jf. Exp. Biol. (1966), 45, 269-278 2 6 9With 6 text-figures

Printed in Great Britain

THE EVAPORATION OF WATER FROM HELIX ASPERSA

IV. LOSS FROM THE MANTLE OF THE INACTIVE SNAIL

BY JOHN MACHIN

Department of Zoology, University of Toronto, Toronto 5, Canada

{Received 27 April 1966)

INTRODUCTION

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

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 evaporationfrom the exposed surface of the mantle is reduced following withdrawal of the snailto the inactive state. That this species of snail is able to ' regulate' evaporation is ofconsiderable physiological and ecological importance. The present work represents acritical examination of the physiological mechanisms which operate to modify waterevaporation from the exposed integument of inactive snails.

MATERIALS AND METHODS

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

Evaporation measurements

The epiphragm was removed from each snail before measurement. Snails wereplaced on a Metier single-pan analytical balance reading to o-i mg. Air was con-tinuously drawn through the weighing chamber of the balance and passed through aCambridge Systems dewpoint hygrometer. Complete air replacement occurred every10 min. Water loss by evaporation could thus be measured gravimetrically. Correc-tions were made for the slight but constant beam deflexion, equivalent to between 2and 3 mg., caused by air movement through the weighing chamber. Temperature andhumidity measurements were recorded at 15 min. intervals.

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 Vial diameter and the depth to which they were filled matched the evaporatingconditions found in the withdrawn snail as closely as possible.

Experiments were performed to test whether or not the lowering of surface vapourpressure during inactivity was permanent or reversible. Measurements of evaporativeloss were made following a short period in which the snail was suspended in humidair over distilled water.

270 JOHN MACHIN

Determination of vapour pressure

Mucus was collected after slight mechanical stimulation of the mantle, and bloodwas 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 aFiske osmometer. These measurements were then used to calculate vapour-pressurevalues from data according to Frazer, Taylor & Crollman (1926). The vapour pressureof 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 toequilibrate at 22°±o-oi° C. The vapour pressure was then obtained from dewpointmeasurements of the air bubbled through each solution.

Measurement of integumental permeability

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

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

(b) Measurements with isolated sections of dorsal body wall. Mantle tissue proved tobe unsatisfactory for measurements of integumental permeability. Alternatively,sections of integument from the body wall were excised, and mounted as a diaphragmbetween 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 bothsolutions was effected with teflon-coated magnetic stirring bars (Fig. 1). Rates of waterdiffusion across the membrane were then measured volumetrically. Generally, goodreadings were obtained 2-3 hr. after the addition of the solutions. There was nomeasurable interference due to aeration of the solutions.

RESULTS AND DISCUSSION

Measurements with intact snails

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

Perhaps the most convincing evidence that the snail is able to regulate evaporationwas provided by weight records of snails which had died naturally. It was found that

The evaporation of water from Helix aspersa. IV 271

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 theinactive condition. In fact it is well known that terrestrial snails are able to survivemany months, even years (Mead, 1961), without food and water. Ward (1897) hasreported that specimens of Helix aspersa were still alive after 13 months inactivity. Itis surprising that this commonly observed fact has not aroused interest earlier, since

Air-

Stirring barMagnet

Volumeadjustment

Integument

Fig. 1. Apparatus for measuring osmotic permeability of isolated snail integument.

Table 1. Rates of evaporation from open shell aperture in still airat 220 C. and 34% relative humidity

Evaporation rate(mg./cm.'/hr.)

Control: empty shell filled with distilled water io-6Inactive snail, artificially stimulated 11-4Dead snail 9-9Normal inactive snail (mean of 110 determinations) 0-48

relatively minor changes in evaporating surface area after death could not in themselvesaccount for such wide differences in evaporation. However, failure to recognize thesignificance 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.

Surface vapour pressure claculations

Since the measurements summarized in Table 1 were made under identicalconditions, the observed decrease in evaporation rate from the inactive snail must have

272 JOHN MACHIN

been caused by a reduction in the vapour pressure gradient between the mantle andair. Evaporation rates may be conveniently expressed (Ramsay, 1935; Machin, 19645)in terms of a simplified form of Fick's law (1955):

(1)

where E is the rate of evaporation per unit area of surface, k is the coefficient of diffu-sion of water vapour in air expressed in appropriate units, p0 is the vapour pressure ofthe evaporating surface,pd is the vapour pressure of 'free' air, and D is the boundary-layer thickness, i.e. the distance separating p0 and pd. In effectively still air, the equa-tion may be reduced to the following (Ramsay, 1935):

E oc (po-pd). (2)

It follows that the relationship between evaporation rates under two different circum-stances designated by suffixes 1 and 2 becomes

(3)

The vapour pressure values given in Table 2 together with the evaporation data inTable 1 can be inserted into the formula to calculate the remaining unknown pOt. Avalue of 7-29 mm. Hg for the vapour pressure of the superficial mucus during theperiod of reduced evaporative loss was obtained.

