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Osmotic balance in gastropod molluscs—II. The brackish water gastropod, Hydrobia ulvae pennant
Comp. Biochem. Physiol., 1965, VoL 16, pp. 143 to 153. Pergamon Press Ltd. Printed in Great Britain
OSMOTIC BALANCE IN GASTROPOD MOLLUSCS--II. THE BRACKISH WATER GASTROPOD, HYDROBDI ULV.4E PENNANT A N G E L A C. AVENS* Department of Zoology, University of Exeter
(Received 12 March 1965) Abatract--1. Blood concentrations of H. ulvae were determined in normal, dilute and concentrated sea-waters, and on exposure and reimmersion. 2. The species proved to be poikilosmotic and very euryhaline, and had a very effective means of volume control. 3. Equilibration to new media took about an hour in active animals, but retraction delayed the process at extreme salinities so that it took several days at least. 4. Increases in blood concentration of up to 100 per cent were permissible before death by desiccation on exposure. 5. These results are similar to those previously found for littoral species although there seems to be a greater tolerance of blood concentration changes in H. u/vae. 6. Small size, salt and water permeability and volume control are discussed in relation to euryhalinity. INTRODUCTION IN ^ previous paper (Avens & Sleigh, 1965) an account was given of an investigation of the osmotic balance between marine and littoral Gastropods and their environments under conditions of varying salinity and exposure. T h e small estuarine snail Hydrobia ulvae is similarly treated in this account, which is to be followed by a report of findings on freshwater and terrestrial snails in later papers. It was hoped that these studies would help in understanding the physiological adaptations involved in the dispersal of Gastropods away from the sea. A study of H. ulvae is interesting not only because it permits comparison with the species previously described, but also because it may indicate features intermediate between marine and freshwater species. H. ulvae lives on estuarine mudflats and on salt marshes, never out of direct reach of the sea. On mudflats it occurs at mid-tide level and upwards, and is consequently covered at almost every tide. Such a habitat is probably only brackish periodically; brackish water being considered to have a salinity between 0"5~oo and 30%o, which is equivalent to having a freezing point depression of between 0.05°C and 1.62°C. On salt marshes, living conditions are probably more extreme, although the animals tend to congregate in damp hollows. Only during the spring tides are such areas covered, thus allowing time for more extreme conditions to * Present address: West Buckland School, Near Bamstable, North Devon, England. 143
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develop. Precipitation and run-off from the land can dilute the available water, whilst evaporation, the inflow of sea water or the freezing of some water out as ice can raise the salinity. There is therefore much variation in living conditions, and indeed Nicol (1935) found that conditions on a salt marsh can vary more in 24 hr than in the sea over a period of 6 months. In a recent paper T o d d (1964) has reported observations similar to some of those described here on H. ulvae. By measuring urine concentrations, which were assumed to be close to the blood concentrations of the animals sampled, she found that H. ulvae was slightly hyperosmotic on dilution from 100 to 50% sea water, but that the urine was sometimcs markedly hyperosmotic in 25% sea water and always hyperosmotic in fresh water. In these latter cases the animals were withdrawn into the shell, but experiments on phenol red uptake indicated that there was some exchange of materials between H. ulvae and its surroundings when the animal was withdrawn. MATERIALS AND METHODS Animals were collccted from a salt marsh at Budleigh Salterton, South Devon, and were kept submerged until required either in Plymouth sea water or in natural water from the salt marsh. Salt-marsh animals were used since they tend to be larger than estuarine ones (Rothschild & Rothschild, 1939). The methods used were essentially those described more fully in the previous paper. Total osmotic concentrations of the blood and sea water samples were measured by the microcryoscopic method of Ramsay & Brown (1955), and results are given as the depression of freezing point, A°C. Temperatures could be r e a d to the nearest 0-005°C, and readings for the same sample are reproducible to + 0.005°C. The animals were stuck, operculum downwards, in slightly hardened plasticene on the stage of a binocular microscope. After cleaning the shell, a hole about 1 m m diam. was bored on the dorsal side of the penultimate whorl with a dentist's drill. Through this hole a blood sample was sucked from the visceral sinus into a fine silica glass pipette of the type used in the freezing-point apparatus. Almost all of this sample was the n blown out under paraffin oil for storage, a little being retained to reduce evaporation errors during sampling. The samples contained regularly distributed blood corpuscles; those with many inclusions or those of yellowish colour being rejected on the grounds of probable contamination. Extraction of blood samples took less than a minute, and A measurement was done within the next half-hour (and usually within 10 min of extraction) so that increases in A on storage were kept to a minimum. Blood AA were measured for animals in natural waters, in dilute and concentrated sea-waters and on exposure and reimmersion. Dilute sea-water solutions were made by adding distilled water to Plymouth sea water, and concentrated sea waters were mixed from boiled and fresh Plymouth sea waters. T h e per cent sea water figures given here are only approximate; A readings were always taken to find the exact concentration.
