Osmotic and Ionic Regulation in a Terrestrial Snail, Pom Ati As Elegans (Gastropoda, Prosobranchia) with a Note on Some Tropical Pomatiasidae

J. Exp. Biol. (1972). 57. 205-2is 205 With 1 text-figure friinted in Great Britain

OSMOTIC AND IONIC REGULATION IN A TERRESTRIAL SNAIL, POM ATI AS ELEGANS (GASTROPODA, PROSOBRANCHIA) WITH A NOTE ON SOME TROPICAL POMATIASIDAE

BY T. J. RUMSEY Department of Zoology, University of Bristol, Bristol BSS 1 UG

(Received 4 January 1972)

The mechanisms of ionic regulation in terrestrial snails of the subclass Pulmonata have stimulated interest for some time (Duval, 1930; Fischer, 1931; Arvanitaki & Cardot, 1932; Pusswald, 1948; Vorwohl, 1961; Burton, 1966, 1968a, b, 1971); but relatively little work has been done on the terrestrial Prosobranchia. The latter have colonized land either via fresh water (Fam. Cyclophoridae) or by migration from the intertidal zone (Fam. Pomatiasidae). This study involves the latter group, concentrat- ing on the British species Pomatias elegans (Miiller). The Pomatiasidae are restricted to calcareous soils (Kilian, 1951; Avens, 1964; C. Little, personal communication) and P. elegans is only found in damp environments (Kilian, 1951), never being active below a R.H. of 95 % (Meyer, 1925). From the restricted distribution of pomatiasids (Kerney, 1968; Rumsey, 1971) it would be expected that adaptation to the terrestrial habitat is minimal, and that this will be partly reflected in their homeostatic mechanisms.

MATERIALS AND METHODS Material P. elegans was collected from Brockley Combe, south of Bristol, and in scrub wood- land on chalk downland near Westbury, Wiltshire. The density of snails was far less than that reported by Kilian (1951), but this perhaps is to be expected in an animal at the northern limit of its range (Kerney, 1968). After collection the snails were kept in large glass tanks containing a layer of beech-leaf litter, twigs and pieces of card- board, with limestone rocks at the bottom; the leaf litter was dampened every 2 weeks, and replaced every 2 months. The temperature was kept at about 25 °C throughout the year. Pomatiasids were also collected from Madagascar (Tropidophora cuvieriana; T.fulvescens- two varieties here named A and B), South Africa (Tropidophora ligata) and Jamaica (Annularia sp., Tudor a interrupta (Link), Licina (Colobostylus) nuttii (Pils.) and Parachondria angustae rufilabris). The areas of collection closely correlate with calcareous soils (Rumsey, 1971). These snails were kept under the same conditions as P. elegans in Bristol. 206 T. J. RUMSEY

Sampling methods A hole was filed in the shell above the heart and kidney (in the first whorl near to the umbilicus) care being taken not to puncture the body wall. Haemolymph was withdrawn from the rectal sinus, and urine from the kidney spaces, using siliconed Pyrex pipettes, tip diameter 0-02-0-05 mm, into which heavy liquid paraffin had first been drawn. The fluid sample was then transferred either to a watch glass, or to a Polythene centrifuge tube under liquid paraffin, and stored for a maximum of 2 min. The haemolymph was centrifuged for 2^ min at 1400-1700 rev/min in a Beck- man 152 microfuge to remove all cells and debris. The volume of urine collected was usually too small for centrifugation so that whole urine had to be used.

Analytical methods Estimation of osmotic pressure (o.P.) Osmotic pressure was measured by ' the freezing-point method of Ramsay & Brown (1955), samples for these determinations being frozen within 5 min after removal from the animal (Little, 1965). The apparatus was calibrated with standard solutions of sodium chloride, and osmotic pressure is expressed as mM/1 NaCl.

