TISSUE AND CELL, 1994 26 (3) 421-429 0 1994 Longman Group Ltd.

DAVID J. PRIOR, TIM K. MAUGEL” and MARlLEE SELLERS

MORPHOLOGICAL CORRELATE OF REGIONAL PARTITIONING OF INTEGUMENTAL WATER ABSORPTION IN TERRESTRIAL SLUGS

Keywords: Skin, terrestrial slugs, foot surface, mollusc. water absorption

ABSTRACT. An ultrastructural study of the foot surface of the terrestrial mollusc, Limaw maximus, has revealed a correlation of epithelial cell type with the functional partitioning of the surface. The lateral absorptive bands of the foot are comprised exclusively of microvillar epithelial cells, while those of the medial locomotor band are all ciliated. Thus, there is a clear partitioning of epithelial cell types between areas of the foot surface with distinct functional roles.

Consistent with the proposed role for paracellular absorption, varying states of hvdration are shown to affect the extent of the intercellular spaces. but not the intracellular architecture.

Introduction

Due to their highly permeable integument, terrestrial gastropods can experience severe dehydration when exposed to drying con- ditions (Dainton, 1954 a, b; Machin, 1975; Prior et al., 1983). However, via contact- rehydration behaviour, they can rapidly rehydrate themselves by absorption of water through surface of the foot (Burton, 1966; Prior, 1982, 1984; Prior and Uglem, 1984).

In slugs, the process of contact-rehydration is mediated by a well-regulated behavioural sequence during which dehydrated slugs move unto a moist surface, assume a flat- tened posture and remain stationary while water is absorbed through the integument of the foot (Prior, 1984). Once rehydrated, they move off the moist surface thus terminating contact-rehydration. The rapid absorption of water during contact-rehydration (7.3 UL cm - ? min - ’ in Limax maximus; Prior, 1984) includes bulk flow of water and solutes

Physiology and Functional Morphology Group, Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011. USA.

* Department of Zoology, University of Maryland, Collcgc Park, MD 20742, USA.

Received 31 January 1994 Accepted 8 February 1994

through an epithelial paracellular pathway in the integument of the foot (Prior and Uglem, 1984; Uglem et al., 1985). Results from ultra- structural studies have likewise suggested the existence of a paracellular pathway in the foot epithelium. For example, Ryder and Bowen (1977) have reported that the extra- cellular marker, horseradish peroxidase, was found to have moved through the integument of the foot of Arion into the basolaterai region of the epithelial cell layer.

The ventral surface of the foot of terrestrial slugs is divided into three major longitudinal bands (Fig. lA, B). The rhythmic muscular contractions that power locomotion are pro- duced only in the medial band. Thus there is a discrete functional difference between the medial and the lateral bands of the foot. Recent observations by Uglem et al. (1985) have suggested that the rapid absorption of water during contact-rehydration may occur primarily through the lateral bands of the foot in Limax. In that study, dehydrated slugs were placed on filter paper saturated with a 1% solution of Blue Dextran (approxi- mate M.W. = 2 x 106). Because the para- cellular pathway acts as a sieve, excluding solute molecules larger than 10,000 Daltons (Uglem et al., 1985), it was expected that following contact-rehydration, Blue Dextran

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422 PRIOR ET AL.

Fig. 1. (A) is a scanning electron micrograph of the surface of the foot of Limax mnrimus. Note the three distinct longitudinal bands; lateral absorptive bands and a medial locomotor band that generates the waves of muscular contractions during locomotion. The regional pattern of the absorptive function of the foot is illustrated in (B). The ‘foot print’ a slug leaves when contact-rehydration occurs on filter paper moistened with a 1% solution of Blue Dextran. The large molecular weight dye is excluded during water absorption, leaving a pattern that shows absorptive activity to be partitioned to the lateral bands.

would be deposited on the pad below the slug. The Blue Dextran was only found

descriptions of the various types of epithelial cells, a correlation of cell structure with the

beneath the lateral bands of the foot (see Fig. 1B). This observation indicated that the

regional variation in function has not been

influx of water during contact-rehydration is made. The present ultrastructural obser- vations establish a functional correlation

primarily through the lateral bands. between the lateral and medial bands of the Although ultrastructural analyses of gas- foot of Limax maximus.

tropod foot integument have provided

MORPHOLOGICAL CORRELATE OF REGIONAL PARTITIONING 421

Methods and Materials

A laboratory culture of Limax maximus (Linnaeus, 1758) was maintained in vented refrigerator boxes lined with moist paper towels. Before use, slugs were fasted 5-7 days in high humidity conditions in order to establish a stable relationship between body weight and hydration (see Prior et al., 1983).

