The mucus producing glands and the distribution of the cilia of the pulmonate slug Limax...

1 J. Zool., Lond. (1983) 201,97-116

The mucus producing glands and the distribution of the cilia of the pulmonate slug Limax pseudofiavus

A N T H O N Y COOK A N D R A J S H R E E S H I R B H A T E School of Biological and Environmental Studies, New University of Ulster, Coleraine,

Londonderry, N. Ireland

(Accepted H March 1983)

(With 2 plates and 9 figures in the text)

The gross anatomy and histochemistry of the mucus-producing glands of Lirnux pseudo- .flavus Evans were investigated. The body mucus can be divided into three areas. The dorsal body surface is covered with a sulphated acid mucopolysaccharide/protein mixture secreted largely by five cell types. The pedal mucus is a mixture of neutral mucopolysaccharide from the suprapedal gland. The dorsal and pedal mucus sheets are separated by the peripodal groove whose cells secrete a weakly acid mucus. The duct of the suprapedal gland, the epidermis around the pneumostome, the ventral surface of the peripodal groove and the centre of the underside of the foot are ciliated. The dorsal and pedal mucus remain stationary relative to the body and the substrate respectively and the only rejection currents seen in the mucus are around the pneumostome.

It is suggested that the pedal mucus is formed by the mixture ofthe products ofthe suprapedal gland and the mucoprotein secreting gland in the leading edge ofthe foot, thus producing a mucus suitable for locomotion. Many areas ofthe animal (e.g. the head, pneumostome, sole and the leading edge of the foot) are capable of producing both a fluid (neutral or weakly acid) and a viscous (acid) mucus. It is postulated that such an arrangement allows for both adhesion and lubrication at different times.

Introduction . . .. Materials and methods . . Results . . . . . .

Dorsal body surface . . Peripodal groove . . . . Sole . . . . . . Pneumostome .. Suprapedal gland . . Leading edge of the foot Semper’s organ .. Ciliated areas . . . .

Discussion . . .. Summary . . . . References . . . .

Contents .. . . .. .. . . . . . . . . . . .. . . . . .. . .

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. .

. . . .

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Page . . . . .. . . 97 .. . I .. .. 98 . . . . . . . . 101 .. . . . . . . 102 . . . . .. . . 105 .. . . .. . . 105 .. . . .. . . 105 . . . . .. . . 106 .. . . I . . . 107 . . . . .. .. 108 . . .. . . . . 109 . . . . .. . . 109 . . . . .. . . 114 . . .. . . . . 115

Introduction

The gastropod epidermis interacts with its external environment through a layer of mucus. Since different areas of epidermis are involved in different functions it is to be expected that the nature of the superficial secretion will vary with these functions.

97 0022-5460/83/010097 +20 $03.00/0 0 1983 The Zoological Society of London

98 A. COOK AND R. SHIRBHATE

Some attempts at functional interpretations of the distribution of mucus producing cells in Patella vulgata L. and Acmaea tessulata (Muller) (Grenon & Walker, 1978) have been made but although the mucus-producing glands of several terrestrial gastropods have been examined histochemically, no extensive study since that of Campion (1 96 1 ) has been aimed at elucidating function.

The integument of a slug may be divided into two functional areas: first the dorsal epidermis which is covered by a secretion from local cells and second the pedal epidermis which is covered by a secretion largely from the suprapedal gland. These two areas are separated by a peripodal groove which runs around the foot on its ventrolateral margin.

In a study of the suprapedal gland of Laevicaulis alte Ferrusac (a veronicellid slug) Dalal & Pandya (1976) concluded that it secreted a sulphated acid and a neutral mucopolysaccharide. Similar conclusions have been reached by Chetail & Binot (1967) for Arion rufus (an arionid slug) but with the inclusion of a lipid component. There is evidence from the work of Denny (1 980) that the pedal mucus of Ariolimax columbianus (Gould) (an arionid slug) contains both protein and carbohydrate,

The epidermal glands have been more extensively studied. Campion (1 96 1 ) described four different mucus producing glands and three other cells containing protein, lipid and calcium in the epidermis of Helix aspersa L. Arcadi (1963) on the other hand found only two cell types in Lehmania poirieri (a limacid slug). The comparative paucity of cell types in slugs is born out by Barr (1 928) and Wondrak (1 969) who described two cell types from the dorsal surface (protein and mucopolysaccharide), one from the peripodal groove and one from the sole of Arion ater L. and by Newel1 (1 973) who made similar findings in Deroceras (= Agrio- limax) reticulatum Muller (a limacid slug). Opposed to this simple view is the finding of Chetail & Binot (1967) of three cell types in the suprapedal gland and four in the sole of Arion rufus. Zylstra (1 972) has shown there to be 1 1 gland cell types on the external surface of Lymnaea stagnalis L. and eight in Biomphalaria glabrata Say, both of which are aquatic basommatop horans.

From these studies it would appear that freshwater snails have a greater diversity of mucus- producing cells than terrestrial ones and that terrestrial snails.have a greater diversity than terrestrial slugs. Slugs then seem to have an anatomically simple mucus-secreting system. The functions of epidermal secretions in slugs on the other hand appear to be at least as diverse as those of terrestrial snails, aside from the secretion of the shell and epiphragm. Certainly slugs are more dependent on their mucus, having no shell into which to withdraw and more dependent on behaviours such as huddling (Cook, 198 I ) and homing (Cook, 1979) which have as their basis the pheromonal qualities of their mucus. The present work describes the distribution and outlines the contents of gland cell types contributing to the surface coating of mucus of the limacid slug, Limaxpseudoflavus Evans, and attempts some functional interpretation of this distribution.

