Dorsal and ventral epithelium of the terrestrial slug, Incilaria fruhstorferi, is simple and consists of five cell types: microvillous, ciliated, round mucous, tubular mucous and channel. Microvillous cells were similar to human intestinal epithelial cells morphologically and functionally. At the base of microvilli, pinocytic vesicles which ultimately fused to form larger vacuoles, or multivesicular bodies were present. At the edge of tail or mouth, ciliated epithelial cells possessed the typical axonemes (9 plus 2 arrangement of microtubles). Mucous secretory cells were either tubular or round and their granules were membrane-bound and secreted by exocytosis. Granules of round mucous cells were proteinaceous but those of tubular cells were acidic mucopolysaccharides. Channel cells were elongate U-shaped and the central lumen was filled with a large amount of fluid (hemolymph). The function of channel cells is thought to remove hemolymph accumulated during hyperhydration. Our experiments of some markers-injection revealed that the fluid containing large molecules passed transcellularly from the hemolymph, across the basal or side region of the cell and into the central lumen. These results suggest that channel cell of the slug skin and vertebrate nephron showed some parallels in structure and function.
Although the body surface of the terrestrial slug is covered with only simple columnar epithelial cells, the surface is prevented from evaporation and is protected from mechanical injuries. The reason is that the surface skin of the slug is covered with a large amount of mucus which is a characteristic secretion. Moreover, in spite of this fragile structure and moist conditions of cutaneous tissue, fairly resistant to invading microorganisms.
Many molluscs secrete a variety of substances which may have protective function such as skin pigments, lectins, opsonins, lysin etc. (Fountain and Campbell, 1984; Fountain, 1985; Iguchi et al., 1982, 1985; Kubota et al., 1985; Tripp, 1992; Kisugi et al., 1992a, b; Furuta et al., 1995). According to Furuta et al. (1995), the water soluble fraction of body surface mucus from Incilaria fruhstorferi plays an important role in the slug's immunodefense system. This fraction exerts opsonic and agglutinating activity for non-self materials, recognizing N-acetyl galactosamine (GalNAc). Moreover, Simkiss and Wilbur (1977) found that a range of large molecules such as beef hemoglobin (MW 68 Kd) passed out from the hemocoel onto the surface of the skin when it was injected into Helix aspersa. And we often observed that soon after injecting carbon particles into slugs, the particles appeared onto the skin's surface. According to Deyrup-Olsen and Martin (1982), the slug, Ariolimax columbianus could also exude large quantities of blood when stimulated physically, electrically, or by the intravascular injection of hypotonic fluids.
In order to elucidate internal defense system of molluscs, however, it is necessary to establish the structure of the terrestrial molluscan epidermis which plays a role as a barrier facilitating internal homeostasis and preventing the entry of pathogens from the environment into the body. The present paper provides histochemical and ultrastructural descriptions of the epidermal cells in the skin of the land slug, I. fruhstorferi.
MATERIALS AND METHODS
The largest slug in Japan, I. fruhstorferi, weighing 4.5–10 g ±1.0 g was maintained in the laboratory at room temperature for at least a month before use and fed on sweet potato, lettuce or mushroom.
Injection of several markers:
To estimate the capacity of surface epithelium to transfer particles (carbon, fluosphare, yeast) or large molecules (FITC-dextran, Keyhole Limpet hemocyanin) from the hemocoelic space, these materials were injected into the hemocoel of slugs. They served as markers for particle or molecule sieving in fluorescence or electron micrographs.
The carbon (Pelican ink) and yeast particles were washed 5 times with 10 volumes of PBS (pH 7.4). After washing, they were diluted with same buffer. The fluosphares (red latex beads, 0.03 and 5.0 μm in diameter, Molecular Probes. Inc. USA) were directly diluted with 20 mM PBS (pH 7.4) and the FITC-dextran (MW 70 Kd, Molecular Probe. Inc. USA) and hemocyanin (BGN Co. Ltd. USA) were diluted with the same buffer.
Each solution or suspension was administered to each slug by injection into the hemocoel of 400 μg/g body weight in 20 mM PBS (pH 7.4). Each slug was then kept in a glass dish, for 3, 20 and 24 hr at room temperature.
Immunohistochemistry using anti-hemocyanin monoclonal antibody.
After excising the dorsal and ventral skins, samples were fixed in phosphate-buffered 10% formalin for 4 days, then dehydrated in a graded series of ethanols, embedded in paraffin, sectioned at 5 μm and mounted on precleaned glass slides. The following procedures were performed at room temperature.