Tissue samples were obtained from slugs that were either fully hydrated (100% initial body weight, IBW). dehydrated to about 65% IBW or were in the process of contact- rehydration (postured on moist filter paper). Tissue samples used for transmission electron microscopy were fixed initially by placing them directly into 2% glutaraldehyde in 0.2 M sodium cacodylate buffer, pH 7.4, for 1 hr at 4°C. After three rinses with cacodylate buffer the samples were post-fixed in osmium tetroxide in cacodylate buffer at 7.4 pH, 4°C for l-2 hr. Further rinsing, and dehydration in a graded serios of ethanol, preceded embedding in Epon resin. Thin sections were cut and placed on uncoated 75/300 mesh copper grids. The sections were doubly stained in 2% aqueous uranyl acetate for 5 min followed by 0.1% lead citrate (Venable and Coggeshall, 1965) for l-2 min before examination with Zeiss 10 CA transmission electron microscope.

For scanning electron microscopy, slugs were relaxed with CO* for 5 min at 0°C and then immersed in 4% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.4) at room temperature. After 24 days in fixative at 4°C. surface mucus was removed by washing in cacodylate buffer (2) and sonication for l(!-20s. This was followed by soaking the specimens in 2 mM EGTA, during which excess mucus was teased away with fine probes. Further washing (10) with jets of 0.2 ,um filtered distilled water (FDW) and a second soaking (X2) in EGTA preceded final washing (X5) in FDW. The cleaned speci- mens were post-fixed for 90min in a 1% solution of osmium tetroxide in FDW (pH 7.4) at room temperature. After rinsing in FDW (X.5). the slugs were dehydrated

through an ethanol series (75-100%) before drying with liquid CO2 by the critical point method. The specimens were coated with gold/palladium alloy before examination in an AMRay 1OOOA scanning electron micro- scope.

Results

Examination of the ventral surface of the foot of Limax maximus by means of scanning electron microscopy (SEM) revealed distinct differences between the medial and lateral bands. The two lateral bands are clearly sep- arated from the medial band by two deep longitudinal grooves (Fig. 1A). The lateral bands are divided throughout their extent by a regular pattern of major horizontal grooves, while the infoldings of the medial band are irregular. When viewed at higher magnification, one can observe the large pores of mucous glands on the surface of both the medial and lateral bands (Fig. 2). The pores are from 1-8 pm in diameter and are especially obvious on the medial band because they are outlined by the dense cilia of the adjacent epithelial cells.

The entire surface of the medial band of the foot is composed of thickly ciliated epi- thelial ceils. The cilia in all regions of the medial band are approximately the same dimensions (5 pm length, 0.2 ,um diameter). The density of the cilia appeared to be the same throughout the medial band. In contrast, with the exception of the ‘lateral fringes’ (Fig. 3), the epithehal cells of the lateral bands were without cilia. The cells of the lateral bands were completely covered with microvilli. Amid these microvillous cells was an occasional tuft of 2-8 tightly grouped cilia (Fig. 2C.D). The most distinctive fea- ture of the lateral bands was the absence of ciliated epithelial cells (Fig. 2C,D).

Due to the absence of cilia on the cells of the lateral band, the contours of the apical surfaces of the individual cells could be easily visualized (Fig. 3C). Although irregularly shaped (possibly due to fixation), the apical

Fig. 2. Scanning electron micrographs show the surface of the medial band of the foot of Climax maximus, (A) final magnification = x28.50: (B) final magnification = x9600) and the surface of the lateral band (C) final magnification = X3230: (D) final magnification = xX640).

426 PRIOR ET AL.

Fig. 4. Transmission electron micrographs illustrate the structure of the epithelial cells in the lateral band ((A) x6000) and medial band ((B) x22,500) of the foot of Limax maximus.

surfaces of individual cells were approxi- a distinct transition from non-ciliated to mately 10 pm in diameter. The ciliated cells of the medial band were decorated with both

ciliated epithelial cells (Fig. 3B,C,D). In this

cilia and interspersed microvilli. transition region no partially ciliated cells were observed. Rather, at the border there

Examination of the outboard edge of the lateral band of the foot surface revealed

were fully ciliated cells directly adjacent to non-ciliated cells.

Fig. 3. The transition zone between the regions of ciliated and microvillar epithelial cell types of the medial and lateral bands of the foot of Limax maximus is illustrated: (A) = ~48; (B) = x340; (C) = x2240; (D) = x6000.

MORPHOLOGICAL CORRELATE OF REGIONAL PARTITIONING 427

Fig. 5. The effects of varying states of body hydration on the cellular architecture of the foot of Limar marimus are illustrated. (A) is an electron micrograph of tissue from a dehydrated animal (~4150); (B) is tissue from an animal fixed during contact-rehydration (x5100).