Materials and methods

Adult and juvenile Limax pseudqflavus were collected from Londonderry and Coleraine and immediately fixed. Zenker’s fixative was used routinely since it provokes the minimum of mucus secretion. Zenker’s is not the ideal fixative for all the histochemical tests used. It gives however, a good general fixation of gastropod tissues and allows the comparison of serial sections each stained with a different technique. This avoids the problem of identifying the same cell type when fixed differently but inevi-

SLUG MUCUS PRODUCING GLANDS AND CILIA 99

T A B I . ~ I .4 surnmarj, ofthe histochemical procedures used

Procedure Demonstrates Control Authority Comments

Mallory’s triple stain

General

Protein amino groups Deamination

Grimstone & Skaer ( 1 972)

Mercuric bromophenol blue

Bonhag (1955)

Diazot izat ion coupling

Protein Tyrosine Glenner & Lillee (1959)

Toluidene blue Metachromasia Kramer & Windrum (1955) metachromasia

Air dried to preserve

Alcian Blue (pH 0.5)leosin

Sulphated MPS

Alcian Blue (pH O.S)/Alcian yellow (pH 2.5)

Alcian blue (pH 2,S)/PAS

Differentiation of sulphated and carboxylated MPS

Ravetto ( I 964)

Differentiation of neutral and acidic M PS

Mowry ( 1 963)

Periodic Acid Schiffs (PAS)

Periodic acid diamine (PAD)

Alizarin Red S

Vic-glycol groups Absence of periodic acid

Pearse ( 1 980)

Neutral MPS Spicer (1 965)

Calcium

Lipids

Extraction in HCI Dahl (1952) Fixed in 95% alcohol

Sudan Black B Chloroform/ Chiffelle & Wax sections fixed in methanol extract- Putt ( I 95 1 ) Elftman’s and frozen Lipase at 37°C sections

MPS, Mucopolysaccharide.

tably leads to uncertainty in the interpretation of histochemical procedures. Fixatives containing formaldehyde (either buffered or with 0.5% cetyl pyridinium chloride) result in the production of an extremely thick mucous coating. This is not due to an active secretion since it persists if the slugs are killed by freezing before fixation. Exceptions to the use ofZenker’s fixative had to be made for material used to locate calcium (fixed in 95% alcohol) (Dahl, 1952), and lipid (10% formaldehyde for frozen sections and Elftman’s fixative (Pearse, 1980) for wax sections). Tissues were dehydrated in alcohol and cleared in methyl benzoate before being embedded in wax. Sections of 7 p m were cut o n a Cambridge Rocker microtome. Frozen sections were cut with a Bright freezing microtome.

Adult animals (over 5 cm) were used for all histochemical procedures. Juveniles (about 1 cm) were used for the serial reconstruction of gross anatomy.

Staining procedures were diverse and are summarized in Table I. Specimens were prepared for the

100 A. COOK A N D R. SHIRBHATE

I 1

+ - + I

I I

m

I 1

I I

I 1

I I

I I

f l m

+ - + I m

* I + + n

+ .- + I

+ I + a:

I I

+ + + I I I I I + + + I a : S &

+ + + + I I I + cr: I & a ! + l + z

+ + + + + + I I A Z + + + + I

m * ,';a!!-

I

t + i I I;+ + + + + + + I W

z o z

I

+ + 2

I

I

I

I

I

I

I

I

>. + + n

+ + + n

S

S

I


scanning electron microscope by fixation for 1 h in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer ( p H 7.2) before being wiped t o remove superficial mucus. T h e y were then dehydrated in acetone, critical point dried and coated with gold.

Results

Fourteen different secretory cell types can be distinguished using the histochemical tests described. These cells vary in content, position, shape and size. Table I1 gives their staining reactions, Table 111 their position, size, shape and content.

The animal can be considered as four different areas as far as its mucus is concerned, i.e. the dorsal and lateral body surfaces down to the peripodal groove; the peripodal groove itselc the leading edge of the foot; and the sole. There are also three specialized galandular areas-the pneumostome, the suprapedal gland and Sernper’s organ in the face. These areas are all indicated in Fig. 1

. rABLE 111 The properties ofthe muciis cell types in Limax pseudoflaws

Cell type Position Shape Texture Size Contents

1

2

3

4

5

6

I

8

9

10

1 1

12

13

14

Dorsal surface

Dorsal surface

Dorsal surface

Ubiquitous

Semper’s organ

Suprapedal gland

Ubiquitous

Peripodal groove

Sole and groove

Sole, head and flanks

Pneumostome and lung

Median foot gland

Inferior foot gland

Superior foot gland

Goblet

Clavate

Spatulate

Elongate

Polygonal

Polygonal

Slender

Oval

Round

Oval

Round

Oval

Polygonal

Oval

Fibrous

Smooth

Granular

Coarse Granular Granular

Reticulated

Solid

Finely

Granular

Granular

Solid

Granular

Granular

Solid

granular

130-360 x 60-1 40 pm

30-60 prn

35-80 pm

13-50 ym 30 ym

200-600 x

150-550 x

130-600 x

500-600 x 300-350 pm

3-1 prn 14-2 5 ym

15-30 pm

15-30 ym

15pm

15pm

15-40 pm

20 pn

20-150 x

Sulphated and carboxylated M PS

Protein

Protein

?

Sulphated, carboxylated and

Neutral MPS

Sulphated and carboxylated MPS

Carboxylated and neutral MPS

Neutral MPS

Neutral MPS

Carboxylated MPS

Carboxylated and neutral MPS

Mucoprotein

Neutral MPS

neutral MPS

I02

dbs I

A. COOK AND R. SHIRBHATE

m

FIG. I . A diagrammatic view of L p~czido/(ai,ui showing the areas of the body possessing mucus-producing glands. Ciliated areas are shown dotted. dbs, Dorsal body surface; ig, inferior gland; m, mantle; mg, median gland; p, pneumostome; ppg, peripodal groove; s, sole; sg, superior gland; so, Semper’s organ; spd, suprapedal duct: spg, suprapedal gland; sps; suprapedal sinus.