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The degree of euryhalinity of the snails was investigated by preparing a series of diluted and concentrated sea-waters and transferring animals directly from 100% sea water to each of the concentrated solutions, and to each of the diluted solutions in which no ill-effects were seen. When ill-effects appeared, mainly prolonged retraction, transfer of active animals was made step by step along the series. Two or 3 days were allowed for equilibration in each solution, except where the animals retracted ; then it was necessary to wait and see if the animals resumed activity or died. Lifting or tapping the operculum with a pin would normally elicit a response should the animal be alive, but in doubtful cases observations were either extended for a further period, or the snail was returned to a more favourable medium in which living animals would become active. In experiments to follow the change in weight of H. ulvae under different conditions, it was necessary to clean and dry the shell with a tissue before weighing. Here results are usually expressed as the per cent change in live weight, i.e. total weight including the shell. Only in a few cases was it possible to extract the animals from their shells and determine the per cent change in wet tissue weight, i.e. live weight minus the dry weight of the shell and operculum. The shell normally forms about 50 per cent of the live weight. RESULTS
1. Blood concentrations in media of different concentrations The blood was isosmotic with the salt-march water; the average blood A of nine animals being 1.809°C (1.775-1.865°C) in a medium of A 1.760°C. The slight hyperosmoticity shown here is not held to be significant since the animals and samples were very small, and evaporation errors would therefore be greater than in experiments with marine and littoral species. The smallest A recorded for natural salt marsh water was 1.04°C, which is equivalent to about 60% sea water. This isosmoticity of the blood and the external medium was maintained in active animals at all tolerated concentrations of the external medium (Fig. 1). In some cases, however, where the concentration change was so great as to cause retraction, the blood concentrations several days after transfer were intermediate between the concentrations of the initial and test media (Fig. 1). This is clearly demonstrated in an experiment on a group of animals transferred directly from 100-20% sea water (A 1.890°C-A 0.350°C); all of these animals retracted, and blood AA were measured each day over a period of 11 days (Fig. 2). A slow drift towards the A of 20% sea water was apparent, similar to the rather more rapid drift found in a corresponding experiment with Littorina saxatilis as mentioned in the previous paper. Since Todd (1964) found that salt-mal'sh H, ulvae whose urine was hyperosmotic to the medium can remain retracted in 25% sea water for 29 days, it is not surprising to find the drift does not go far towards the A level of 20% sea water in only 11 days. Nevertheless, all of the three animals that resumed activity on being brought into a warmer laboratory for blood extraction (21°C as opposed to 10-15°C) were isosmotic with the medium (Fig. 2). This drift must result from exchange between the animal and the test medium. Todd has in fact
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shown that phenol red can pass into the tissues of retracted H. ulvae in fresh water within 48 hr. Such exchange may arise from an ill-fitting operculum or from the necessity to open the shell periodically for respiratory and/or excretory purposes, although such opening has not been observed, but it could not entirely result from exchange in the initial period of activity often seen on immersion in media that cause retraction. x
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FIG. 3. Effects on the blood osmotic concentrations of active animals of transfer (a) from 60% sea water (A = 1.09°C) to 100% sea water (A -- 1.91°C), and (b) from 100% sea water (A = 1.95°C) to 60% sea water (A = 1.07°C).
When animals remain active after transfer, however, equilibrium is achieved very quickly; after a change f r o m 60 to 100% sea-water and vice versa, the blood reached an equilibrium level in a little over an hour (Fig. 3).
2. Weight changes on transference to different media T h e live weights of groups of snails (usually 10) were measured before transfer to a new m e d i u m and at intervals between 15 rain and 4 hr after transfer. Changes
in live weight did not exceed some 3 per cent, and were generally about 1 per cent whether the change was f r o m dilute to normal sea-water or vice versa, or f r o m concentrated to normal sea-water. Such small changes as did occur were not consistent, nor did they accord with the laws of osmosis. Control experiments
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indicated that errors of this size could arise from differences in the thoroughness of drying. Weight changes of the magnitude found for Littorina saxatilis (Avens & Sleigh, 1965) were not observed here.
3. Tolerance of dilute and concentrated media Field records of the salinities at which H. ulvae were found are available from many sources for dilute media. In a brackish water loch on North Uist, Nicol (1936) found H. ulvae at a salinity of 10%o (A = 0.53°C approx.), and the closely related H. ventrosa at 24%0 (A = 1-29°C approx.). Rather lower values have been reported for the Baltic by Remane (1958), who found H. ulvae at salinities of 6-8%0 (A = 0.32-0-43°C approx.), and Fretter & Graham (1962) quote the work of Jessen (1918) and Johansen (1918), both working at Randjersfjord, who found H. ulvae in water of 1%o and fresh water respectively. McMillan (1948) suggested that there may be different biological races in H. ulvae. Animals that she took from Burton Marsh in Cheshire behaved normally in 7-5% sea water, 5% sea water proving to be lethal, whereas snails from an estuary 9 miles away and from a brackish ditch in Co. Down were active in 25% sea water but retracted in 22% sea water. It is important to note that variation in the experimental temperature can affect results. Ellis (1925) found that at room temperatures rising to 25°C, H. ulvae could not tolerate dilution beyond 2q °/ /'O sea water (A = 0.48°C) whilst at temperatures from 25-30°C they could withstand 15% sea water (A = 0.29°C). T o d d (1964) reported, moreover, that survival of retracted animals at the low salinities of 25% sea water and fresh water was more prolonged at 5°C than 15°C, although adaptation to media of low salinity within the viable range was quicker at the higher temperature. H. ulvae from an Essex salt marsh were active in a medium of A = 0.53°C, but retracted in a medium of A = 0.26°C (Robson, 1920). Snails from Budleigh Salterton salt marsh behaved in a similar manner. In experiments with several dozen animals no ill-effects were seen on dilution to 40c}'~, sea water (A = 0.765°C). 'Salt shock' or retraction was seen at dilutions of 35, 30 and 25% sea waters (AA = 0.650, 0-585 and 0.485°C), but the animals resumed normal behaviour in a few days. In a medium of A = 0.390°C (20% sea water) this resumption of activity was followed in a few more days by death, whilst in 15C}/o sea water (A = 0.295°C) all the snails remained retracted until death. Tolerance of concentrated media has not attracted the same attention. In this study, salt shock was found in a medium of A = 3.680°C, but activity was resumed in a few days. A medium of A = 5.135°C must have been close to the upper tolerance limit, for only one snail out of ten became active, the others remaining retracted for over 8 days.