Slides were deparaffinized and redehydrated. For immunohistochemical detection of hemocyanin pathway in the slug skin, specimens were treated with 3% H2O2 in distilled water (DW) for 15 min, then 2N HCl for 2 hr, and were then soaked in 20 mM phosphate buffer (pH 7.4) containing 110 mM NaCl (PBS). After treatment with 10% normal rabbit serum in PBS for 10 min, they were then incubated with primary antibodies, i.e. goat anti-hemocyanin (from Keyhole Limpet) monoclonal antibody (BGN Co. Ltd. USA, dilution of 1:200) in 50 mM tris buffer (pH 7.2) for 60 min. After rinsing with PBS, they were treated with biotinylated rabbit anti-goat IgG (Nichirei kit, Nichirei Co. Ltd. Tokyo, Japan) for 30 min. They were then rinsed with PBS and treated with streptavidin-horse radish peroxidase conjugates (Nichirei kit) for 30 min. After rinsing with PBS, sections were immersed for 10 min in diaminobenzidine containing H2O2 (Nichirei kit). As a control experiment, sections were treated with normal rabbit serum instead of anti-hemocyanin monoclonal antibody. No control sections showed any signal.
Several histochemical procedures were performed to reveal carbohydrates. Acidic mucosubstances were demonstrated by alcian blue (pH 2.5)(Bonet and Huguet, 1985) or by toluidine blue, which stains the acidic mucus metachromatically (Gabe, 1968). Moreover, we examined the staining pattern of the epidermis (dorsal and ventral sides) after incubation with seven lectins. The following procedures were performed at room temperature. After deparaffinization, specimens were treated with 0.3% H2O2 in DW for 15 min to block the endogenous peroxidase activity and rinsed with PBS (pH 7.4). They were treated with 0.1% normal bovine serum albumin (BSA) in 0.1 M Hepes buffer (HB, pH 7.5) for 30 min and rinsed again with HB. Sections were incubated with one of the following seven biotinylated lectins (25 μg/ml) in 0.1% BSA-HB for 30 min, purchased from Vector Laboratories, USA: concanavalin A (Con A), Dolishos biflorus agglutinin (DBA), peanut agglutinin (PNA), Ricinus communis agglutinin (RCA), soybean agglutinin (SBA), Ulex europeus agglutinin (UEA-1) and wheat germ agglutinin (WGA). After incubation with lectin, the sections were rinsed with HB and then incubated with avidin-biotin-peroxidase complex (ABC kit, Vector) in 0.1% BSA-HB for 30 min. After rinsing with HB, the sections were reacted with a mixture of 0.05% 3,3′-diaminobenzidine-4HCl (Wako, Tokyo, Japan) and 0.01% H2O2 in 0.05M Tris buffer (pH 7.2) for 5 min. They were rinsed with DW, dehydrated and mounted with Entellan New (Merck, Darmstadt, Germany). The staining intensities were scored as - negative staining, ± weak staining and + positive staining. As a control procedure, to confirm the specificity of the histochemical reactions, several sections were preincubated with appropriate hapten sugars and then incubated with biotinylated lectins in the presence of hapten sugars. Sugars used were; 0.2 M α-metyl-D-mannosid (Sigma, St. Louis, USA) for Con A, 0.2 M N-acetyl-D-galactosamine (Aldrich, Wisconsin, USA) for DAB and SBA, 0.1 M D-galactose (Wako) for PNA, 0.2 M lactose (Wako) for RCA-1, 0.1 M L-fucose (Aldrich) for UEA-1, and 0.2M N-acetyl-D-glucosamine (Wako) for WGA. Nuclei were counterstained with hematoxylin
For transmission electron microscopy, the skin from dorsal or ventral surface was fixed for 1.5 hr in 1.6% paraformaldehyde - 3% glutaraldehyde in 0.1M cacodylate buffer (pH 7.4). Fixed tissues were rinsed with the same buffer (pH 7.4), postfixed with 1% OsO4 for 1.5 hr, dehydrated, and embedded in Epon 812 resin. Ultrathin sections were obtained with a Super Nova ultramicrotome (Reichert-Jung, Austria), double stained with uranyl acetate and lead citrate, and examined in a JEM-1210 electron microscope (JEOL, Tokyo Japan). Semithin sections, approximately 1–2 μm thick, stained with an alkaline solution of toluidine blue, were also stained for light microscopy. For scanning electron microscopy, the double fixed tissues were dehydrated, critical point dried, sputter-coated with Au-Pd and examined with a S-650 scanning electron microscope (Hitachi, Tokyo Japan).
Epidermal cells of the land slug, I. fruhstorferi, depending on the region of the body, vary from cuboidal to columnar in form. The cells between dorsal and ventral skin, i.e. at the edge of tail or mouth, are cuboidal. The external surface of the epidermis was composed of a simple microvillous cell layer which held in place the covering of mucus. The cell layer is supported by a mat of connective tissue through which muscle fibers ran. As shown in Fig. 1, five main types of cells were distinguished: (1) microvillous, (2) ciliated (see Fig. 4), (3) round mucous, (4) tubular mucous and (5) channel.