Dorsal body surface

The general dorsal body surface is thrown into short folds which in section appear as short humps. The mantle of the animal is lightly wrinkled but this pattern is not noticeable in sections. An active, hydrated animal is swollen and mucus tends to accumulate between the body folds giving the whole animal a glossy appearance. A dehydrated inactive animal is contracted and has little mucus between these folds and appears velvety.

The mantle shield and general dorsal body surface contain five main cell types. The largest of these is the type 1 cell which is widespread over the exposed surface of the animal. It is goblet shaped with fibrous contents which stain strongly for both sulphated and carboxylated acid mucopolysaccharides (Plate I(a)). Cell type 7 can easily be mistaken for the ducts of type 1 cells. The staining reactions of type 7 cells are very similar (Table 11) but these cells occur in much greater numbers and also in areas devoid of type 1 cells (e.g. the sole and the folds near the peripodal groove). Cell type 10 is rare in the dorsal surface and will be considered later (Plate I (a)). There are also two protein-containing secretory cells. The type 2 cells are widespread but only rarely found in the epidermis of the head. These cells are straplike with contents which are smooth in texture but which tend to shrink and split horizontally during preparation (Plate I (b)). Type 3 cells lie deeper in the epidermis, have a spatulate shape and finely granular contents. Both types stain positively for protein and not at all for carbohydrates (Table 11). Cell types 2 and 3 also stain with Sudan Black B after Elftman’s fixative. This reaction persists after chloroform/methanol extraction and after overnight treatment with a 5% solution of Lipase at 37°C. These cells do not stain with Sudan black in frozen sections though their staining with mercuric bromophenol blue persists. In addition the yellow granular cells (type 4) are widespread on the dorsal surface and never stain with any of the procedures used. They are visible after fixation with Zenker’s or Elftman’s fixative but not after fixation with formaldehyde. The dorsal mucus of Lirnax

I04 A. COOK AND R. SHIRBHATE

pseudoflaws is yellow and the pigment concerned is soluble in water and absolute alcohol. The yellow granular cell may be the source of the yellow coloration. Yellow granular cells also occur very infrequently in the sole. A test for calcium (Alizarin Red S) showed no positive responses from any secretory cell.

The general body mucus therefore consists mainly of acidic (both sulphated and carboxylated) mucopolysaccharides with the addition of protein and the products of the granular yellow cells.

PLATE 11. The mucus-secreting glands and cilia of Lirnax pseudqflavus. (a) A longitudinal section through the head of a slug stained in PAS and Alcian Blue (pH 2.5). so, Semper’s organ; sp, superior gland, mg, median gland: ig, inferior gland; dpg, duct of the pedal gland; spg, suprapedal gland. The scale represents 1 mm. (b) A scanning electron micrograph of the anterior of a slug. m. Mouth: i , insertion of the anterior tentacle: I. lip: dpg, duct of the pedal gland: ppg, periopodal groove. (Magnification x 40.) (c) A scanning electron micrograph of the pneumostome indicating the extent of ciliation (c). (Magnification x 30.) (d) The boundary between the ciliated and unciliated region around the pneumostome indicated in Plate I1 (c). (Magnification x 730).

SLUG MUCUS PRODUCING GLANDS AND CILIA I05

The peripodal groove This groove may be viewed as being continuous with the duct of the suprapedal gland

which flattens out at the leading edge of the foot (Plate I1 (b)). Some of the cell types found in this groove have affinities with both those on the leading edge of the foot and with else- where in the sole. Only cell type 8 is found exclusively in the groove (Plate I (c)). Its staining responses to Alcian Blue/Alcian Yellow and PAD suggest that it contains a mixture of weakly acidic (carboxylated) and neutral mucopolysaccharides. In any one 7 pm section there are only approximately a dozen cells of this type but they occur regularly down the length of the groove (Fig. 2). Cell type 9 occurs in the sole and the groove. It contains neutral muco-substances (Table 111). Cell type 10 has a similar distribution but differs in that it stains strongly with PAS whereas type 9 does not.

The mucus in the peripodal groove therefore contains a diversity of mucus, none of which is strongly acidic.

The sole The sole contains cell types 9 and 10 described above (Fig. 3). Cell type 9 forms a subepi-

dermal layer in which occasional type 10 cells occur. The most superficial and most common of the mucus-producing glands of the sole are the type 7 cells. These contain a sulphated mucopolysaccharide (Plate I (e)).

The major contributor to the mucus found under the sole is the suprapedal gland. The sole itself contributes a range of mucopolysaccharides from highly sulphated to neutral.

The pneumostome The external structure of the pneumostome is shown in Plate I1 (c). The area around the

0

0 Irnrn - FIG. 2. A cross-section through the peripodal groove showing the distribution of secretory cells and the extent

of ciliation. Striped cells, type 8; open cells, type 10; dotted cells, type 9; solid cells, type 7.