4. Effects of exposure and reimmersion On salt marshes particularly, but also in estuaries, H. ulvae must be able to tolerate the desiccating effects of exposure as well as great salinity changes. Lambert (1930) found animals living in a ditch connected to the T h a m e s estuary to be
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exposed for some 20 hr each day, and the time of exposure may often be even longer on a salt marsh. Behavioural adaptations of the animals may well reduce the actual exposure to which they are subjected. Thus Newell (1962) states that H. ulvae soon buried themselves once they were exposed by the falling tide on a mudflat. Moreover, animals at Budleigh Salterton tended to congregate amongst the plant roots where conditions were very moist. We need to know more of the microclimate of the species before the importance of an ability to tolerate exposure can be assessed. Blood concentrations increased rapidly on exposure in a dry container at room temperatures of about 20°C. Five animals taken from a medium of A = 1.925°C had a blood A of 1.985°C, and after exposure for 4-5 days the blood concentration had increased by about 90 per cent, for four animals of the same batch had an 3"0 --
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Fic. 4. Effect of exposure on the blood osmotic concentrations of animals kept initially in salt-marsh water of A = 1.37°C. The animals were placed in an open, dry jar at 16-20°C. average blood A of 3.50°C (range 3.395-3.710°C). In a similar experiment deaths tended to occur after the blood concentration had risen by 90-100 per cent (Fig. 4), although the salt-marsh water from which the animals were taken was hypoosmotic to normal sea water, and although the blood concentrations reached were less than those recorded in other experiments. It appears that the amount of water lost (i.e. the extent of dehydration), rather than the maximum blood concentration, is the important factor in survival. In this experiment, all the animals died between the sixth and the eighth days; it is probable that they would have survived longer had they not been tested daily for irritability, since the tactile stimulation involved
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sometimes caused the loss of fluid. O f the H. ulvae kept in a dry jar at 15.6-29.4°C b y Stopford (1951), 15 per cent survived a fortnight, with the first deaths occurring on the fourth day and the last after 17 days. W h e n exposed animals are reimmersed in sea-water, they quickly take advantage of the presence of free water. In the experiment shown in Fig. 5(a) snails from saltmarsh water (A = 1.08°C), were exposed for 5 days before being immersed in
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FIG. 5. Effects of exposure and reimmersion in sea water hyperosmotic t o the blood (a) on blood osmotic concentrations and (b) on total live weight of eight animals. In (a) animals kept in salt-marsh water of A = 1-08°C were exposed for 5 days before reimmersion in 100% sea water (A = 1"92°C). In (b) animals kept in salt-marsh water of A = 1"15°C were reimmersed after exposure in 100% sea water (A = 1"90°C). normal sea water (A = 1.92°C). On exposure the blood A increased to about 1.25°C, and on immersion in sea-water equilibrium between the blood and the m e d i u m was achieved within an hour. I n a similar experiment, weight lost during exposure was regained within about an hour (Fig. 5b) in spite of the fact that the blood concentration rose on immersion in sea-water.
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DISCUSSION H. ulvae resembles the marine and littoral species in being poikilosmotic, the blood concentration lying close to the concentration of the external medium in active animals. Moreover, like the littoral species Littorina saxatilis, H. ulvae can delay the attainment of equilibrium with unfavourable media by retraction into the shell, thereby enabling the animals to avoid excessive variations in internal concentration in short periods of extremely high or low salinity. The hyperosmotic condition of the urine seen in salt marsh and estuarine H. ulvae by Todd (1964) in 25% sea water and in fresh water, is held to result from the slow rate of equilibration in retracted animals. Here too there is evidence of a drift towards the A of the new external medium for the average urine A of summer estuarine animals in 25% sea-water was 1.19°C after 8 days, and 0.67°C after 9 days. There is a difference between H. ulvae and the marine and littoral species, however, in that no significant weight changes in H. ulvae occurred on transfer to new media, although they are present in the marine and littoral forms. H. ulvae is not a unique brackish water animal in this respect either. Freidrich (1937) found that Alderia modesta remained healthy and showed no significant weight changes on dilution of the medium from 17~ooto 5~oosalinity; although below 5~oo there was a marked weight increase, this was associated with a physiological deterioration of the animal. Freidrich concluded that A. modesta actively extruded salts when transferred to dilute media of higher salinity than 5~o. Results for H. ulvae similarly suggest that salts are lost in hypo-osmotic media and gained in hyperosmotic media, and that salt movements take place so quickly that neither swelling nor shrinkage can be seen on transfer to a new medium. Under similar conditions definite weight changes occur in Littorina saxatilis (Averts & Sleigh, 1965); there water movements are evidently more rapid than the movements of salts. It is also interesting that exposed and dehydrated H. ulvae gained weight when placed in sea-water hyperosmotic to the blood (Fig. 5b); this gain ill weight and the accompanying increase in blood concentration indicate the entry of both water and salts. In a similar experiment L. saxatilis lost weight on equilibration since it loses water more quickly than salts are gained. The Pulmonate slug, Onchidiurn chamaeleon, which lives in brackish mangrove swamps, was found to show weight changes similar to those of the marine and littoral species in spite of the fact that the animal had been proved permeable to urea and magnesium ions (Dakin & Edmonds, 1931); evidently the permeability to water is still higher than that to salts. The difference in relative permeabilities to salts and water in different snails may partly reflect a difference in size, and the relatively larger surface area to volume ratio of the smaller species. If the integument is the site of salt exchange, a salt balance could be reached more quickly in small species where diffusion paths are shorter. H. ulvae and A. modesta are small species, the latter with a particularly large surface area because of its cerata, whilst O. chamaeleon is a much larger species. Some support for this idea comes from Mayes (1962) who found that the weight changes in the small littoral snail, Littorina neritoides, were much less than those in the larger British Littorina sp. ;
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movements of salts were held to be responsible for the better volume control shown by this species. It is interesting that the other British estuarine snails Hydrobia ventrosa, Pseudamnicola confusa, Truncatella subcylindrica, Assiminea grayana and Phytia myosotis are all of small size. Perhaps the smaller species with better volume control are more fitted to invade brackish habitats than the larger species. Attempts to measure blood sodium concentrations in animals equilibrated at different salinities were not successful, and so there are no indications as to possible changes in blood protein concentrations during equilibration. It seems likely that salt movements rather than changes in protein concentration are the primary causes of changes in osmotic pressure. Certainly the cells must be able to tolerate great changes in blood concentration. H. ulvae was in fact the most euryhaline Gastropod encountered in this study; active animals in equilibrium with the medium being taken from media of A = 0.350°C-5.135°C, calculated as a salinity range of 6-94y/ooo(35%0 is taken as equivalent to a A of 1.907°C). Tolerance of exposure by H. ulvae is comparable with that found in Littorina saxatilis; both species will tolerate an increase in blood concentration of about 100 per cent as a result of desiccation, and both are capable of retraction for long periods. It is not known whether respiratory and/or excretory adaptations are associated with the ability to remain retracted for long periods. The enlargement of the kidney in H. ulvae could be associated with tolerance of exposure or tolerance of dilution. H. ulvae appears to be adapted to life in brackish conditions because of its extreme euryhalinity, implying tolerance of large changes in blood concentration at cellular level in this poikilosmotic species. The control of body volume that is thought to result from rapid salt movements is important, and so are the behavioural adaptations involved in retraction and burrowing in mud, which enable the animal to avoid extremes of salinity and desiccation. These adaptations are similar to those seen in the littoral Gastropods (described in Avens& Sleigh, 1965), but they are carried to a greater extreme in H. ulvae.
