Press
.
Carriker
,
M. R.
(
1946
).
Morphology of the alimentary system of the snail Lymnaea stagnalis oppressa Say
.
Trans. Wis. Acad. Sci. Arts Lett
.
38
,
1
88
.
Carriker
,
M. R.
&
Bilstad
,
N. M.
(
1946
).
Histology of the alimentary system of the snail Lymnaea stagnalis oppressa Say
.
Trans. Am. microsc. Soc
.
65
,
250
75
.
Chaisemartin
,
C.
(
1965
).
La teneur en calcium de l’eau et l’équilibre calcique chez l’Ecrevisse Astacus pallipes Lereboullet en période d’intermue
.
C. r. Séanc. Soc. Biol
.
159
,
1214
17
.
Clark
,
A. M.
(
1957
).
The distribution of carbonic anhydrase in the earthworm and snail
.
Aust. J. Sci
.
19
,
205
7
.
Collip
,
J. B.
(
1921
).
A further study of the respiratory processes in Mya arenaria and other marine molluscs
.
J. biol. Chem
.
49
,
297
310
.
Dall
,
W.
(
1965
).
Studies on the physiology of a shrimp, Metapenaeus sp. (Crustacea: Decapoda: Penaeidae) V. Calcium metabolism
.
Aust. J. mar. Freshwat. Res
.
16
,
181
203
.
Dotterweich
,
H.
&
Elssner
,
E.
(
1935
).
Die Mobilisierung des Schalenkalkes für die Reaktions-regulation des Muscheln (Anodonta cygnaea)
.
Biol. Zbl
.
55
,
138
63
.
Dugal
,
L. P.
(
1939
).
The use of calcareous shell to buffer the product of anaerobic glycolysis in Venus mercenaria
.
J. cell. comp. Physiol
.
13
,
235
51
.
Freeman
,
J. A.
&
Wilbur
,
K. M.
(
1948
).
Carbonic anhydrase in molluscs
.
Biol. Bull. mar. biol. Lab., Woods Hole
94
,
55
9
.
Greenaway
,
P.
(
1970
).
Sodium regulation in the freshwater mollusc, Limnaea stagnalis (L.) (Gastropoda : Pulmonata)
.
J. exp. Biol
.
53
,
147
63
.
Greenaway
,
P.
(
1971
).
Calcium regulation in the freshwater mollusc, Limnaea stagnalis (L.) (Gastropoda : Pulmonata). I. The effect of internal and external calcium concentrations
.
J. exp. Biol
.
54
,
199
214
.
Horiguchi
,
Y.
(
1958
).
Biochemical studies on Pteria (Pinctada) martensi (Dunker) and Hyriopsis schlegeli (Martens). IV. Absorption and transference of 45Ca in Hyriopsis schlegeli (Martens)
.
Bull. Jap. Soc. scient. Fish
.
23
,
710
15
.
Horiguchi
,
Y.
,
Miyake
,
M.
,
Yoshii
,
G.
,
Okada
,
Y.
,
Inoue
,
Y.
&
Miyamura
,
M.
(
1954
).
Biochemical studies with radioactive isotopes on Pteria (Pinctada) martensi (Dunker) and Hyriopsis schlegeli (V. Martens). I. Ca metabolism by 45Ca tracer in Hyriopsis schlegeli (V. Martens
.)
Bull. Jap. Soc. scient. Fish
.
20
,
101
6
.
Kapur
,
S. P.
&
Gibson
,
M. A.
(
1968a
).
A histochemical study of the development of the mantle-edge and shell in the freshwater gastropod, Helisoma duryi eudiscus (Pilsbry)
.
Can. J. Zool
.
46
,
481
91
.
Kapur
,
S. P.
&
Gibson
,
M. A.
(
1968b
).
A histochemical study of calcium storage in the foot of the fresh-water gastropod, Helisoma duryi eudiscus (Pilsbry)
.
Can. J. Zool
.
46
,
987
90
.
Kawai
,
D. K.
(
1954
).
The metabolism of the pearl-oyster Pinctada martensii. II. Variations of the carbonic anhydrase of various tissues in relation to age and season, with special reference to the deposition of calcium carbonate
.
Physiol. Ecol. (Japan)
6
,
23
7
.
Kawai
,
D. K.
(
1955
).
Carbonic anhydrase in pearl oyster. II. Changes of the enzyme activity in relation to growth and seasons
.
Publs Seto mar. biol. Lab
.
5
,
89
94
.
Little
,
C.
(
1965a
).
Osmotic and ionic regulation in the prosobranch gastropod mollusc, Viviparus viviparus Linn
.
J. exp. Biol
.
43
,
23
37
.
Little
,
C.
(
1965b
).
The formation of urine by the prosobranch gastropod mollusc, Viviparus viviparus Linn
.
J. exp. Biol
.
43
,
39
54
.
Numanoi
,
H.
(
1939
).
Distribution of calcium in the soft parts of the freshwater bivalve Cristariaplicata
.
Jap. J. Zool
.
8
,
353
6
.
Potts
,
W. T. W.
(
1954
).
The inorganic composition of the blood of Mytilus edulis and Anodonta cygnea
.
J. exp. Biol
.
31
,
376
85
.
Schoffeniels
,
E.
(
1951a
).
Mise en évidence par l’utilisation de radiocalcium d’un mécanisme d’absorption du calcium à partir du milieu extérieur chez l’Anodonte
.
Archs int. Physiol
.
58
,
467
8
.
Schoffeniels
,
E.
(
1951b
).
Utilisation du radiocalcium pourl’étude de la diffusion et duremplacement du calcium au niveau des tissus de l’Anodonte
.
Archs int. Physiol
.
58
,
469
72
.
Schoffeniels
,
E.
(
1951c
).
Utilisation du 45Ca pour l’étude de l’état physico-chimique du calcium sanguin échangé au niveau des tissus, chez l’Anodonte
.
Archs int. Physiol
.
59
,
137
8
.
Tsujii
,
T.
&
Machu
,
A.
(
1953
).
Histochemical observation of carbonic anhydrase on shell and pearl formation
.
Kagaku
23
,
148
.
Vacge
,
L. E.
(
1952
).
Quantitative studies of calcium metabolism in Helix aspersa
.
J. exp. Zool
.
120
,
311
42
.
Wilbur
,
K. M.
(
1964
).
Shell formation and regeneration
.
Physiology of Mollusca
, Volume
1
.
London
:
Academic Press
.

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 m1m2. 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 (m1m2) and the efflux, m3. The accompanying diagram illustrates the movement of labelled and unlabelled calcium ions between the compartments (Fig. 5).

Fig. 5.

Diagram illustrating movements of labelled and unlabelled calcium ions between calcium compartments. For explanation for symbols see text.

Fig. 5.

Diagram illustrating movements of labelled and unlabelled calcium ions between calcium compartments. For explanation for symbols see text.

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

Therefore
Hence
Multiplying each side by
and integrating,
(c = integration constant).
When t = 0, n2 = 0. Therefore
Hence
Therefore
Therefore
But the specific activity of the blood, S.A.2, is , and that of the medium (s.A.1) is .
Therefore,