106 A. COOK A N D R. SHIRBHATE

A 0-lrnrn U

FIG. 3. Mucus-secreting in the sole. (A) A low power view of the area between the central and peripheral bands of the foot. (B) A high power view of the central region. (C) A high powever view of the peripheral region. Solid cells. type 7; open cells. type 10; dotted cells, type 9; cells filled with open circles, type 4.

aperture is equipped with the normal complement of mucus-producing cells found on the dorsal surface, with the addition of cells of type 10. These occur sporadically over the dorsal surface but are concentrated in the area of the pneumostome. Type 1 1 cells are found in the pneumostome. They are subepidermal and are found commonly in the lung itself and deep in the channel leading from the lung to the exterior. Near the surface the type 1 1 cells are replaced by type 10 cells lying under a layer of type 7 cells, a configuration which also occurs on the head (Fig. 4). The epidermal surface around the pneumostome is ciliated (Plate I1 (d)). In transverse section the pneumostome is surrounded by an annulus of large cells for which no ducts were found. They stain positively for protein and with Sudan Black in both frozen and wax sections. Their function may be to serve as an elastic annulus around the pneumostome.

The suprapedal gland This is the largest mucus-producing gland in the body and the only one with a discrete

duct. It lies in the foot musculature from just behind the head to about three-quarters of the way down the body. There are very few muscle strands in the gland itself and the duct is separated from the body cavity by a thin dorsal membrane. The duct has a central groove on its ventral floor containing long cilia. This groove is bounded by two densely staining lateral prominences bearing short cilia. Only one cell type is present (cell type 6). These cells contain a neutral mucopolysaccharide. The cells are large, round and each appears to

SLUG MUCUS PRODUCING GLANDS AND CILIA

, 107

OO 0 U *-

0 I mm

FIG. 4. High power view of (A) the lining of the duct of the pneumostorne and (B) the epidermis of the head. Both areas are served by type 7 epidermal cells (solid) overlying an area of type 10 cells (open) producing a less acid mucus.

0.1 rnm

FIG. 5 . The duct of the suprapedal gland in a juvenile L. psoudoflaviis. The central groove, whose cells stain only faintly, support long, poorly organized cilia whereas its more densely staining lateral prominences are equipped with short cilia. Most of the duct is unciliated. I , Lumen; Ip, lateral prominence; cg, central groove.

have an extension to the central groove of the duct of the suprapedal gland (Fig. 5 and Plate I (4).

The duct ofthe suprapedal gland is uniform down its length except for the extreme anterior portion. In the head the duct broadens and the ciliated lateral prominences and the central groove are replaced by a more generally ciliated ventral floor. This floor is raised in the centre (Fig. 6).

The leading edge of the foot This area of the animal contains three cell types in three discrete blocks. For convenience

A. COOK AND R. SHIRBHATE

4 3 2 I

B I \

.- ........ - ..d ......... - c2

FIG. 6. (A) A longitudinal section of a juvenile L. pseudoflavus showing the plane of section and the position of “landmarks” such as the eyes, the buccal mass and the root of the mantle. (B) I , 2, 3 , 4 Sections through the duct of the suprapedal gland showing changes in its shape and in the extent of ciliation (dotted region). (C) I , 2, 3 , 4 . Transverse sections of whole animals showing the position and shape of the suprapedal gland and the suprapedal duct. so, Semper’s organ; sps, suprapedal sinus; spg, suprapedal gland.

they will be called the superior gland (type 14) the median gland (type 12) and the inferior gland (type 13) (Fig. 1 and Plate I1 (a)).

The largest of these three glandular areas in median longitudinal sections is the inferior gland. The ducts of these cells open on to the foot just before it would normally make contact with the ground. The staining properties of these cells indicate that they contain a mucoprotein (Table 11).

The median gland (type 12) produces carboxylated and neutral mucopolysaccharides. These cells are similar to the type 9 cells of the foot in their contents, their morphology and in their position. There are differences in the intensity of staining by PAD, PAS and in Toluidene Blue but this may not represent a significant change in their contents. The superior gland (type 14) is on the dorsal surface of the foot at the exit of the suprapedal duct. It produces a neutral mucopolysaccharide (Table 11).

The mucus produced under the foot is highly complex, being composed of the products of at least seven cell types (i.e. 6, 7 ,9 , 10, 12, 13, 14).

Semper ‘s organ This organ is in the face of the animal (Fig. 1) and the ducts of its cells (type 5) empty

on to an area roughly bounded by the two pairs of tentacles (Plate I (6). The mucus stains positively for sulphated, carboxylated acid and neutral mucopolysaccharides. The gland


stains in a mottled, inconsistent fashion probably indicating considerable diversity of mucus production.

Ciliated areas Figure 1 shows the areas of the body which are ciliated. The duct of the suprapedal gland

is also partially ciliated (Fig. 5) . Figure 7 shows the movements of small particles of chalk blown on to the surface of the slug. The pneumostome is the only part of the exposed body to be ciliated (Plate I1 (c), (d)) and it is only in this area that small particles are moved in the mucus. Irritants (e.g. 0-5°/o Methylene Blue) applied to the surface of the body result in an increased mucus production and writhing movements culminating in the washing off of the irritant. When applied to the head, irritants are removed by the withdrawal of the head under the mantle and the inversion of the tentacles. On re-extension the Methylene Blue is confined to a small blob of mucus which is moved down below the peripodal groove and is left behind.

/-/ .. . . . . . . . .

. . . . . . :

t . .

.. . . . c c c - c C C C + - - C C c c .-.-

FIG. 7. The movement of chalk particles blown on to the surface of L. pseudoflavus. Particles radiate away from the pneumostome and accumulate laterally and dorsally. Particles moved ventrally are swept down below the peripodal groove. Particles below the peripodal groove move backwards with respect to the animal but remain stationary with respect to the ground.

