The Evaporation of Water from Helix Aspersa: IV. Loss from the Mantle of the Inactive Snail

When a snail withdraws into its shell the only parts of the body left exposed to the air are a number of fleshy extensions of the mantle ; the so-called mantle collar. This area of the body is a potentially serious source of water loss in the inactive terrestrial snail.

The evaporation of water from active specimens of the common garden snail, Helix aspersa, is nearly identical to that from a free water surface (Machin, 1964a). However, preliminary findings (Machin, 1965) indicate that the rate of evaporation from the exposed surface of the mantle is reduced following withdrawal of the snail to the inactive state. That this species of snail is able to ‘regulate’ evaporation is of considerable physiological and ecological importance. The present work represents a critical examination of the physiological mechanisms which operate to modify water evaporation from the exposed integument of inactive snails.

The snails used in the experiments were Helix aspersa imported from biological supply houses in England.

The epiphragm was removed from each snail before measurement. Snails were placed on a Metier single-pan analytical balance reading to 0·1 mg. Air was continuously drawn through the weighing chamber of the balance and passed through a Cambridge Systems dewpoint hygrometer. Complete air replacement occurred every 10 min. Water loss by evaporation could thus be measured gravimetrically. Corrections were made for the slight but constant beam deflexion, equivalent to between 2 and 3 mg., caused by air movement through the weighing chamber. Temperature and humidity measurements were recorded at 15 min. intervals.

Control measurements were made using glass vials filled with saturated salt solutions. Vial diameter and the depth to which they were filled matched the evaporating conditions found in the withdrawn snail as closely as possible.

Experiments were performed to test whether or not the lowering of surface vapour pressure during inactivity was permanent or reversible. Measurements of evaporative loss were made following a short period in which the snail was suspended in humid air over distilled water.

Mucus was collected after slight mechanical stimulation of the mantle, and blood was collected from the body cavity by way of the lung floor (Machin, 1962). Freezing-point depression data were obtained from 50 mg. samples of blood and mucus with a Fiske osmometer. These measurements were then used to calculate vapour-pressure values from data according to Frazer, Taylor&Crollman (1926). The vapour pressure of the saturated-solution controls was determined by a more direct method. Gas-washing bottles containing the solutions were placed in a water bath and allowed to equilibrate at 22°±0·01° C. The vapour pressure was then obtained from dewpoint measurements of the air bubbled through each solution.

Movement of water through the integument of the snail is probably due to the osmotic or activity gradient across it. The present measurements, made on the basis of varying activity gradients, should not be confused with those obtained using heavy water, where the osmolarity can be identical on both sides of the membrane. In the present investitation two methods were employed to determine integumental permea-bility.

(a) Measurements with intact snails. Extended snails were weighed both before and after a short period of immersion in saline solutions (Cardot, 1921) of different osmotic pressures. Animals were then sacrificed and the freezing-point depressions were determined for the blood and external medium. This method proved to be relatively inaccurate, because of the difficulty of measuring total skin area and the possibility that snails were drinking water (see Machin, 1962).

(b) Measurements with isolated sections of dorsal body wall. Mantle tissue proved to be unsatisfactory for measurements of integumental permeability. Alternatively, sections of integument from the body wall were excised, and mounted as a diaphragm between two layers of shellac-coated wire gauze, held in circular clamps (Machin, 1964 a). Saline solutions of differing osmotic pressure were then placed in 20 ml. Plexiglass (Perspex) chambers on either side of the membrane. Mixing of both solutions was effected with teflon-coated magnetic stirring bars (Fig. 1). Rates of water diflfusion across the membrane were then measured volumetrically. Generally, good readings were obtained 2-3 hr. after the addition of the solutions. There was no measurable interference due to aeration of the solutions.

Results of measurements made with intact snails in different physiological states under the same atmospheric conditions are summarized in Table 1. It is clear that in controlling evaporation from the mantle surface some change is involved in the properties of mucus, freshly produced by stimulation. The results also show that this change disappears with the death of the snail.

