Water Exchange in the Mantle of A Terrestrial Snail During Periods of Reduced Evaporative Loss

Active terrestrial snails normally maintain a thick water-rich layer of mucus on their external surfaces. The rate of evaporation from this layer in natural conditions is frequently high and practically indistinguishable from that of distilled water (Machin, 1964). Once the snail becomes inactive and withdraws into the shell, however, water loss from the remaining exposed surfaces, the mantle collar, usually begins to decrease rapidly (Machin, 1965). After several hours evaporation may be reduced to only 2 % of the rate characteristic of freshly produced mucus.

In an earlier study (Machin, 1966), this phenomenon was observed in the snail Helix aspersa over a restricted ambient humidity range. Low rates of loss or decreased evaporative cooling in other terrestrial helicids, H. pomatia (Hogben & Kirk, 1944), Otala lactea, Sphincterochila boissieri (Machin, 1967) and Helicella virgata (Pomeroy, 1968) suggest the capacity to reduce evaporation within the mantle is more widespread. In the present study measurements on Otala lactea go beyond those obtained on Helix aspersa, and represent the next step in an attempt to understand the mechanism by which evaporation is regulated.

Cultures of Otala lactea, imported from Malta and obtained from local food markets, were kept in dark temperature-controlled rooms at 15–16 °C, 50–70% R.H. Food and water were provided infrequently, so that animals were inactive most of the time. Small batches of animals were transferred to laboratory conditions (19–23 °C) and allowed to become acclimated for at least 2 weeks. During that period occasional animals which consistently became active during the night were discarded.

The techniques and apparatus, with the exception of the chamber lid, were essentially the same as those described earlier (Machin, 1969). For experimentation single snails were fixed in Plasticene, aperture uppermost, to a platform attached to the chamber lid. The position of the snail once the chamber was closed was such that the air exit tube was directed towards the centre of the shell aperture, level with its rim (Fig. 1). One junction of a pre-calibrated copper-constantan thermocouple was made to rest lightly on the mantle surface, while the other remained free a few cm away. The water bath round the chamber was maintained at 25 0 ±0·1 ·C and air was passed through the apparatus at a rate of 270 ml/min. Snails frequently became active immediately after transfer to the apparatus or during the following night. Others secreted an epiphragm over the aperture which usually resulted in removing the thermocouple from the mantle surface. Fortunately some snails remained unaffected by the handling. After the first few days activation rarely occurred. It is remarkable that, once acclimated to the experimental chamber, snails remain inactive throughout experiments lasting several weeks, even though they are exposed to humidities ranging from 1·5% R.H. to saturation.

As in previous studies it is necessary to express the surface temperature depression in terms of evaporation rate. Calibration runs were performed using empty shells of Otala which were packed with cotton wool soaked in distilled water. Evaporative loss was calculated from the difference in the amount of water in the air streams entering and leaving the experimental chamber. Absolute humidities were determined by weighing the water absorbed by silica gel and by calculating it directly from the dew point of the air stream.

Evaporation from distilled water was found to be proportional to depression of surface temperature and to the vapour pressure gradient between the water and the incoming air. Mean rates of evaporation obtained were 17·1 mg/cm²/h/°C temperature depression or 3–16 mg/cm²/h/mmHg.

By contrast steady-state losses, which were maintained apparently indefinitely by the snail in constant humidity, were very low. Temperature depressions of the mantle surface did not exceed 0·07 °C (2·5 μV) even at the lowest humidities. These temperature differences are of the same magnitude as the fluctuations observed in the surrounding water bath, and perhaps in the air entering the chamber. The correct expression of surface temperature depression in terms of evaporative loss is therefore open to some error. Accordingly the practice, assuming that the electrical zero and effective temperature zero of the thermocouple were identical, was abandoned and the point of zero evaporation determined instead by extrapolation of a leastsquares regression to zero vapour gradient, as in Fig. 2. This method also reduces possible errors due to temperature differences arising from metabolic heat production by the animal itself.

The practice of introducing corrections for vapour pressure gradients in the air (Machin, 1969) into calculations of mantle permeability was found unnecessary. Using data obtained from a snail exposed to the full range of experimental humidites, calculated discrepancies in vapour pressure between ambient air and that on the surface varied between 0·05 and 0·12 mmHg. Since these are negligible in relation to the total gradients existing between blood and air the permeability of the mantle tissues can be obtained directly from the slope of the line in Fig. 2. A value of 0·016 mg/cm²/h/ mmHg was obtained.

Records of surface temperature show that once steady state is reached, the difference between ambient and surface vapour pressures is always very slight. If comparatively large changes in ambient vapour pressure, of the order of 2 or 3 mmHg were made, corresponding changes at the surface would be expected. From a consideration of the vapour pressure gradients existing during a change, Machin (1969) has concluded that a net gain or loss of water should take place depending on the direction of the humidity change. It can be seen in Fig. 3 that there are marked changes in surface temperature following a humidity change. The peaks obtained are much more prominent than those obtained from toad skin. Since evaporative loss following a humidity decrease is overwhelmingly greater than loss under steady-state conditions, there must be a net loss of water from the surface to the air. Conversely, increases in ambient humidity produce a temperature rise indicating condensation and a net gain of water from the atmosphere (Fig. 3). Identical traces were obtained from mucus which had been removed from the mantle and spread on a glass coverslip.