SUMMARY 1. Blood extracted from the visceral sinus in active animals was isosmotic with the medium at all tolerated salinities. 2. Equilibration in active animals following transfer from 100% to 60% sea water and vice versa took about an hour, but retraction delayed the process so that it took several days at least following transfer from 100%-20% sea water. 3. Effective volume control is present, since no significant weight changes were found on transfer from normal to dilute sea water and vice versa and from concentrated to normal sea water. 4. H. ulvae proved to be a very euryhaline species, active animals being found in equilibrium with media with AA from 0.350°C to 5.135°C, a salinity range calculated as about 6-94%o.
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5. H. ulvae can tolerate a 100 per cent increase in blood concentration through desiccation prior to death. On reimmersion in sea water (even if it was hyperosmotic to the blood), active animals reached osmotic equilibrium and regained lost weight within an hour. 6. T h e s e results are similar to those found in littoral species (Avens & Sleigh, 1965), except that there is complete volume control in H. ulvae. T h e relations between salt and water permeabilities, volume control, small size and euryhalinity are discussed. Behavioural adaptations, particularly the use of the shell as a shelter from adverse conditions, and extreme euryhalinity involving cellular tolerance of wide changes in blood concentration are stressed as important adaptations to life in brackish habitats. Acknowledgonents--I am very grateful to Dr. M. A. Sleigh, now at the Zoology Department of the University of Bristol, for all his sustained help and advice. This work was undertaken during the tenure of a Scholarship from the Department of Scientific and Industrial Research, and to them I should like to express my appreciation.
REFERENCES AWNS A. C. & SLEIGH M. A. (1965) Osmotic balance in Gastropod molluscs. I. Some marine and littoral Gastropods. Comp. Biochera. Physiol. |6, 103-123. DAmN W. & EDMONDSE. (1931) The regulation of the salt contents of the blood of aquatic animals and the problems of the permeabifiry of the bounding membranes in aquatic invertebrates..4ust.~. Exp. Biol. Med. Sd. 18, 169-187. ELLIS A. E. (1925) Experimental acclimatisafion of Sabanea ulvae Pennant to fresh water. Ann. Mag. Nat. Hist. 15, 496-497. Fm~mRICH F. (1937) Einige Beobachtungen uber das Verhalten der .4lderia modesta Lov. im brackwasser. Biol. Zbl. 57, 101-104. F m ~ R V. & GRAHAMA. (1962) BHtish Prosobranch Molluscs. Bartholomew, London. LAMBERT F. J. (1930) Animal life in the marsh ditches of the Thames estuary. Proc. Zool. Soc. Lond. ll, 801-808. MAYES P. A. (1962) Comparative investigations of the euryhaline character of LittoHna and the possible relationship to intertidal zonation. Nature, Lond. 195, 1269-1270. McMILLAN N. F. (1948) Possible biological races in Hydrobia ulvae Pennant and their varying resistance to lowered salimty..7. Conch. 23, 14--16. NEWELL R. (1962) Behavioural aspects of the ecology of Perinsia ( = Hydrobia) ulvae (Pennant), Gastropoda, Prosobranchia. Pro¢. Zool. Soc. Lond. 138, 49-75. NlCOL E. A. T. (1935) The ecology of a salt marsh..7. Mar. Biol. Ass. U.K. 20, 203-261. NICOL E. A. T. (1936) The brackish water lochs of North Uist. Pro¢. Roy. Soc. Edinb. 56, 169-195. RAMSAY J. A. & BROWN R. H. J. (1955) Simplified apparatus and procedure for freezing point determinations upon small volumes of fluid..7. Sci. Inst~um. 32, 372-375. RSMANE A. (1958) Okologie des brackwassers. D/e Biolosie des Bracktcassers (Edited by R~MANE A. & ScmJsIPER C.), Teil I, pp. 1-216. Stuttgart. ROBSON G. C. (1920) Observations on the succession of the Gastropods Paludestrina ulvae and P. vemrosa in brackish water. Ann. Mas. Nat. Hist. 9, 525-529. ROTHSCHILDA. & ROTHSCHILDM. (1939) Some observations on the growth of Perinsia ulvae (Pennant 1777) in the laboratory. No,it. Zool. 41, 240-247. STOPFOm~ S. C. (1951) An ecological study of the Cheshire foreshore of the Dee estuary. .7. Anita. Ecol. 20, 103-122. TODD M. E. (1964) Osmotic balance in Hydrobia ulvae and Potamopyrs~j~hi~i (Gastropoda: Hydrobiidae)..y. Exp. Biol. 41, 665-677.