Table 2. Equivalent concentrations of corresponding pairs of blood and mucus calculatedfrom determinations of freezing-point depression together zcith their calculated vapourpressures at 220 C.

Blood concentrationNaCl equivalent

(g./ioo ml.)

1 1 9I-OI1-301 1 6

i-341 03

Mean±s.E. I - I 6 ± O - O 5

Vapour press.(mm. Hg)

197019-72196919701968I9-7I1970

Mucus concentrationNaCl equivalent

(g./ioo ml.)

1-441 031 44I-2O1 4 91 091-28 ±008

Vapour pressure(mm. Hg)

196719-731967197019-66I9-7I1969

There are a number of pratical reasons why this calculation may be subject to error:(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;(b) assumptions permitting the simplification of equation (1) to (2) may not be validin this case, E may vary independently with (po —pd).

To serve as a control, the evaporation data of various standard saturated salt solutionswere used to calculate p0 by the same method as used on snails. Observed andcalculated vapour pressures are given in Table 3. It can be seen that they agree fairlywell. Therefore, calculations of surface vapour pressures from evaporation results maybe considered valid.

The evaporation of water from Helix aspersa. IV 273

Mechanism underlying decrease in mantle vapour pressure

One of the fundamental physiological differences between active extended andinactive regulating snails lies in the functioning of the mucus glands. Observations(see Machin, 1965) suggest that periods of low evaporative loss coincide with acessation or reduction of the muscular activity of the mantle which normally bringsabout mucous extrusion. Several unsuccessful attempts to measure the thickness of

Table 3. Observed and calculated vapour pressures of saturated salt solutions at 22-0° C

Saturated saltsolutions

ZnSO4

Na,SO«NaClKNO,CaCl,

Observed vapourpressure

(mm. Hg)

18421842iS-481080

6-73

Calculated vapourpressure

(mm. Hg)

192418231509

9-457-08

Theoretical curve

10 20 30 40 50 60 70

H,O lost from sample (%)

Fig. 2. Graph showing relation between evaporation rate and water contentof mucus (O) and 1 % NaCl ( • ) samples.

the mucous layer were made. The appearance of the pattern of reflected highlights(Machin, 1964a), however, does indicate that this layer is much thinner duringregulation. It is suggested that decrease in volume is due to continued evaporation inthe absence of further replacement of mucus. The resulting increase in solute con-centration could be the cause of low surface vapour pressure during regulation.

274 JOHN MACHIN

This working hypothesis was tested by measuring the change in evaporation rateresulting from known amounts of water loss in isolated mucus samples. The resultsplotted in Fig. 2 show that the lowering of vapour pressure by solute concentration inthe mucus follows the same course as a similar sized drop of 1 % NaCl. This is to beexpected since Machin (1962) and Burton (1965) have shown that mucus is rich inelectrolytes. Discrepancies between observed and theoretical curves are almost certainlydue to unavoidable decreases in the surface area of the experimental samples as they

25/ahick 100/fthick 2S0/< thick SOO/zthkk

Amount of H,O lost (mg./cm.1)

Fig. 3. Change in evaporation rate of an intact snail at the onset of regulation (O) comparedwith isolated mucus samples of different size. The calculated thicknes* of each sample if evenlyspread over 1 cm.* of mantle surface is also given.

dried up. Repeated measurements of freezing-point depression made over a periodof 24 hr. using the same sample kept at room temperature failed to show any chemicaldecomposition. Increase in the number of solute molecules could also have reducedthe vapour pressure.

In Fig. 3 it can be seen that the change in the evaporation rate of an artificiallystimulated snail could be reasonably explained by the progressive dehydration of aninitially 100/i 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 atfirst and then diverge (shaded area). As dehydration progressed the vapour pressuregradient between blood and mucus in the intact snail would initiate outward diffusionand slow down the decrease in evaporation rate.

In the absence of glandular extrusion the vapour pressure of the mucus ultimatelydepends on the delicate balance between diffusion and evaporation. It is possible to

The evaporation of water from Helix aspersa. IV 275

test whether this dynamic equilibrium exists by measuring evaporation immediatelyfollowing exposure to humid air. High external vapour pressure should reduce theevaporation component of the system almost to nil and permit the superficial mucusto rehydrate. In Fig. 4 initially high rates of evaporation, once the normal humidityis 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.

14

12

J_ 10

i-•g

II 6•sS 4

34%R.H

I I20 30 40 500 10 20 30 0 10

Time (min.)

Fig. 4. Observed evaporation rates of regulating snails following a period in humid air.