Perhaps the most convincing evidence that the snail is able to regulate evaporation was provided by weight records of snails which had died naturally. It was found that it takes just 4 days to dehydrate the body completely by uncontrolled evaporative loss. By contrast the same individuals before death had survived several months in the inactive condition. In fact it is well known that terrestrial snails are able to survive many months, even years (Mead, 1961), without food and water. Ward (1897) has reported that specimens of Helix aspersa were still alive after 13 months inactivity. It is surprising that this commonly observed fact has not aroused interest earlier, since relatively minor changes in evaporating surface area after death could not in themselves account for such wide differences in evaporation. However, failure to recognize the significance of the above may have been due to an earlier assumption that the epi-phragm was more important in reducing water loss than recent measurements (Machin, 1966) now show.

The vapour pressure values given in Table 2 together with the evaporation data in Table 1 can be inserted into the formula to calculate the remaining unknown p_o2. A value of 7·29 mm. Hg for the vapour pressure of the superficial mucus during the period of reduced evaporative loss was obtained.

There are a number of pratical reasons why this calculation may be subject to error:

1. (a) surface temperature measurements were not feasible, hence the ‘real’ value of p₀ at the temperature of the evaporating surface, instead of the air, could not be used ;

2. (b) assumptions permitting the simplification of equation (1) to (2) may not be valid in this case, E may vary independently with (p_o—p_d).

To serve as a control, the evaporation data of various standard saturated salt solutions were used to calculate p_o2 by the same method as used on snails. Observed and calculated vapour pressures are given in Table 3. It can be seen that they agree fairly well. Therefore, calculations of surface vapour pressures from evaporation results may be considered valid.

One of the fundamental physiological differences between active extended and inactive regulating snails lies in the functioning of the mucus glands. Observations (see Machin, 1965) suggest that periods of low evaporative loss coincide with a cessation or reduction of the muscular activity of the mantle which normally brings about mucous extrusion. Several unsuccessful attempts to measure the thickness of the mucous layer were made. The appearance of the pattern of reflected highlights (Machin, 1964a), however, does indicate that this layer is much thinner during regulation. It is suggested that decrease in volume is due to continued evaporation in the absence of further replacement of mucus. The resulting increase in solute con-centration could be the cause of low surface vapour pressure during regulation.

This working hypothesis was tested by measuring the change in evaporation rate resulting from known amounts of water loss in isolated mucus samples. The results plotted in Fig. 2 show that the lowering of vapour pressure by solute concentration in the mucus follows the same course as a similar sized drop of 1 % NaCl. This is to be expected since Machin (1962) and Burton (1965) have shown that mucus is rich in electrolytes. Discrepancies between observed and theoretical curves are almost certainly due to unavoidable decreases in the surface area of the experimental samples as they dried up. Repeated measurements of freezing-point depression made over a period of 24 hr. using the same sample kept at room temperature failed to show any chemical decomposition. Increase in the number of solute molecules could also have reduced the vapour pressure.

In Fig. 3 it can be seen that the change in the evaporation rate of an artificially stimulated snail could be reasonably explained by the progressive dehydration of an initially 100 μ thick layer of mucus on the mantle. It would be expected that de-crease in evaporation rate of intact snails and isolated samples would be similar at first and then diverge (shaded area). As dehydration progressed the vapour pressure gradient between blood and mucus in the intact snail would initiate outward diffusion and slow down the decrease in evaporation rate.

In the absence of glandular extrusion the vapour pressure of the mucus ultimately depends on the delicate balance between diffusion and evaporation. It is possible to test whether this dynamic equilibrium exists by measuring evaporation immediately following exposure to humid air. High external vapour pressure should reduce the evaporation component of the system almost to nil and permit the superficial mucus to rehydrate. In Fig. 4 initially high rates of evaporation, once the normal humidity is restored, followed by the eventual establishment of the original low rate of evapora-tion, indicate the presence of a dynamic equilibrium between blood, mucus and air.