Estimation of the concentrations of individual ions The concentrations of ions are expressed as mM/1, this term being synonymous with mg ion/1. Haemolymph and urine samples were pipetted out and placed under liquid paraffin, after centrifugation. From these drops of fluid samples of known volume were removed with Polythene micropipettes. (1) Chloride. Chloride was estimated by the electrometric method of Ramsay, Brown & Croghan (1955) using a volume of approximately 1 /A. (2) Sulphate. Sulphate was determined by a modification of the method of Spencer (i960). The concentration was measured colorimetrically on a Pye Unicam SP 500 spectrophotometer. Sodium sulphate (Na2SO4) was used as a standard solution. (3) Bicarbonate. This ion was determined by the method of Little & Ruston (1970). The volume of fluid used for the estimation was approximately 1-5 /*1. (4) Sodium, potassium, calcium and magnesium. Determinations of these ions were made with a Pye Unicam SP 90 atomic absorption spectrophotometer, using the emission mode for sodium and potassium and the absorption mode for magnesium and calcium. The original centrifuged samples were diluted with glass-distilled water for the determination of ions in the following ratios: sodium 1:2000; potassium 1:100; calcium 1:2oo; magnesium 1:2ooo. No interference was found for any of these ions at these concentrations.

RESULTS Haemolymph composition of Pomatias elegans Yearly cycle Pomatias showed a yearly cycle of haemolymph concentration during the study period, which was from September 1970 to August 1971 (Fig. 1). Two maxima of O.P. Osmotic and ionic regulation in Pomatias elegans 207

210

200 190 g 180 If 170 J" 160 I 150 f 140 M 130 u •S 120 *° 110 -*-V d 100t Sept. Oct. Nov. Dec. • Jan. Feb. Mar. Apr. May June July Aug. •*- 1970 i 1971-*- Fig. 1. The monthly variation in O.P. of the haemolymph of Pomatias elegans kept in. terraria at 25 °C. The points represent means, or individual samples. The vertical lines represent + 1 S.E. of the means. No active or active and feeding snails were found between January and April 1971. • •, Active snails; • •, active and feeding snails; A—.—A, inactive snails. were found: one between December and February, and the other between June and July. During March and April 1971 minimum values for osmotic pressure of the haemolymph (104-129 mM/1 NaCl) were obtained. This cycle reflects patterns of activity in Pomatias which are partly intrinsic. The snails hibernated over the winter (as found also by Kilian, 1951) and aestivated in the summer, although they were kept under constant conditions. The maximum osmotic pressure of the haemolymph was obtained in January 1971 (202 mM/1 NaCl), by which time some snails had been inactive for 2 months. In February the haemolymph concentration began to decrease, and by the end of March most Pomatias were fully active. In April almost all the snails were active for most of the time (observed in the labora- tory terraria and in the field). Pomatias became less active in May, and during June and July many more inactive (aestivating) snails were found. These cycles of haemolymph concentration are similar to those found by Burton (1968) in Helix aspersa and Cepaea nemoralis.

Concentration related to activity The composition of the haemolymph of P. elegans was found to vary with the activity of the snail (Table 1). At the time of each estimation the snails were divided into three categories, and classed as 'active', 'active and feeding' or 'inactive'. At all times of the year (see Fig. 1) inactive snails had a higher O.P. of the haemolymph than active animals, the difference being 20-40 mM/1 NaCl. The o.p. of feeding snails is the most variable and can be higher or lower than that of inactive snails at the same time of year, but is always higher than the haemolymph o.p. of active non- 208 T. J. RUMSEY

Table i. The total ionic composition of the haemolymph of Pomatias elegans Concentrations expressed as mM/1.

O.P. Na K Ca Mg Cl so4 HC03 Active ± S.E. 140 no 60 165 2-5 106-0 3-2 II-O ±i-5 ±10 ±0-2 ±i-o ±0-2 ±0-5 ±O-2 ±0-5 Inactive + S.E. I7S-4 125-6 6-5 20-2 2-56 I2O-O 3'7 4-57 ±2-5 ±60 ±O-2 ±20 ±0-31 ±i-o ±o-i ±0-2 No. of samples = 5.