Discussion

The physical properties of a mucous coating are determined by at least three factors. First the presence of acid mucopolysaccharides is indicative of a high classical viscosity (Hunt, 1973); second, the presence of protein will provide a matrix conferring some solid properties to the mucus (Denny & Gosline, 1981; Hunt, 1973); finally the presence of calcium ions will increase the viscosity of protein/polysaccharide matrices (Gray, 1926). Of these factors, the localized production of calcium appears to play no great part in Limax pseudoflavus since little was found. Although the techniques used allow the descriptions of different cell populations they cannot give an exact chemical description of cell contents. This is especially true since the fixative routinely used (Zenker’s) contains potassium dichromate which tends to oxidise carbohydrates and increase their responses to Alcian stains (Zylstra, 1972). For these reasons the properties of the contents of each cell type is not known exactly.

The possible functions of mucus in a slug can be divided into three broad categories: locomotion; cleansing; and communication. Further possible minor functions include cryopro- tection, the prevention of desiccation and excretion.

Clearly the neutral mucopolysaccharide produced by the suprapedal gland (cell type 6) will be involved in locomotion. Denny & Gosline (1 98 1) describe in great detail the physical properties of the pedal mucus of Ariofimax columbianus. It varies from being an elastic solid when relaxed to being a viscous fluid when under sheer forces. The mucus these workers


used was collected from a crawling animal and consisted of both protein and carbohydrate but since no histochemical study has been conducted on the suprapedal gland of Ariolimux there is no certainty that both components were derived from this gland.

The mucus of the suprapedal gland must have two types of properties; those suited to free movement down the duct and those ofa diphasic gel under the foot. These two requirements may not be compatible. There are few muscles in the suprapedal gland of L. pseudoflaws and only a thin membrane separating the duct and the body cavity (Fig. 5 and Plate I (d)). Movements within the duct therefore must be by the activity ofthe cilia on the ventral floor. The neutral contents of the suprapedal gland cells (type 6) indicate that the contents of the suprapedal duct will not be highly viscous. Further the lack of protein in the secretion will not allow the sort of sulphydryl cross bonding envisaged by Denny & Gosline (1 98 1) as being involved in the sol/gel properties of the pedal mucus of Ariofimux. If there is protein in the pedal mucus of L. pseudoflaws then it must be added to the suprapedal gland secretion after it leaves the duct. The type 13 cell in the leading edge of the foot contribute a mucoprotein (Table 11) to the suprapedal secretion as it moves over this area of the foot. It may be the products of these gland cells which give the pedal mucus the properties described by Denny & Gosline (198 1). Chetail & Binot (1 967) describe the suprapedal gland of Arion rufus. Neu- tral mucopolysaccharide is secreted in the posterior of the gland and is joined by a more acid secretion from the front. The leading edge of the foot has not been examined. This may be a similar system to that proposed for L. pseudoflaws where an initially free-flowing mucus receives additives at the front of the animal in order to acquire the properties required under the foot. Cellular separation of the synthesis of a protein/polysaccharide complex in the hypobranchial gland of Buccinium undutum L. has also been demonstrated (Hunt, 1973).

The suprapedal duct broadens out as it approaches its exit. The central area also rises. This will tend to spread the mucus out over the dorsal surface ofthe anterior foot by pushing it towards the edges. This area is also covered with cilia and receives the secretion of the type 14 cells in the superior gland. These produce a neutral, non-metachromatic mucopolysaccharide which should therefore have a low viscosity. A mucus of this type at this place may assist in the spreading of the products of the suprapedal gland over the foot. The type 12 cells lie immediately below these type 14 cells. Type 12 cells secrete a carboxylated mucopolysaccharide and since both in their position and their product they are closely allied to the type 9 cells of the sole their role may not be specifically associated with being located at the leading edge of the foot. Despite the minor differences in staining properties, this median gland might be better viewed as an extension of the layer of type 9 cells in the sole. This layer is penetrated by the large inferior gland (type 13 cells) giving the impression of two separate cell populations (Fig. 8).

The whole of the leading edge of the foot is ciliated and so is the central band of the tripar- tite foot. During locomotion the pedal waves are also confined to this central band. The cilia in the peripheral bands peter out at a level corresponding to the back of the head. The wide distribution of cilia on the anterior foot probably serves to further distribute the mucus to the edges. The presence of cilia in the central band may also be associated with the distribution of mucus by moving it backwards whilst it is in its sol phase during locomotion thus countering the peristaltic effect of the pedal waves moving it forward. The lack of cilia in the peripheral bands of the foot may therefore be associated with the lack of pedal waves in these areas.

In a stationary animal the mucus must allow both for adhesion and for detachment of

SLUG MUCUS PRODUCING GLANDS AND CILIA 1 1 1

Lubricont - - L O W vlscoslty polysacchoride

9

10 7 4

4

\

I I Adhesive or lubricont

FIG. 8. The suggested formation of pedal mucus. Suprapedal gland mucus is spread out over the leading edge of the foot aided by a lubricant from the superior gland. Protein is added to form a biphasic sol/gel system. Mucus from cell types 9 (and 12) and 10 allow for the detachment ofthe foot. Mucus from cell type 7 aids adhesion.

the foot from the substrate. Gastropods adhere to their substrate by Stefan type adhesion (Grenon & Walker, 1978). This form of adhesion depends upon the apposition of two flat surfaces separated by a thin layer of fluid. The strength of the adhesion increases with the viscosity of the fluid. Type 7 cells are widespread over the body but specially common in the sole and produce the most highly sulphated products found in this region. It would be expected therefore that the secretions of these cells would increase the viscosity of the pedal mucus and thereby promote adhesion. The type 9 cells on the other hand secrete a neutral mucopolysaccharide which would be expected to be less viscous and therefore aid in releas- ing the foot from the substrate. Similarly the type 12 cells at the leading edge of the foot also secrete a neutral mucopolysaccharide and might therefore be involved in unsticking the anterior foot. These suggested functions for the pedal mucus imply a control over mucus production in the sole which has yet to be demonstrated. This interpretation ofthe formation and functi‘on of the pedal mucus is summarized in Fig. 8.