OSMOTIC BALANCE IN GASTROPOD MOLLUSCS--II. THE BRACKISH WATER GASTROPOD, HYDROBDI ULV.4E PENNANT A N G E L A C. AVENS* Department of Zoology, University of Exeter
(Received 12 March 1965) Abatract--1. Blood concentrations of H. ulvae were determined in normal, dilute and concentrated sea-waters, and on exposure and reimmersion. 2. The species proved to be poikilosmotic and very euryhaline, and had a very effective means of volume control. 3. Equilibration to new media took about an hour in active animals, but retraction delayed the process at extreme salinities so that it took several days at least. 4. Increases in blood concentration of up to 100 per cent were permissible before death by desiccation on exposure. 5. These results are similar to those previously found for littoral species although there seems to be a greater tolerance of blood concentration changes in H. u/vae. 6. Small size, salt and water permeability and volume control are discussed in relation to euryhalinity. INTRODUCTION IN ^ previous paper (Avens & Sleigh, 1965) an account was given of an investigation of the osmotic balance between marine and littoral Gastropods and their environments under conditions of varying salinity and exposure. T h e small estuarine snail Hydrobia ulvae is similarly treated in this account, which is to be followed by a report of findings on freshwater and terrestrial snails in later papers. It was hoped that these studies would help in understanding the physiological adaptations involved in the dispersal of Gastropods away from the sea. A study of H. ulvae is interesting not only because it permits comparison with the species previously described, but also because it may indicate features intermediate between marine and freshwater species. H. ulvae lives on estuarine mudflats and on salt marshes, never out of direct reach of the sea. On mudflats it occurs at mid-tide level and upwards, and is consequently covered at almost every tide. Such a habitat is probably only brackish periodically; brackish water being considered to have a salinity between 0"5~oo and 30%o, which is equivalent to having a freezing point depression of between 0.05°C and 1.62°C. On salt marshes, living conditions are probably more extreme, although the animals tend to congregate in damp hollows. Only during the spring tides are such areas covered, thus allowing time for more extreme conditions to * Present address: West Buckland School, Near Bamstable, North Devon, England. 143
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develop. Precipitation and run-off from the land can dilute the available water, whilst evaporation, the inflow of sea water or the freezing of some water out as ice can raise the salinity. There is therefore much variation in living conditions, and indeed Nicol (1935) found that conditions on a salt marsh can vary more in 24 hr than in the sea over a period of 6 months. In a recent paper T o d d (1964) has reported observations similar to some of those described here on H. ulvae. By measuring urine concentrations, which were assumed to be close to the blood concentrations of the animals sampled, she found that H. ulvae was slightly hyperosmotic on dilution from 100 to 50% sea water, but that the urine was sometimcs markedly hyperosmotic in 25% sea water and always hyperosmotic in fresh water. In these latter cases the animals were withdrawn into the shell, but experiments on phenol red uptake indicated that there was some exchange of materials between H. ulvae and its surroundings when the animal was withdrawn. MATERIALS AND METHODS Animals were collccted from a salt marsh at Budleigh Salterton, South Devon, and were kept submerged until required either in Plymouth sea water or in natural water from the salt marsh. Salt-marsh animals were used since they tend to be larger than estuarine ones (Rothschild & Rothschild, 1939). The methods used were essentially those described more fully in the previous paper. Total osmotic concentrations of the blood and sea water samples were measured by the microcryoscopic method of Ramsay & Brown (1955), and results are given as the depression of freezing point, A°C. Temperatures could be r e a d to the nearest 0-005°C, and readings for the same sample are reproducible to + 0.005°C. The animals were stuck, operculum downwards, in slightly hardened plasticene on the stage of a binocular microscope. After cleaning the shell, a hole about 1 m m diam. was bored on the dorsal side of the penultimate whorl with a dentist's drill. Through this hole a blood sample was sucked from the visceral sinus into a fine silica glass pipette of the type used in the freezing-point apparatus. Almost all of this sample was the n blown out under paraffin oil for storage, a little being retained to reduce evaporation errors during sampling. The samples contained regularly distributed blood corpuscles; those with many inclusions or those of yellowish colour being rejected on the grounds of probable contamination. Extraction of blood samples took less than a minute, and A measurement was done within the next half-hour (and usually within 10 min of extraction) so that increases in A on storage were kept to a minimum. Blood AA were measured for animals in natural waters, in dilute and concentrated sea-waters and on exposure and reimmersion. Dilute sea-water solutions were made by adding distilled water to Plymouth sea water, and concentrated sea waters were mixed from boiled and fresh Plymouth sea waters. T h e per cent sea water figures given here are only approximate; A readings were always taken to find the exact concentration.
O S M O T I C BALANCE I N H Y D R O B 1 A U L V A E
145
The degree of euryhalinity of the snails was investigated by preparing a series of diluted and concentrated sea-waters and transferring animals directly from 100% sea water to each of the concentrated solutions, and to each of the diluted solutions in which no ill-effects were seen. When ill-effects appeared, mainly prolonged retraction, transfer of active animals was made step by step along the series. Two or 3 days were allowed for equilibration in each solution, except where the animals retracted ; then it was necessary to wait and see if the animals resumed activity or died. Lifting or tapping the operculum with a pin would normally elicit a response should the animal be alive, but in doubtful cases observations were either extended for a further period, or the snail was returned to a more favourable medium in which living animals would become active. In experiments to follow the change in weight of H. ulvae under different conditions, it was necessary to clean and dry the shell with a tissue before weighing. Here results are usually expressed as the per cent change in live weight, i.e. total weight including the shell. Only in a few cases was it possible to extract the animals from their shells and determine the per cent change in wet tissue weight, i.e. live weight minus the dry weight of the shell and operculum. The shell normally forms about 50 per cent of the live weight. RESULTS
1. Blood concentrations in media of different concentrations The blood was isosmotic with the salt-march water; the average blood A of nine animals being 1.809°C (1.775-1.865°C) in a medium of A 1.760°C. The slight hyperosmoticity shown here is not held to be significant since the animals and samples were very small, and evaporation errors would therefore be greater than in experiments with marine and littoral species. The smallest A recorded for natural salt marsh water was 1.04°C, which is equivalent to about 60% sea water. This isosmoticity of the blood and the external medium was maintained in active animals at all tolerated concentrations of the external medium (Fig. 1). In some cases, however, where the concentration change was so great as to cause retraction, the blood concentrations several days after transfer were intermediate between the concentrations of the initial and test media (Fig. 1). This is clearly demonstrated in an experiment on a group of animals transferred directly from 100-20% sea water (A 1.890°C-A 0.350°C); all of these animals retracted, and blood AA were measured each day over a period of 11 days (Fig. 2). A slow drift towards the A of 20% sea water was apparent, similar to the rather more rapid drift found in a corresponding experiment with Littorina saxatilis as mentioned in the previous paper. Since Todd (1964) found that salt-mal'sh H, ulvae whose urine was hyperosmotic to the medium can remain retracted in 25% sea water for 29 days, it is not surprising to find the drift does not go far towards the A level of 20% sea water in only 11 days. Nevertheless, all of the three animals that resumed activity on being brought into a warmer laboratory for blood extraction (21°C as opposed to 10-15°C) were isosmotic with the medium (Fig. 2). This drift must result from exchange between the animal and the test medium. Todd has in fact
ANGELA C. AVENS
14.6 6--
i0m0cine/I
5 ~
4
U
3-"o
m
x ,,,,"" _~x.d , 0
I
2
3
External medium,
4
5
A°C
FIG. 1. T h e osmotic concentration of the blood in active animals ( O ) and in animals still retracted several days after transfer to test media ( x ), in relation to the osmotic concentration of the external m e d i u m . 2"0 o o
o
o 0
1.5
8
o
g
0
0 o
o
o
o
o 0
o f~ .