Table 2. The relationship between ionic percentages in the haemolymph of active, feeding and inactive animals throughout the year (O.P. = 100%) August 1971 activity Cations (%) Anions (%)

Na K Ca Mg Cl S0< HCO3 Mean + S.E. Mean ± S.E. Mean + S.E. Mean ± S.E. Mean + S.E. Mean ± S.E. Mean + S.E. Active 79-72 ±1-24 4-45 + 0-28 n-76±o-8 i-62±o-i4 8I-I±I-O 3-510-2 8-66 + 0-62 (n = 9) (n = 7) (n = 9) (n = 10) (n = 5) (n = 6) (>i = 4) Feeding 77-75 ±1-25 4-5410-27 10-4511-7 — (n = 9) (n = 4) (n = 3) Inactive 68-2O±3-o 5-oo±o-74 11-0 + 0-33 i-88±o-i4 3-olo-i 3-5510-4 (n = 5) (n = 3) (»i = 4) (n = 4) (n = 3) (n = 4) Significance of difference between active and inactive snails t 7-S c-5 0-91 2-08 — 0-42 9-6 n 12 8 II 12 7 6 P < O-OOI > o-io > 0-05—0-10 — > o-io < o-ooi The figures for '% cations' or '% anions' are calculated using the values for osmotic pressure in mM/1 NaCl as 100% of the cations and 100% of the anions. Haemolymph O.P. for active Pomatias was 140 mM/1; for feeding Pomatias 158 mM/1 and for inactive Pomatias 175-4 mM/1. Numbers in parentheses show the number of samples. feeding snails. It is suggested that Pomatias can become active with a high haemolymph O.P., but that this rapidly falls when the snail has become fully active. The cycles of haemolymph O.P. in relation to activity complement weight changes found in this species (Rumsey, 1971). When the animals are inactive, the weight is low and haemolymph O.P. high, and when animals are active the weight is high and the haemolymph O.P. low. This relationship has also been found in pulmonates (Duval, 1930; Burton 1965). The ionic composition of the haemolymph for Pomatias analysed in August 1971 can be seen in Table 2. The most striking feature is the high concentration of the ions in this snail, especially calcium, which is higher than most figures previously recorded for both pulmonates and prosobranchs, giving values between 16-5 and 20-2 mM/1 Ca2+ for the haemolymph. When the relative proportions of the various ions in the haemolymph are compared in active and inactive snails (Table 2) significant differences are found. The figures for active and feeding snails do not differ significantly from one another for any ion (P > o-io). In inactive snails magnesium rises in percentage concentration (P < 0001) and bicarbonate is halved (P < 0001). Figures for potassium, calcium Osmotic and ionic regulation in Pomatias elegans 209

Table 3. The ionic composition of haemolymph and urine in Pomatias elegans Concentrations expressed in mM/1.

O.P. Na K Ca Mg Mean + s.E. Mean + s.E. Mean + s.E. Mean ± s.E. Mean + s.E. Haemolymph (n = 6) 175-42 ±2-5 125-6 ±6-o 6-o±o-2 20-2 + 2-0 2-46 + 0-31 Urine (n = 5) 172-50 + 3-9 n7-9±5'6 6-o 180 2-64 + 0-21 No. in parentheses show the number of samples.

Table 4. The relationship between ionic percentages in the haemolymph and mantle fluid of active Pomatias elegans Na K Ca Mg Mean ± s.E. Mean + s.E. Mean + s.E. Mean + s.E. Haemolymph 79-72 ±1-24 4-45 + 0-28 n-76±o-8 I-62±O-I6