The surface morphology of the epithelial cells observed with SEM was compared with that observed in transmission electron micro- graphs of sections from both the lateral and medial bands. The epithelial cells in both bands were generally polygonal in cross- section with irregularly shaped nuclei, which were usually surrounded by clusters of mito- chondria (Fig. 4). The cells were separated by extensive intercellular spaces that were continuous with the hemocoel. Cells in both the lateral and medial bands were bound

together by elaborate junctional complexes (Fig. 4B). At the apical borders of the cells the lateral plasma membranes are thickened, forming a junction 04-0.8 ,nm in length with a typical belt desmosome configuration. Immediately beneath the region of des- mosome the adjacent plasma membranes appeared thickened and tightly opposed, having the striated appearance of typical invertebrate smooth septate junctions (Fig. 4B). The length of this junctional region varied considerably, but was usually from

428

2-6 ,um in tissue from hydrated slugs in which the intercellular spaces were distended (Figs. 4A, B).

Central to the septate junctional regions the extent of the intercellular spaces between adjacent cells was found to be highly variable. In normally hydrated tissue, there were numerous ‘blisters’ or ‘bubbles’ where the adjacent plasma membranes were sep- arated, thus creating obvious intercellular spaces (Figs. 4, 5). The extent of the inter- cellular spaces has been found to depend upon the hydration state of the slug from which the tissue was taken (see Prior and Uglem, 1984). In tissue from dehydrated slugs there is essentially no observable inter- cellular space (Fig. 5A), while in tissue from slugs in the process of contact-rehydration (Fig. 5B), the intercellular spaces were exten- sive. It is in such hydrated tissue that the structural role of the septate junction becomes especially apparent. Even though the intercellular spaces were distended, the junctions remained intact.

Variations in body hydration had no appar- ent effect on the density of the cytoplasm or distribution of organelles in the epithelial cells. At the ultrastructural level, it appeared that the only effect of hydration was on the extent of distention of the intercellular spaces.

PRIOR ET AL.

we find to be a thin boundary region between the major lateral bands of the foot and the dorsal body wall (Fig. 3).

It has been suggested that the lateral bands of the foot surface are the principle sites of integumental water absorption during con- tact-rehydration (Uglem et al., 1985; see Fig. 1). Consistent with this suggestion, we have observed that, except for the ‘lateral fringes’, the lateral bands are composed completely of microvillous epithelial cells that resemble those of absorptive surfaces in numerous other systems (see Phillips and Lewis, 1983). In contrast, the medial locomotor band is composed exclusively of ciliated epithelial cells. Thus the morphological differences between the epithelia of the lateral and medial bands are well correlated with func- tional partitioning.

It has been shown that during contact- rehydration there is a dramatic increase in epithelial permeability of the foot of Agri- olimax, Lehmania and Limax (Uglem et al., 1985; Ryder and Bowen, 1977; Prior, 1984; Prior and Uglem, 1984). Nevertheless, the only ultrastructural variation observed in the foot epithelium during contact-rehydration was the extent of the intercellular spaces (Fig. 5). During contact-rehydration there is extensive distension of the intercellular spaces, but not measurable change in the structure of the junctional regions or dis- tribution of cytoplasmic inclusions. These results are similar to those of Lord and DiBona (1976) who observed that osmotically driven water movement across the body wall of the planarian, Dugesia tugrina, caused distension of intercellular spaces but no change in the structure of the intercellular septate junctions.

The present results are consistent with the proposal that contact-rehydration in Limax is mediated by an increase in epithelial per- meability of the lateral bands of the foot epithelium. It appears that the foot surface is partitioned into a medial band specialized for locomotion and lateral bands that are specialized for absorptive functions.

Discussion

The present observations provide a description of the regional distribution of the cell types comprising the epithelium of the foot of Limax maximus. These observations demonstrate a morphological correlate of the regional variation in water absorptive func- tion proposed by Uglem et al. (1985). The basic epithelial cell types in the foot of Limux correspond with those seen in the mantle of the snail, Otala (Newell and Appleton, 1979) and the dorsal body wall and foot of the slugs, Arion hortensis and Agriolomax reticulatus (Newell, 1977; Ryder and Bowen, 1977). In the foot epithelium, as in dorsal skin, mucus secreting cells and mucous glands are inter- spersed among ciliated and non-ciliated microvillous cells. It appears that the ‘lateral bands’ of ciliated cells described by Newell (1977) correspond to the ‘lateral fringe’ that

Acknowledgement

This work was supported by the N.I.H. (2503RR3401).

MORPHOLOGJCAL CORRELATE OF REGIONAL PARTITIONING 419

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