The dorsal mucus consists of a highly sulphated acid mucopolysaccharide from the type 1 cells with the addition of protein from the type 2 and 3 cells (Type 11). The staining of cell types 2 and 3 with Sudan Black after Elftman’s fixative but not in frozen sections is confusing. Chetail & Binot (1 967) report a similar staining pattern for four cell types in Arion rufus (see Table IV). The dorsal mucus of L. pseudoflavus will be a viscous fluid and the addition of protein may provide a matrix to lend coherence. At the tail end of the animal the dorsal mucus sometimes comes away in sheets and/or strings which would support the view that it has mechanical properties not to be expected ofa viscous fluid. The high viscosity and cohesion of the dorsal mucus may be seen as a water-saving device in the sense that it does not flow off the animal rapidly and therefore does not need frequent replacement. This mucus may also serve as a cryoprotectant (Hargens & Shabica, 1973).

The type 4 cells (yellow granular) have an unknown product but could be the source of the yellow pigment (probably a flavone-Campion, 1961) seen in the dorsal mucus of agitated animals. The dorsal mucus produced in response to mechanical and/or chemical stimulation is very watery and will favour the removal of foreign bodies from that surface. In a normal unirritated animal the dorsal mucus hardly moves at all (Fig. 7) and since the dorsal surface is not ciliated any ejection of matter from this area must be accomplished by flooding rather than by discrete rejection currents.

The area around the pneumostome and the peripodal groove have two features in common. First, they are both ciliated and second they both contain cells which produce a neutral


TABLE IV A comparison o f m u c ~ ( . ~ cell types in terrestrial pulmonates

Nearest L. pxudo- Author/species Cell type Position PAS AB Tol. B. Prot. Lipid ,/lavus equivalent

Type 1 Type 10 Type 9

? Type 2 +* ?

- - + + + +

Campion (1961) A Mantle -

Helix uspersa C Sole -

Calcic Mantle - Protein Mantle - - - Lipid Mantle - - - -

- - B Mantle + + + + D Sole + + + - - Type lo?

- - -

- - - - - + +

Chetail & al Binot ( 1 967) a2

b Arion rufus I

I1 111 IV

Pedal g Pedal g Pedal g Sole Sole Sole Sole

+ + + + + + + + + + +

+ + - -

- - +

Dalal & I Pandya (1 976) Luevicaulis alte

Pedal g + + +

*No controls performed with Sudan black; PAS. periodic acid Schiffs; AB, Alcian blue: Tol. B., Toluidene Blue: Prot., Protein.

or carboxylated mucopolysaccharide (pneumostome type 10 and 1 1 and groove type 8). The area round the pneumostome is cleaned in two ways. First if large irritants are placed in the pneumostome the area will be flushed out with pallial water. Second, small particulate matter is moved radially out from the pneumostome either to end up lodged in the dorsal mucus or to be moved down on to the foot mucus. Cell types 10 and 1 1 presumably provide the mucus for this rejection current. This is supported by the observation that cell type 10 is more frequent on the right side (pneumostome) than on the left side (non-pneumostome) between the bottom of the mantle and the peripodal groove. In the channel of the pneumostome type 10 cells form a deep sub-epidermal band beneath a layer of type 7 cells. This is a configuration which also occurs on the dorsal surface of the head. The mucus from type 7 cells may provide for adhesion between two areas (edges of the pneumostome or between head and lower mantle) which that from type 10 or 1 1 will release. Adhesion between adjacent areas of the body will prevent excessive water loss.

The peripodal groove produces a weakly acid (type 8) and neutral mucopolysaccharide (types 9 and 10). This area has to be viewed in the context of the mass mucus movements in the crawling animal. The peripodal groove is an extension of the suprapedal duct and all mucus above it is therefore stationary relative to the animal and all below it stationary relative to the ground. To facilitate the movement of one mass of mucus relative to the other it is reasonable to suggest that a fluid mucus is inserted between them. The cilia in the groove


Static

mucus viscous

Lubricant fluid mucus

Stattc biphasic mucus

Substrate

FIG. 9. The two mucous sheets on the surface of a slug. The dorsal mucus remains static and the pedal mucus remains attached to the substrate. Both sheets are normally viscous and the movement of one relative to the other is aided by the insertion of a fluid mucus from the peripodal groove.

do not provide a rejection current since particles in that area of the foot remain stationary in one or other of the mucous sheets. The function of the cilia in the groove may be to aid in the insertion of the fluid mucus between the more viscous dorsal and pedal mucous sheets (Fig. 9).

Semper’s organ is a large gland pouring mucus on to the front of the head. The contents of this gland are diverse and therefore any functional interpretation is necessarily speculative. Mucus on the face of the animal may assist in feeding by sticking together particulate food, but this would be a function more normally associated with salivary secretions (Moreno et af., 1982). It is likely that it serves to clean the head and the sensory epithelia ofthe tentacles during their withdrawal. Rapid production of large quantities of mucus on the head forms a blob of mucus containing any irritants which is then removed by wiping the head on the underside of the mantle. Treating the head with methylene blue produces a response like this but of course it is not known from where the mucus comes.

Whilst there appears to be a large volume of information available on which to basexom- parisons many studies are not strictly comparable. Previous studies on slugs have either been conducted on a narrow range of tissues (e.g. Ghose, 1963; Dalal & Pandya, 1976; Barr, 1926, 1928) or .have been conducted with the electron microscope with little regard to the chemistry of the contents of the cells or to their distribution over the surface of the animal (Newell, 1973, 1977; Wondrak, 1969).