Mantle fluid 8o-o±3-i4 2-3i±o-28 7'03±i-4 o-66±o-i2 (» = 4) t 0-084 9'9 2"84 81-5 n 11 11 10 11 P > o-io < o-ooi > o-02-o-oi < o-ooi The figures for ' % cations' are calculated using the values for osmotic pressure in min/1 NaCl as 100% of the cations, o.p. of haemolymph was 140 mM/1. Mantle fluid o.p. was 115 mM/1. and sulphate are not significantly different between active and inactive snails (P > 010). Various changes in the ionic constituents of the haemolymph in relation to activity have been reported for other snails. Duval (1930) found a fall in bicarbonate concentration when the total haemolymph concentration in H. pomatia increased, and vice versa. He suggested a bicarbonate store that could be used when the snail became active. The low bicarbonate concentration of 6-5 mM/1 was found in two individuals of Pomatias just coming out of aestivation, which indicates that a system similar to that in Helix could be present. A rise in potassium ions in feeding snails as found by Burton (1965, 1968) for Helix was not found in Pomatias. However, a rise in magnesium in the haemolymph of inactive snails as suggested for Pila (Meenakshi, 1956) was shown in Pomatias (P = o-05-o-io). The kidney and mantle fluid Urine Table 3 shows the relationship between the O.P. and ionic content of the haemolymph and urine of inactive snails, analysed in August 1971. It can be seen that they have the same O.P. and the same concentration of the cations measured. The same situation is found in active and feeding snails at all times of the year. Avens (1964) suggested that the urine of Pomatias is hyposmotic to the haemolymph, but the present results do not verify this.

14 EXB 57 Table 5. Composition of the body fluids from a number of tropical Pomatiasidae

Tropidophora cuvieriana A. Haemolymph 99.12 f 1.55 (4) A. Urine IOI.IO+ 1.10 (3) T.fulvescens A A. Haemolymph 138.oof 2.60 (4) A. Mantle fluid - T.fulvescens B A. Haemolymph 103.12 f 2.90 (4) A. Urine 99.83 f 2.40 (4) A. Mantle fluid 95.0 (2) T. ligata A. Haemolymph 147.00 f 4.10 (5) A. Urine 133.60f 2.90 (3) A. Mantle fluid 123.0 Tudora interrupta A. Haemolymph 161.00 f 2.00 (3) Parachondria angustae rujilabris A. Haemolymph 102~00fo.50 (5) A. Urine 102.00 f 2.00 (5) Licina nuttii A. Haemolymph I 18.00 f 2.00 (4) A. Urine 114.0 I. Haemolymph 180.0 (2) I. Urine 181.oof 4.00 (3) Annularia sp. I. Haemolymph 94.00 f 1.00 (5) I. Urine 96.50f 2.00 (5) Numbers in parentheses show the number of samples: A. = active snails; I. = inactive snails. Osmotic and ionic regulation in Pomatias elegans 211

\lanlle fluid This fluid shows more variation in O.P. at any one time of the year than does the haemolymph. It may show a value higher or lower than that of the haemolymph of the snail from which it was obtained. In most cases however, the concentration of the mantle fluid is the lower, except when the snail has just emerged from aestivation. No mantle fluid is produced from inactive snails, and no fluid was produced in animals used for analysis between October 1970 and March 1971. When comparison is made between the percentages of ions in the haemolymph and in the mantle fluid (Table 4) it becomes apparent that ions are being reabsorbed. Of the cations measured, sodium was the only one not to have changed as a percentage concentration. Potassium (P < o-ooi), calcium (P < 0-02-0-01) and magnesium (P < o-ooi) are all proportionately lower in concentration in the mantle fluid.

Tropical Pomatiasidae Analyses of haemolymph, urine and mantle fluid were made on several tropical members of the Pomatiasidae. These came from Madagascar, South Africa and Jamaica (see Materials). The osmotic and ionic composition of the body fluids of some of these is seen in Table 5. The total concentration varies between species, although they are all found in damp habitats. The two varieties of T. fulvescens have a significantly different haemolymph concentration (P < o-ooi). Within any one species haemolymph and urine are isosmotic and have the same ionic concentration. The mantle fluid is hyposmotic to the haemolymph and has lower ionic concentrations, as generally found in P. elegans. It appears from the Jamaican species that there is some relationship between the haemolymph concentration and the amount of rainfall experienced throughout the year in the normal habitat. Both Tudora interrupta and Licina nuttii have a high haemolymph concentration; the O.P. is 161 ± 2-0 mM/1 NaCl in the former, and 118 ± 2-0 mM/1 NaCl in the latter. These species are found in areas where the rainfall is less than 50 in/year. (Scientific Research Council of Jamaica, 1963). P. angustae rufilabris and Annularia sp. are found in areas with 50-100 in of rain/year (S.R.C. Jamaica, 1963), and these species have lower haemolymph O.P. All the tropical species have a fairly constant haemolymph concentration throughout the year as they do not hibernate.