I-0
o
0"5
0
I
0
I
2
I
4
I
I
6
Time after transfer,
8
I
I0
I
12
days
Osmotic concentration of the blood in retracted ( O ) and active animals ( x ) after transfer from 100% sea-water (A = 1.89°C) to 20% sea-water (A = 0.35°C). T h e solid line denotes the concentration of the external m e d i u m . FIG. 2.
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OSMOTIC BALANCE I N H Y D R O B 1 A U L V A E
shown that phenol red can pass into the tissues of retracted H. ulvae in fresh water within 48 hr. Such exchange may arise from an ill-fitting operculum or from the necessity to open the shell periodically for respiratory and/or excretory purposes, although such opening has not been observed, but it could not entirely result from exchange in the initial period of activity often seen on immersion in media that cause retraction. x
2-0
xx (e)
x t.5 o o rn External medium I-0
I
0
40
80
T i m e after transfer,
2.0
-
External medium x
x/
- ~.5
"o o o
1.0
0
min
X X
X--Xo
x
I
40
Time ofter
I
I
80
transfer,
12C rain
FIG. 3. Effects on the blood osmotic concentrations of active animals of transfer (a) from 60% sea water (A = 1.09°C) to 100% sea water (A -- 1.91°C), and (b) from 100% sea water (A = 1.95°C) to 60% sea water (A = 1.07°C).
When animals remain active after transfer, however, equilibrium is achieved very quickly; after a change f r o m 60 to 100% sea-water and vice versa, the blood reached an equilibrium level in a little over an hour (Fig. 3).
2. Weight changes on transference to different media T h e live weights of groups of snails (usually 10) were measured before transfer to a new m e d i u m and at intervals between 15 rain and 4 hr after transfer. Changes
in live weight did not exceed some 3 per cent, and were generally about 1 per cent whether the change was f r o m dilute to normal sea-water or vice versa, or f r o m concentrated to normal sea-water. Such small changes as did occur were not consistent, nor did they accord with the laws of osmosis. Control experiments
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ANGELAC. AVENS
indicated that errors of this size could arise from differences in the thoroughness of drying. Weight changes of the magnitude found for Littorina saxatilis (Avens & Sleigh, 1965) were not observed here.
3. Tolerance of dilute and concentrated media Field records of the salinities at which H. ulvae were found are available from many sources for dilute media. In a brackish water loch on North Uist, Nicol (1936) found H. ulvae at a salinity of 10%o (A = 0.53°C approx.), and the closely related H. ventrosa at 24%0 (A = 1-29°C approx.). Rather lower values have been reported for the Baltic by Remane (1958), who found H. ulvae at salinities of 6-8%0 (A = 0.32-0-43°C approx.), and Fretter & Graham (1962) quote the work of Jessen (1918) and Johansen (1918), both working at Randjersfjord, who found H. ulvae in water of 1%o and fresh water respectively. McMillan (1948) suggested that there may be different biological races in H. ulvae. Animals that she took from Burton Marsh in Cheshire behaved normally in 7-5% sea water, 5% sea water proving to be lethal, whereas snails from an estuary 9 miles away and from a brackish ditch in Co. Down were active in 25% sea water but retracted in 22% sea water. It is important to note that variation in the experimental temperature can affect results. Ellis (1925) found that at room temperatures rising to 25°C, H. ulvae could not tolerate dilution beyond 2q °/ /'O sea water (A = 0.48°C) whilst at temperatures from 25-30°C they could withstand 15% sea water (A = 0.29°C). T o d d (1964) reported, moreover, that survival of retracted animals at the low salinities of 25% sea water and fresh water was more prolonged at 5°C than 15°C, although adaptation to media of low salinity within the viable range was quicker at the higher temperature. H. ulvae from an Essex salt marsh were active in a medium of A = 0.53°C, but retracted in a medium of A = 0.26°C (Robson, 1920). Snails from Budleigh Salterton salt marsh behaved in a similar manner. In experiments with several dozen animals no ill-effects were seen on dilution to 40c}'~, sea water (A = 0.765°C). 'Salt shock' or retraction was seen at dilutions of 35, 30 and 25% sea waters (AA = 0.650, 0-585 and 0.485°C), but the animals resumed normal behaviour in a few days. In a medium of A = 0.390°C (20% sea water) this resumption of activity was followed in a few more days by death, whilst in 15C}/o sea water (A = 0.295°C) all the snails remained retracted until death. Tolerance of concentrated media has not attracted the same attention. In this study, salt shock was found in a medium of A = 3.680°C, but activity was resumed in a few days. A medium of A = 5.135°C must have been close to the upper tolerance limit, for only one snail out of ten became active, the others remaining retracted for over 8 days.