Comparisons with marine prosbranchs (Grenon & Walker, 1978) are extremely difficult. First, the functional anatomy of the animals is very different and secondly the distribution and role of the mucus is different. In general, however, a limpet is covered with a mixture of polysaccharide and protein just like a slug and at the periphery of the foot there is a band of cells (Grenon & Walker’s (1978) type 5 ) producing non-sulphated fluid mucus. Limpets


therefore seem to conform to the pattern of mucus sheets separated by a more fluid mucus seen in L. pseudoflavus.

Where detailed work has been carried out some comparisons are possible (Table IV) but it must be stressed that because of variations in techniques exact correspondence of staining properties is unlikely.

Only one cell type described in other work is radically different from those in L. pseudqflavus. These are the calcic glands in Helix aspersa (Campion, 196 1). Clearly the role of calcium in a slug is different from that in a snail. The major store of calcium in any pulmonate is the digestive gland (Wagge, 195 1) and specific cells in the digestive glands of L. pseudqflavus also stain strongly with Alizarin Red S. No cutaneous gland cells, however, stain in a similar fashion. The dorsal mucus of some pulmonates is milky (e.g. Helix, Deroceras reticulaturn) due to the presence of calcium. That of L. pseudogavus is never milky.

Both Campion (1961) and Chetail & Binot (1967) demonstrate lipid in some cell types though only after wax impregnation. These findings must be of dubious validity since in neither case were the tests accompanied by adequate controls (e.g. lipase or chloroform/ methanol extraction). Lipid does appear in gastropod mucus (Wilson, 1968) but it may appear as a consequence of the release of membrane bound vesicles or as a result of the mucus being partly an ultrafiltrate of the haemolymph (Burton, 1965) rather than by deliberate secretion.

Mucus is used extensively by gastropods as a means of communication and navigation, e.g. in prey-finding (Paihe, 1963), mating (Quick, 1960), homing (Cook, 1979) and trail following (Cook, 1977) and there is some evidence of there being close control over mucus production (e.g. in Onchidiurn-McFarland, 1980) for these purposes. The commonest speculation as to the nature of the pheromones in mucus is that they are lipids (Bousefield et al., 198 1 ; Chase & Boulanger, 1978). The present observations unfortunately add nothing to these speculations.

One pair of the cell types described is very similar. This is types 12 and 9. Even amalgamating these two types there are 13 different mucus-secreting cell types in L. pseudoflavus. Four of these are largely from the dorsal surface (1, 2, 3 and 4), two are widespread (7 and lo), two from the leading edge of the foot (13 and 14) and one each from the foot (12=9), the pneumostome (1 l ) , the peripodal groove (8), the suprapedal gland (6) and Semper’s organ ( 5 ) . This implies that the mucus-secreting system of this slug is at least as complex as those of the terrestrial snails which have been studied.

We wish to acknowledge the technical assistance given by Joan Taggart and Ken Thompson and the expertise of Steve Lowry both with the stereoscan and in the preparation of the plates. Mary Crisp kindly criticized a n early draft. Sam Irwin at the Ulster Polytechnic assisted us in the provision of a cryostat.

SLUG MUCUS PRODUCING GLANDS AND CILIA

Summary

I I5

T h e mucus-producing glands of Limax pseuiioflavirs can be considered in six groups: (a) T h e dorsal surface secretes a sulphated mucopolysaccharide mixed with protein. (b) T h e suprapedal gland secretes a neutral mucopolysaccharide. (c) The sole is capable of secreting sulphated and neutral mucopolysaccharides. (d) T h e leading edge of the foot secretes three different products; two neutral mucopolysaccharides

and a mucoprotein. (e) The epidermis around the pneumostome produces a sulphated and a carboxylated mucopolysac-

charide. Cells within the pneumostome and lung produce a weakly carboxylated mucopolysaccharide. (0 Semper’s organ (in the face) produces a variety of polysaccharides. T h e dorsal and pedal mucus form sheets, the former being stationary relative to the slug’s body,

Functional speculations are made based on the distribution of cell types and the known consistency the latter stationary relative t o the ground. These are separated by the products o f t h e peripodal groove.

of the mucus.

REFERENCES

Arcadi, J. A. (1963). Some mucus producing cells of the garden slug (Lehmania poirieri). Ann. N . Y. Acad. Sci.

Barr, R. A. (1926). Some observations on the pedal gland of Milau. Q. J l microsc. Sci. 70: 647-667. Barr, R. A. (1928). Some notes on the mucous and skin glands o f Arion ater. Q. J l microsc. Sci. 71: 503-525. Bonhag, P. F. (1955). Histochemical studies ofthe ovarian nurse tissue and oocytes ofthe Milkweed bug, Oncopelfus

Bousefield, J. D., Tait, A. I . , Thomas, J . D. & Towner-Jones, D. (1981). Behavioural studies on the nature of the

Burton, R. F. (1965). Relationships between the cation contents of slime and blood in the snail Heli-v aspersa L.

Campion, M. (1961). The structure and function ofthe cutaneous glands in Helix aspersa. Q. JI microsc. Sci. 102:

Chase, R. & Boulanger, C. M. (1978). Attraction of ths snail Achatina,firlicu to extracts of conspecific pedal glands.

Chetail, M. & Binot, D. (1967). Particularites histochemiques de la glande et de la sole pedieuses d’ilrion rufus

Chiffelle, T. J. & Putt, F. A. (1951). Polypropylene and ethylene glycol as solvents for Sudan IV and Sudan Black

Cook, A. (1977). Mucus trail following by the slug Li/nax grosstri Lupu. Anim. Behav. 22, 774-78 I . Cook, A. (1979). Homing by the slug Limax pseudqfluvus Evans. Anirn. Brhav. 27: 545-552. Cook, A. (1981). Huddling and the control o f water loss by the pulmonate slug Limaxpseudqflavus. Anim. Behav.