DISCUSSION The Pomatiasidae, whether from a temperate (P. elegans) or tropical (Madagascan, S. African and Jamaican species) climate are all found in areas of calcareous soil. Kilian (1951) states that the minimum concentration necessary for Pomatias is 7% calcium. The haemolymph O.P. is higher, in all species of Pomatiasidae, than is generally found in terrestrial snails. In the Pulmonata the range of values is from 35 ITIM/1 NaCl for Orthalicus undatus (Burton, 1971) to 75 mM/1 NaCl for Helix aperta (Burton, 1968). 14-2 [2 T. J. RUMSEY

Table 6. The bicarbonate concentration in the haemolymphof various molluscs Haemolymph HCO3 concentration in mM/1 Habitat Condition (mean + s.E.) Author Prosobranchia Littorina littorea S.W. Active 4-10 Rumsey (1971) S trombus gigas S.W. Active 10-16 + 0-36 Little (1967) Viviparus viviparus F.W. Active 11-0+1-4 Little (1965) V. japonicus F.W. Active 14-0 Rumsey (1971) Pomacea lineata F.W. Active 23-4±i-7 Little (1968) P. lineata F.W. Aestivating 25-64 + 4-69 Little (1968) Theodoxus fluviatilis F.W. Active 11-3+0-3 Little (1971) Poteria yallahsensis Terrestrial Active 11-70 + 0-9 Little (personal communication) P. lineata Terrestrial Active 1370 Little (personal communication) Eutrochatella tankervillei Terrestrial Active 125 Little (1971) Pomatias elegans Terrestrial Active n-o±o-5 Present study P. elegans Terrestrial Aestivating 4-57 ±0-2 Present study Tropidophora cuvieriana Terrestrial Active I3-5O±I-22 Present study Pulmonata Lymnaea stagnalis F.W. Active 27-0 Duval (1928) L. stagnalis F.W. Active 14-0 Chaismartin et al. (1967) L. stagnalis F.W. Active J2-O±O-4 Rumsey (1971) Planorbis corneus F.W. Active 340 Duval & Portier (1928) Helix pomatia Terrestrial Active 5-71 + 0-36 Sorokina & Zelenskaya (1967) H. pomatia Terrestrial Active 15-34 Burton (1968) calculated Lamellibranchia Anodonta cygnea F.W. Active I2-O Potts (1954)