4. Effects of exposure and reimmersion On salt marshes particularly, but also in estuaries, H. ulvae must be able to tolerate the desiccating effects of exposure as well as great salinity changes. Lambert (1930) found animals living in a ditch connected to the T h a m e s estuary to be
149
O S M O T I C BALANCE I N H Y D R O B I A U L V A E
exposed for some 20 hr each day, and the time of exposure may often be even longer on a salt marsh. Behavioural adaptations of the animals may well reduce the actual exposure to which they are subjected. Thus Newell (1962) states that H. ulvae soon buried themselves once they were exposed by the falling tide on a mudflat. Moreover, animals at Budleigh Salterton tended to congregate amongst the plant roots where conditions were very moist. We need to know more of the microclimate of the species before the importance of an ability to tolerate exposure can be assessed. Blood concentrations increased rapidly on exposure in a dry container at room temperatures of about 20°C. Five animals taken from a medium of A = 1.925°C had a blood A of 1.985°C, and after exposure for 4-5 days the blood concentration had increased by about 90 per cent, for four animals of the same batch had an 3"0 --
X
2"5
x
u <] ° 2-0 eA
I-5
I
--X--
E x t e r n o l medium
Dead animals
found here
1"0
I
0
I
2
Exposure time,
I
4
6
8
cloys
Fic. 4. Effect of exposure on the blood osmotic concentrations of animals kept initially in salt-marsh water of A = 1.37°C. The animals were placed in an open, dry jar at 16-20°C. average blood A of 3.50°C (range 3.395-3.710°C). In a similar experiment deaths tended to occur after the blood concentration had risen by 90-100 per cent (Fig. 4), although the salt-marsh water from which the animals were taken was hypoosmotic to normal sea water, and although the blood concentrations reached were less than those recorded in other experiments. It appears that the amount of water lost (i.e. the extent of dehydration), rather than the maximum blood concentration, is the important factor in survival. In this experiment, all the animals died between the sixth and the eighth days; it is probable that they would have survived longer had they not been tested daily for irritability, since the tactile stimulation involved
150
ANCELA C. AVENS
sometimes caused the loss of fluid. O f the H. ulvae kept in a dry jar at 15.6-29.4°C b y Stopford (1951), 15 per cent survived a fortnight, with the first deaths occurring on the fourth day and the last after 17 days. W h e n exposed animals are reimmersed in sea-water, they quickly take advantage of the presence of free water. In the experiment shown in Fig. 5(a) snails from saltmarsh water (A = 1.08°C), were exposed for 5 days before being immersed in
2"0
1"8
I
External medium x
x . ~
X X X
X tJ
x o
"~
1"4--
m
(a)
X
x
o
w
~-o 240-
-
x~X
I
0
"\.\
I
I
20
40
60
Time after reimmersion,
rain
• (b) Put into I 0 0 % •%• seawater /
~o, "4 220
25 200
I
0
I
I
I
20 40 60 80 Exposure time,
I00 hr
I
120 140
FIG. 5. Effects of exposure and reimmersion in sea water hyperosmotic t o the blood (a) on blood osmotic concentrations and (b) on total live weight of eight animals. In (a) animals kept in salt-marsh water of A = 1-08°C were exposed for 5 days before reimmersion in 100% sea water (A = 1"92°C). In (b) animals kept in salt-marsh water of A = 1"15°C were reimmersed after exposure in 100% sea water (A = 1"90°C). normal sea water (A = 1.92°C). On exposure the blood A increased to about 1.25°C, and on immersion in sea-water equilibrium between the blood and the m e d i u m was achieved within an hour. I n a similar experiment, weight lost during exposure was regained within about an hour (Fig. 5b) in spite of the fact that the blood concentration rose on immersion in sea-water.
OSMOTIC BALANCE IN HYDROBIA ULVAE
151
DISCUSSION H. ulvae resembles the marine and littoral species in being poikilosmotic, the blood concentration lying close to the concentration of the external medium in active animals. Moreover, like the littoral species Littorina saxatilis, H. ulvae can delay the attainment of equilibrium with unfavourable media by retraction into the shell, thereby enabling the animals to avoid excessive variations in internal concentration in short periods of extremely high or low salinity. The hyperosmotic condition of the urine seen in salt marsh and estuarine H. ulvae by Todd (1964) in 25% sea water and in fresh water, is held to result from the slow rate of equilibration in retracted animals. Here too there is evidence of a drift towards the A of the new external medium for the average urine A of summer estuarine animals in 25% sea-water was 1.19°C after 8 days, and 0.67°C after 9 days. There is a difference between H. ulvae and the marine and littoral species, however, in that no significant weight changes in H. ulvae occurred on transfer to new media, although they are present in the marine and littoral forms. H. ulvae is not a unique brackish water animal in this respect either. Freidrich (1937) found that Alderia modesta remained healthy and showed no significant weight changes on dilution of the medium from 17~ooto 5~oosalinity; although below 5~oo there was a marked weight increase, this was associated with a physiological deterioration of the animal. Freidrich concluded that A. modesta actively extruded salts when transferred to dilute media of higher salinity than 5~o. Results for H. ulvae similarly suggest that salts are lost in hypo-osmotic media and gained in hyperosmotic media, and that salt movements take place so quickly that neither swelling nor shrinkage can be seen on transfer to a new medium. Under similar conditions definite weight changes occur in Littorina saxatilis (Averts & Sleigh, 1965); there water movements are evidently more rapid than the movements of salts. It is also interesting that exposed and dehydrated H. ulvae gained weight when placed in sea-water hyperosmotic to the blood (Fig. 5b); this gain ill weight and the accompanying increase in blood concentration indicate the entry of both water and salts. In a similar experiment L. saxatilis lost weight on equilibration since it loses water more quickly than salts are gained. The Pulmonate slug, Onchidiurn chamaeleon, which lives in brackish mangrove swamps, was found to show weight changes similar to those of the marine and littoral species in spite of the fact that the animal had been proved permeable to urea and magnesium ions (Dakin & Edmonds, 1931); evidently the permeability to water is still higher than that to salts. The difference in relative permeabilities to salts and water in different snails may partly reflect a difference in size, and the relatively larger surface area to volume ratio of the smaller species. If the integument is the site of salt exchange, a salt balance could be reached more quickly in small species where diffusion paths are shorter. H. ulvae and A. modesta are small species, the latter with a particularly large surface area because of its cerata, whilst O. chamaeleon is a much larger species. Some support for this idea comes from Mayes (1962) who found that the weight changes in the small littoral snail, Littorina neritoides, were much less than those in the larger British Littorina sp. ;
152
ANGELAC. AVENS
movements of salts were held to be responsible for the better volume control shown by this species. It is interesting that the other British estuarine snails Hydrobia ventrosa, Pseudamnicola confusa, Truncatella subcylindrica, Assiminea grayana and Phytia myosotis are all of small size. Perhaps the smaller species with better volume control are more fitted to invade brackish habitats than the larger species. Attempts to measure blood sodium concentrations in animals equilibrated at different salinities were not successful, and so there are no indications as to possible changes in blood protein concentrations during equilibration. It seems likely that salt movements rather than changes in protein concentration are the primary causes of changes in osmotic pressure. Certainly the cells must be able to tolerate great changes in blood concentration. H. ulvae was in fact the most euryhaline Gastropod encountered in this study; active animals in equilibrium with the medium being taken from media of A = 0.350°C-5.135°C, calculated as a salinity range of 6-94y/ooo(35%0 is taken as equivalent to a A of 1.907°C). Tolerance of exposure by H. ulvae is comparable with that found in Littorina saxatilis; both species will tolerate an increase in blood concentration of about 100 per cent as a result of desiccation, and both are capable of retraction for long periods. It is not known whether respiratory and/or excretory adaptations are associated with the ability to remain retracted for long periods. The enlargement of the kidney in H. ulvae could be associated with tolerance of exposure or tolerance of dilution. H. ulvae appears to be adapted to life in brackish conditions because of its extreme euryhalinity, implying tolerance of large changes in blood concentration at cellular level in this poikilosmotic species. The control of body volume that is thought to result from rapid salt movements is important, and so are the behavioural adaptations involved in retraction and burrowing in mud, which enable the animal to avoid extremes of salinity and desiccation. These adaptations are similar to those seen in the littoral Gastropods (described in Avens& Sleigh, 1965), but they are carried to a greater extreme in H. ulvae.