Dahl, L. K. (1952). A simple and sensitive method for calcium. Proc. Soc. exp. Bid. Med. 80: 474-479. Dalal, Y. M. & Pandya, G. T. (1976). A histochemical and structural study of the suprapedal gland o f the garden

Denny, M. (1980). Locomotion: the cost of gastropod crawling. Science, Wash. 208: 1288-1290. Denny, M. & Gosline. J . M. (1981). The physical properties of the pedal mucus of the terrestrial slug. .Ariolimax

Chose, K. C. (1963). The pedal gland of.4chatina.fulicu. J . Anim. Morph. Phy.sio1. 10: 80-82. Glenner. G. G. & Lillie, R. D. (1959). Observations o f the diazotization-coupling reaction for the histochemical

Gray, J. (1926). The properties ofan intercellular matrix and its relation to electrolytes. J . exp. Bid. 3: 167-187. Grenon, J-F. & Walker, G. (1978). The histology and histochemistry of the pedal glandular system of two limpets,

Grimstone, A. V. & Skaer, R. J. (1 972). A guidebook to microscupial methods. London: Cambridge University Press.

106: 451-457.

./asciatu~ (Dallas). I Cytology, nucleic acids and carbohydrates. J. Morph. 96: 38 1 4 3 9 .

stimuli responsible for triggering mucus trail tracking by Biomphalaria glabrata. Maluc. Rev. 14: 49-64.

Comp. Biochem. Ph-vsiol. 15: 339-345.

195-2 16.

Behav. Biol. 23: 107-1 1.

(Stylommatophora: Arionidae). Malacologia 5: 269-284.

B. Stain Tech. 26: 51-56.

29: 289-298.

slug, Laevicau1i.r alte (Ferrusac). J . Anim. Murph. Physiol. 23: 46-5 1.

columbianus. J . e.vp. Bid. 88: 375-395.

demonstration of tyrosine metal chelation and lormazan variant. J . Histochem. C.vtochem. 1: 4 16-422.

Patella vulgata and Acmaea tessulnta (Gastropoda: Prosobranchia). J . mar. hiol. Ass. U.K. 58: 803-8 16.

I I6 A. COOK AND R. SHIRBHATE

Hargens, A. R. & Shabica, S. V. (1973). Protection against lethal freezing temperature by mucus in an antarctic

Hunt, S. (1973). Fine structure ofthe secretory epithelium in the hypobranchial gland ofthe prosobranch gastropod

Kramer, H. & Windrum, G. M. (1955). The metachromatic staining reaction. J. Historhem. Cytochem, 3: 227-237. McFarland, I. D. (1980). Trail following and trail searching in the homing of the intertidal gastropod mollusc

Onchidium verraculatum. Mar. Behav. Physiol. 7: 95-108. Moreno, F. J.. Pinero, J., Hildago, J., Navas, P., Azion, J. & Lopez-Campos, J. L. (1982). Histochemical and ultra-

structural studies on the salivary glands of Helix aspersa (Mollusca). J. 2001.. Lund. 196: 343-354. Mowry, R. W. (1963). The special value of methods that colour both acidic and vicinal hydroxyl groups in the

histochemical study of mucins with revised direction of the colloidal iron stain, the use of Alcian Blue GBX in combination with the P.A.S. reaction. Ann. N. Y. Acad. Sci. 106: 402403 .

Newell, P. F. (1973). Etude de I’ultrastructure de I’epithelium dorsal et pedieux des Limaces Arion hortensis Ferrusac et Agriolimax reticulatus (Muller). Haliotis 3: 13 1-1 41.

Newell, P. F. (1977). The structure and enzyme histochemistry ofslug skin. Malacologin 16: 183-195. Paine, R. T. (1963). Food recognition and predation on opisthobranchs by Navunax inermis. Veliger 6: 1-9. Pearse, A. G. E. ( I 980). Histochemistrji: theoretical and applied. (4th ed.). London: Churchill Livingstone. Quick, H. E. ( 1 960). British slugs. (Pulmonata: Testacellidae, Arionidae, Limacidae). Bull. Br. Mus. (Nut. H1st.J

Ravetto, C. (1964). Alcian blue-Alcian yellow: A new method for the identification of different acidic groups. J .

Spicer, S. S. (1965). Diamine methods for differentiating mucosubstances histochemically. J . Historhem. 13:

Wagge, L. E. (195 I ) . The activity of amoebocytes of alkaline phosphatase during regeneration of the shell in the

Wilson, R. A. (1968). An investigation into the mucus produced by Lymnaea truncatula, the snail host of Fasciola

Wondrak, G. (1969). Electronenoptische Untersuchungen der Drusen-und Pigmentzellen aus der Korperdecke von

Zylstra, U. (1972). Histochemistry and ultrastructure ofthe epidermis and the subepidermal gland cells ofthe fresh-

limpet. Cryobiol. 10: 33 1-337.

mollusc, Buccinum undatum. J . mar. hiol. Ass. U.K. 53: 59-72.

ZOO^.) 6: 103-226.

Hisrochem. Cytochem. 12: 44-45.

2 I 1-234.

snail, Helix aspersa. Q. J l microsc. Sci. 92: 307-32 1.

heparica. Comp. Biochem. Physiol. 24: 629433 .

drion rufus (L.) (Pulmonata). 2. mikrosk.-anat. Forsch. 80: 1 7 4 0 .

water snails, Lymnaea scagnalis and Biomphalaria pfiifferi. 2. ZeNforsch. mikrosk. .4nat. 130: 93-1 34.

The mucus producing glands and the distribution of the cilia of the pulmonate slug Limax...

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