Osmotic and Ionic Regulation in a Terrestrial Snail, Pom Ati As Elegans (Gastropoda, Prosobranchia) with a Note on Some Tropical Pomatiasidae
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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 environ- ments (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.ful- vescens- 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 con- centration 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 haemo- lymph 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 haemo- lymph 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.