SUMMARY 1. Blood extracted from the visceral sinus in active animals was isosmotic with the medium at all tolerated salinities. 2. Equilibration in active animals following transfer from 100% to 60% sea water and vice versa took about an hour, but retraction delayed the process so that it took several days at least following transfer from 100%-20% sea water. 3. Effective volume control is present, since no significant weight changes were found on transfer from normal to dilute sea water and vice versa and from concentrated to normal sea water. 4. H. ulvae proved to be a very euryhaline species, active animals being found in equilibrium with media with AA from 0.350°C to 5.135°C, a salinity range calculated as about 6-94%o.
OSMOTIC BALANCE I N H Y D R O B I A U L V A E
153
5. H. ulvae can tolerate a 100 per cent increase in blood concentration through desiccation prior to death. On reimmersion in sea water (even if it was hyperosmotic to the blood), active animals reached osmotic equilibrium and regained lost weight within an hour. 6. T h e s e results are similar to those found in littoral species (Avens & Sleigh, 1965), except that there is complete volume control in H. ulvae. T h e relations between salt and water permeabilities, volume control, small size and euryhalinity are discussed. Behavioural adaptations, particularly the use of the shell as a shelter from adverse conditions, and extreme euryhalinity involving cellular tolerance of wide changes in blood concentration are stressed as important adaptations to life in brackish habitats. Acknowledgonents--I am very grateful to Dr. M. A. Sleigh, now at the Zoology Department of the University of Bristol, for all his sustained help and advice. This work was undertaken during the tenure of a Scholarship from the Department of Scientific and Industrial Research, and to them I should like to express my appreciation.
REFERENCES AWNS A. C. & SLEIGH M. A. (1965) Osmotic balance in Gastropod molluscs. I. Some marine and littoral Gastropods. Comp. Biochera. Physiol. |6, 103-123. DAmN W. & EDMONDSE. (1931) The regulation of the salt contents of the blood of aquatic animals and the problems of the permeabifiry of the bounding membranes in aquatic invertebrates..4ust.~. Exp. Biol. Med. Sd. 18, 169-187. ELLIS A. E. (1925) Experimental acclimatisafion of Sabanea ulvae Pennant to fresh water. Ann. Mag. Nat. Hist. 15, 496-497. Fm~mRICH F. (1937) Einige Beobachtungen uber das Verhalten der .4lderia modesta Lov. im brackwasser. Biol. Zbl. 57, 101-104. F m ~ R V. & GRAHAMA. (1962) BHtish Prosobranch Molluscs. Bartholomew, London. LAMBERT F. J. (1930) Animal life in the marsh ditches of the Thames estuary. Proc. Zool. Soc. Lond. ll, 801-808. MAYES P. A. (1962) Comparative investigations of the euryhaline character of LittoHna and the possible relationship to intertidal zonation. Nature, Lond. 195, 1269-1270. McMILLAN N. F. (1948) Possible biological races in Hydrobia ulvae Pennant and their varying resistance to lowered salimty..7. Conch. 23, 14--16. NEWELL R. (1962) Behavioural aspects of the ecology of Perinsia ( = Hydrobia) ulvae (Pennant), Gastropoda, Prosobranchia. Pro¢. Zool. Soc. Lond. 138, 49-75. NlCOL E. A. T. (1935) The ecology of a salt marsh..7. Mar. Biol. Ass. U.K. 20, 203-261. NICOL E. A. T. (1936) The brackish water lochs of North Uist. Pro¢. Roy. Soc. Edinb. 56, 169-195. RAMSAY J. A. & BROWN R. H. J. (1955) Simplified apparatus and procedure for freezing point determinations upon small volumes of fluid..7. Sci. Inst~um. 32, 372-375. RSMANE A. (1958) Okologie des brackwassers. D/e Biolosie des Bracktcassers (Edited by R~MANE A. & ScmJsIPER C.), Teil I, pp. 1-216. Stuttgart. ROBSON G. C. (1920) Observations on the succession of the Gastropods Paludestrina ulvae and P. vemrosa in brackish water. Ann. Mas. Nat. Hist. 9, 525-529. ROTHSCHILDA. & ROTHSCHILDM. (1939) Some observations on the growth of Perinsia ulvae (Pennant 1777) in the laboratory. No,it. Zool. 41, 240-247. STOPFOm~ S. C. (1951) An ecological study of the Cheshire foreshore of the Dee estuary. .7. Anita. Ecol. 20, 103-122. TODD M. E. (1964) Osmotic balance in Hydrobia ulvae and Potamopyrs~j~hi~i (Gastropoda: Hydrobiidae)..y. Exp. Biol. 41, 665-677.