Approximate estimates of the relative permeabilities of the whole mantle and of the superficial layers subject to net water exchange can be made by comparing peak and steady-state surface temperatures. They show that water leaves these layers 21 times more rapidly and enters 13 times more rapidly than it passes through the mantle as a whole.

Continuous records of the temperature of the mantle surface frequently include smaller thermal events (Fig. 6) resembling cooling peaks previously described except that they are preceded by a brief temperature increase. The cooling phase of the event apparently disappears in saturated air.

The most attractive explanation of these events is that they represent the extrusion of small amounts of hydrated mucus by an otherwise inactive mantle epithelium. Initial heating is perhaps caused by local muscular contraction which is necessary for mucus extrustion (Campion, 1961; Machin, 1964). Cooling then follows as water rapidly evaporates from the mucus until the vapour pressure of the general surface is again reached.

Surface-temperature records were analysed to test this hypothesis by comparing the heights of corresponding heating and cooling peaks (Fig. 7). At a given ambient humidity cooling is roughly proportional to heating. There is considerable variation in the size of the peaks. This might be due to variation in the magnitude in the event itself and also in its distance from the recording thermocouple. The data presented in Fig. 7 are consistent with the mucus-extrusion hypothesis, since for a heating peak of a given size the amount of cooling varies with ambient humidity. Comparisons of mean height of heating peak further suggests that the events tend to increase in size as the humidity becomes less. Measurements of the amount of mucus secreted, using the areas of some of the larger peaks, suggest that they represent the total secretion of up to five giant mucus galnds (Campion, 1961) containing about 12 μg of mucus each.

The frequencies of these events were also calculated from the time the animals were placed in the experimental chamber. Data plotted in Fig. 8 from several animals shows that frequency is high at the beginning of the experiment and decreases some what with time.

Imposed on this is a diurnal rhythm; frequency in the darkened laboratory is, roughly twice that during working hours with the lights on. Frequency is unrelated to ambient humidity.

Results presented here confirm and extend previous observations (Machin, 1965, 1966) that Otala lactea is able to regulate evaporative water loss from the mantle. This remarkable ability extends over a very wide range of ambient humidities, from 1·5 % to near saturation. Experiments show that low rates of loss can be maintained at least for several weeks. Calculated mantle permeability for Otala is about half the previously reported value for Helix aspersa (Machin, 1966). In view of the problems inherent in measuring small temperature differences reliably, quoted permeability values must still be regarded with some reservation until more accurate gravimetric techniques are used. Very low rates of evaporation over a very wide range of ambient humidity suggests the presence of an impermeable barrier. However, it is somewhat embarrassing that an as yet unidentifiable barrier has a permeability approaching that of insect cuticle (Beament, 1959). A previous suggestion (Machin, 1966) about the presence of a cuticle is unfounded since subsequent electron microscopy by the author has shown it to be a brush border made up of tightly packed microvilli of the type described by Lane (1963).

The thermocouple technique has proved to be a remarkably informative experimental tool. Measurements have convincingly demonstrated that the impermeable barrier is located beneath a hygroscopic compartment which exchanges water readily and rapidly with the atmosphere. The principle evidence for this is that gain by or loss from the surface layers, under the same vapour pressure gradients, takes place much more rapidly than diffusion through the total thickness of the mantle tissue. The fact that net water exchange, gain or loss, is virtually the same for a given humidity change and that similar exchanges occur in mucus spread on a glass plate further indicates that in non-steady-state conditions, contributions of water from within are negligible. These observations finally discount any possibility that snail mucus has any significant water-retarding properties of its own.

Calculated solvent volumes of the superficial hygroscopic layer show that the compartment volume changes predictably except at low humidities. Since apparent increases in solvent volume persisted in isolated mucus, this seems to be a property of the mucus itself. Inaccuracies in the method-such as the discrepancy between ambient and mucus vapour pressures, were found to be too small to account for the anomaly. Explanations based on a more tightly bound water compartment which is only released by low ambient humidities is not sufficiently convincing to be proposed seriously without further study. Although the determination of the volume of the superficial compartmental may eventually help to identify the impermeable layer beneath it, it must be emphasized that present estimates relate only to the solvent phase. Since mucus contains macromolecules, the total volume of the compartment, solvent and solute, may be considerably larger.

The presence of intermittent thermal events which seem to represent the activity of single cutaneous mucus glands or small groups of these slightly modifies previous concepts of the state of the mantle during reduced evaporation. The appearance of glandular extrusion as a series of discrete events rather than continuous high-level evaporation which is found when a large number of glands are active, confirm that during regulation mucus production is indeed drastically reduced but not necessarily stopped completely. Under these conditions, the resulting dehydration of superficial layers by humidities as low as 1·5% R.H. raise some interesting physiological questions regarding the state of the mantle epithelium. Machin (1964) has argued that the active maintenance of a superficial layer of water-rich mucus seemed to be an essential precaution against skin dehydration. Now it appears that the mantle epithelium which differs very little from that on other body surfaces of the snail in its histology (Campion, 1961) apparently resists or tolerates desiccation by some other means. It was thought at first that intermittent low-level glandular extrusion represented a contingency insurance against excessive dehydration. Although in general more mucus may be extruded by each event in lower humidities, no correlation between extrusion frequency and ambient humidity was found. It is well known that snails are more commonly active at night. Since extrusion frequency was highest during darkness and after mechanical disturbance during transfer to the experimental chamber, gland activity seems to be related to the sensory status of the snail.

This study was supported by grants from the National Research Council of Canada and the Ontario Tuberculosis Association.