Evidence has been presented which shows that the mantle of the inactive snail is in some way capable of reducing evaporative loss. A central part of the study of the mechanism of reduction must be the measurement of integument permeability. If there is a massive reduction in the vapour pressure of the superficial mucus, the mucus will tend to rehydrate by drawing water from the blood. Low rates of evaporation maintained for long periods of time indicate that the rate of diffusion of water from blood to mucus is slow. Measurements of the permeability of the mantle therefore may be expected to yield low values.

In the virtual absence of the mucus extrusion it is possible to calculate mantle permeability, knowing both the vapour pressure of the blood and of the superficial mucus. Since the vapour pressure of the mucus remains steady during regulation, the amount of water leaving the mucus by evaporation must equal the amount arriving by diffusion from the blood. If the snail loses water by evaporation at a rate of 0·48 mg./cm.²/hr. (Table 1). the permeability of the mantle is 0·039 mg./cm.²/hr. per mm. Hg vapour pressure difference. The evaporation data in Table 1 also permits the mantle permeability of snails which had died from natural causes to be calculated. A perme-ability of 6·2 mg./cm.²/hr. /mm.Hg. was obtained. Mantle permeability of snails which had been killed by immersion in liquid air was also calculated using data given in Fig. 5. Liquid air was found to be the most satisfactory means of killing the snail since it served to keep the animal withdrawn in the shell and caused minimum damage to the mantle. It can be seen that once the dead snail was brought back to room tempera-ture evaporation continued at a rate which was similar to that from freshly extruded mucus in stimulated inactive snails. However, the rate of evaporation decreased, probably as the superficial mucus extruded before death dried up, until a linear fall in evaporation rate was attained. The mantle tissue itself became progressively dessiccated after this. Subsequent decrease in evaporation, not observed in the living snail, was almost certainly due to excessive depletion of the water reserves of the blood and increasing resistance to outward diffusion (Machin, 1964a; Beament, 1961). Assuming equilibrium is established, when the excess mucus has been dried (point marked +), the permeability of the mantle becomes 1·5 mg./cm.²/hr./mm. Hg.

In Fig. 6. results are presented of measurements made with intact animals and isolated preparations. The osmotic permeability of an unknown thickness of intact body wall is 214 mg./cm.²/hr. per mm. Hg. and for a section of isolated integument 0·74 mm. thick 47 mg./cm.²/hr. per mm. Hg. At the same temperature the permea-bility of Rana escalenta skin, 0·07 mm. thick, is 39 mg./cm.²/hr./mm. Hg (recalculated from data of Hevesey, Hofer&Krogh, 1935). In view of the difficulty of estimating the actual extent of the vapour pressure gradient within the skin, values for isolated snail integument and frog skin are probably comparable.

It was found, in calculating tissue permeabilities in air, that small errors in deter-mining evaporation rate or atmospheric humidity could lead to large discrepancies in permeability, whenever the calculated vapour pressure gradient was small. It is possible therefore that the permeability values obtained for dead and non-regulating tissues are comparable, In the regulating snail, where the vapour pressure difference across the mantle is large, much more confidence can be placed in the calculations. Even allowing for error, the mantle during regulation must be at most one-fortieth as permeable as other tissues. It must be concluded that there is a fundamental physio-logical difference between mantle and body wall even though the two tissues closely resemble one another histologically (Campion, 1961). This difference could be attributed to the presence of an active or passive barrier to water. Since tissue desicca-tion was never observed in living snails, even at low evaporation rates, the transition from high internal or low external vapour pressure must take place at or near the integumental surface. This suggests that the barrier is superficial and may occur in a cuticular layer 2 μ thick on the integumental surface (Machin, 1965). An alternative explanation, that the waterproof barrier lies in the mucus itself, is not supported by observation. Earlier studies with isolated mucus samples and intact regulating snails (Machin, 1962) indicate that mucus is readily wettable and therefore easily hydrated by water in contact with it.

I am indebted to Dr D. G. Butler and to my wife for reading the manuscript and providing helpful suggestions. The research was supported by operating grants from the National Research Council (A-1717) and Medical Research Council of Canada (MA-1916).