In the terrestrial Prosobranchia, excluding the Pomatiasidae, the range is from 26-5 mM/1 NaCl for Eutrochatella (Little, 1971) to 36-8 mM/1 NaCl for Poteria (E. Andrews & C. Little, in preparation). As shown above, the values for Pomatia- sidae vary from 86-5 mM/1 NaCl for T. cuvieriana to 167 mM/1 NaCl for Licina nuttii. These high concentrations relate to the evolution of this group from the marine Littorinidae, without passing through fresh water. Most other terrestrial snails have probably evolved from freshwater ancestors, and this is reflected in the low o.P. of their body fluids. The concentration of calcium in pomatiasid haemolymph (especially of Pomatias) is high, a value of 20-2 mM/1 being obtained for one group of Pomatias, and this is higher than any concentration of this ion, for a terrestrial snail, found in the literature. The tropical pomatiasids have a lower concentration of calcium in the haemolymph, but most analyses were of immature animals, in which a fast turnover of this substance might be expected. Urine taken from the kidney is isosmotic with the haemolymph, and has the same concentration of ions. This again probably reflects the evolution of the Pomatiasidae from the Littorinidae, which also have urine with the same composition as the haemolymph (Avens, 1964; Todd, 1964; Rumsey, 1971). Large quantities of salts Osmotic and ionic regulation in Pomatias elegans 213 have therefore to be taken up from the environment to retain a stable haemolymph concentration. This helps to explain the restriction to damp calcareous soils where the microclimate is comparatively stable (Geiger, 1959). The mantle fluid is usually less concentrated than the haemolymph and urine in- dicating that ionic reabsorption is occurring over the mantle epithelium; sodium, potassium, calcium and magnesium are all less concentrated than in the haemolymph. Other terrestrial prosobranchs also show this ability to reabsorb ions over the mantle surface (E. Andrews & C. Little, in preparation; Little, 1971; Rumsey, 1971). Comparison of the haemolymph composition of all the pomatiasids indicates some correlation with habitat. Such a relationship has, in the past, been suggested for pulmonates by Arvanitaki & Cardot (1932) and Pusswald (1948), and disbelieved by Burton (1968, 1971). The pomatiasids with the highest haemolymph concentrations were found to inhabit the driest areas, and vice versa (Rumsey 1971). There is the possibility that a high o.p. reduces evaporative water losses, an adaptive advantage for animals in a dry habitat. Home (1971) suggested that an increased urea concentration in the haemolymph of Bulimulus during aestivation increased the chances of survival during periods of drought. Large variations in the concentration of calcium, bicarbonate and magnesium ions seem to be a general feature of all terrestrial gastropods. The figures for concentrations of calcium are difficult to interpret because of binding with various pro- teins (Manigault, 1939; Tilgner-Peter, 1957; Roach, 1963). In Pomatias levels vary between 8 and 25 mil/1 in active snails. During inactivity the concentration rises, but this rise is proportional to the rise in total O.P. Calcium therefore does not seem to play a large part in the buffering capacity of the haemolymph, as it does in Helix (Burton, 1968). Another ion involved in the buffering of haemolymph is bicarbonate. The concentration of this ion is halved in inactive Pomatias (Table 2) giving a concentration of 4/6 mM/1 compared to the concentration of 12 mM/1 found in active snails. Duval (1930) showed the same situation in Helix pomatia, and Trams et al. (1965) also found it in Cepaea. Table 6 shows that, like calcium, bicarbonate shows a very similar concentration in all classes of gastropod from all habitats. The reason for the high concentration in Pomacea is unknown. Magnesium ions, also involved in buffering (Burton, 1969), have also been found to increase in concentration in the haemolymph of aestivating snails (Meenakshi, 1956, de Jorge et al. 1965) including Pomatias (present study). In Pomatias the concentration rose from 1-62 to i-88% of the cation total (P = 005-0-10). The relative rise in potassium and calcium concentrations after feeding, or inactivity, in various pulmonates (Burton, 1965, 1968) was not found in pomatiasids, although the total O.P. did rise.

SUMMARY 1. The haemolymph concentration in the pomatiasids is higher than in any other terrestrial gastropods so far studied. 2. P. elegans shows a yearly cycle of haemolymph concentration, showing maximum values in January and February 1971, and June and July 1971. Minimal values were obtained between March and May 1971. 214 T. J. RUMSEY 3. The concentration of calcium in the haemolymph (especially of Pomatias) is high, a value of 20-2 mM/1 being obtained for one group of Pomatias. This reflects their dependence on calcareous soils for survival. 4. Haemolymph and urine are isosmotic and isoionic. 5. The mantle fluid is generally less concentrated than the haemolymph and urine, ionic reabsorption presumably occurring over the mantle epithelium. Potassium, calcium and magnesium ions are selectively reabsorbed. 6. The highest haemolymph concentrations in pomatiasids were found in animals that inhabited areas with the least rainfall, and vice versa. 7. During inactivity the percentage ionic composition of the haemolymph of P. elegans altered as the o.p. increased. Sodium decreased, magnesium increased, and bicarbonate decreased. It is suggested that the fall in bicarbonate concentration is due to a change in the buffering capacity of the haemolymph.

This paper forms part of a dissertation for the degree of Ph.D. at the University of Bristol. I am grateful to Dr C. Little for supervising this work, and to Professor H. E. Hinton for providing the facilities in the Department of Zoology. The work was financed by a grant from the Natural Environment Research Council.

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