Integument permeability

Evidence has been presented which shows that the mantle of the inactive snail is insome way capable of reducing evaporative loss. A central part of the study of themechanism of reduction must be the measurement of integument permeability. Ifthere is a massive reduction in the vapour pressure of the superficial mucus, the mucuswill tend to rehydrate by drawing water from the blood. Low rates of evaporationmaintained for long periods of time indicate that the rate of diffusion of water fromblood to mucus is slow. Measurements of the permeability of the mantle thereforemay be expected to yield low values.

In the virtual absence of the mucus extrusion it is possible to calculate mantlepermeability, knowing both the vapour pressure of the blood and of the superficialmucus. Since the vapour pressure of the mucus remains steady during regulation, theamount of water leaving the mucus by evaporation must equal the amount arrivingby diffusion from the blood. If the snail loses water by evaporation at a rate of 0-48mg./cm.2/hr. (Table 1). the permeability of the mantle is 0-039 nig./cm.2/hr. per mm.Hg vapour pressure difference. The evaporation data in Table 1 also permits the mantlepermeability of snails which had died from natural causes to be calculated. A perme-

276 JOHN MACHIN

ability of 6-2 mg./cm.2/hr. /mm.Hg. was obtained. Mantle permeability of snails whichhad been killed by immersion in liquid air was also calculated using data given inFig. 5. Liquid air was found to be the most satisfactory means of killing the snail sinceit served to keep the animal withdrawn in the shell and caused minimum damage tothe 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 extrudedmucus in stimulated inactive snails. However, the rate of evaporation decreased,

Regulating

10 20 30 40 50

Time (hi.)

Fig. 5. Observed rates of evaporation of intact snails following mechanical stimulation orimmersion in liquid air. Assumed equilibrium point in the dead snail is indicated by + .

probably as the superficial mucus extruded before death dried up, until a linear fallin evaporation rate was attained. The mantle tissue itself became progressivelydessiccated after this. Subsequent decrease in evaporation, not observed in the livingsnail, was almost certainly due to excessive depletion of the water reserves of the bloodand increasing resistance to outward diffusion (Machin, 1964a; Beament, 1961).Assuming equilibrium is established, when the excess mucus has been dried (pointmarked +), 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 andisolated preparations. The osmotic permeability of an unknown thickness of intactbody wall is 214 mg./cm.2/hr. per mm. Hg. and for a section of isolated integument0-74 mm. thick 47 mg./cm.2/hr. per mm. Hg. At the same temperature the permea-bility of Rana esculenta skin, 0-07 mm. thick, is 39 mg./cm.s/hr./mm. Hg (recalculatedfrom data of Hevesey, Hofer & Krogh, 1935). In view of the difficulty of estimatingthe actual extent of the vapour pressure gradient within the skin, values for isolatedsnail integument and frog skin are probably comparable.

The evaporation of water from Helix aspersa. IV 277

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 inpermeability, whenever the calculated vapour pressure gradient was small. It is possibletherefore that the permeability values obtained for dead and non-regulating tissuesare comparable, In the regulating snail, where the vapour pressure difference acrossthe mantle is large, much more confidence can be placed in the calculations. Evenallowing for error, the mantle during regulation must be at most one-fortieth as

35

J 30

q 25X

i *

15

a3 10

004 008 0-12Vapour pressure difference (mm. Hg).

0-16

Fig. 6. Rates of water flux under different vapour-pressure gradientsin intact snails (O) and isolated dorsal integument ( • ) .

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 closelyresemble one another histologically (Campion, 1961). This difference could beattributed 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 transitionfrom high internal or low external vapour pressure must take place at or near theintegumental surface. This suggests that the barrier is superficial and may occur in acuticular layer 2/1 thick on the integumental surface (Machin, 1965). An alternativeexplanation, that the waterproof barrier lies in the mucus itself, is not supported byobservation. Earlier studies with isolated mucus samples and intact regulating snails(Machin, 1962) indicate that mucus is readily wettable and therefore easily hydratedby water in contact with it.

18 Exp. BioL 45, 2

278 JOHN MACHIN

SUMMARY

1. Data is presented which suggest that inactive specimens of Helix aspersa areable to regulate evaporative water loss from the mantle.

2. Evaporation is reduced following the cessation of glandular extrusion, by theconcentration of solutes in partially dehydrated superficial mucus.

3. Methods are described for determining vapour pressure gradients across livingand freshly killed mantle tissue and for calculating permeabilities.

4. Osmotic permeability measurements using isolated and intact body wall weremade.

5. The permeability of regulating mantle tissue was 0-039 mg./cm.2/hr. per mm.Hg vapour pressure difference. Living body wall and freshly killed mantle were atleast forty times more permeable to water.

6. Low permeability seems to be a unique property of living mantle tissue.

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

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