(1) Microvillous cells:
These cells ranged from 14–21 μm in height (Figs. 1C, 2) and occasionally protrusions were observed at the tips of the microvilli. The epithelia were confused with the numerous intercellular spaces which were very large and often extended (Fig. 2). The epidermal cells closely clung to each other by zonula adherens (175–225 Å) in the apical region of cells. The zonula adherens is the most prominent in cells with a well-developed brush border. Below the microvilli of the border, the apical cytoplasm is traversed at the level of the zonula adherens by a mat of interwoven filaments the so-called terminal web (Fawcett, 1981). The 17 to 18 nm space between parallel membranes of adjacent cells was filled with continuous layer of dense materials. Below this zonula the membrane interdigitations were present (Fig. 2). The microvillous epidermal cells often possessed fine filaments (actin filaments)(Fig. 2C) which ran toward the tip of the microvilli, these filaments arose from the “terminal web” that was devoid of cell organelles. The terminal web was found besides several fibers and vesicles, these components were similar to core filaments of brush border associated with human intestinal epithelia. Irregularly shaped mitochondria were gathered chiefly in the cell's apical region (Fig. 2). The mitochondria usually had an oval to slightly oblong form; the long axis ran parallel to the length of the cell (Fig. 2). Dense bodies, multivesicular bodies (MB) and several large and small vesicles were interspersed among the mitochondria. The dense bodies which were considered as lysosomes were about 0.5–1.0 μm in diameter. The multivesicular bodies usually contained small vesicles of similar size. Golgi bodies were rarely observed in microvillous cell (Fig. 2B). The endoplasmic reticulum was mainly smooth endoplasmic reticulum (SER), and numerous free ribosomes were observed throughout the cytoplasm (Fig. 2B).
Microfilaments frequently occurred in bundles, especially along the sides of the nucleus and usually ran parallel with the cell axis (Fig. 2C). They extended from the interior of the cell into microvilli. At the base of microvilli, pinocytic vesicles (PV) and some multivesicular bodies (MB) which contained small vesicles were often observed (Fig. 2). The basement membrane appeared to have occasional indentations or thrown into basal folds, and at places broad (Fig. 3).
(2) Ciliated cells:
At the edge of tail or mouth, epidermal cells were ciliated. The cilia possessed very long striated roots, up to 2–9 μm in length and the typical axonemes (9 plus 2 arrangement of microtubles) (Fig. 4). Cell connection of ciliated cells was similar to that of microvillous cells. The cytoplasm was closely packed with many mitochondria and interspersed with glycogen-like deposits.
The presence of mucous glands is well established in molluscs, and the cells typically possess a cell body located in the subepidermal connective tissue and a secretory process extending to the surface of the epidermis.
(3) Round mucous cells:
The round mucous cells were more abundant on the ventral surface than on the dorsal surface (Table 1). They were located directly underneath the epidermal cells and opened onto the surface with the short neck. In our regenerating experiments, the cell body moves more deeply into the dermis as the neck grows (Yamaguchi et al., 1999). The cell was globose, measuring about 10–16 μm in diameter (Fig. 5A) and its neck was anchored to adjacent columnar epithelial cells by means of a junctional complex (Fig. 1C). The oval nucleus located at the center of the cell, measuring about 3.6–5.8 μm in diameter. Membrane-bound secretory granules were present around the nucleus and the cell body became progressively distended with secretory granules becoming more prominent. Chromatins showed heterochromatic granules and a nucleolus was present. Golgi bodies were stacked and consisted of seven to nine lamellae (Fig. 5B). The cytoplasm was rich in rough endoplasmic reticulum (RER). Few mitochondria laid among rich RER. Basement membrane which was found outside the body and the neck of the round mucous cells ran the adjoining columnar epithelial cells. Fine muscle fibers were also observed around the cell body.
Distribution and differences of histological observations of skin mucous cells from land slug, Incilaria fruhstorferi. Detection of mucous mucopolysaccarides was performed by the reaction of 7 kinds of biotinylated lectins.
(4) Tubular mucous cells:
Tubular cells possessed a cell body located in the musculature and a long secretory process extending to the surface of the epidermis (Fig. 5C). The nucleus laid at the base of the cell where Golgi bodies and RER existed along with most of the cytoplasm. And the cell possessed a distal region which extended into the long secretory process packed with membrane-bound mucous products. These mucous granules were secreted by exocytosis (Fig. 5C). Golgi bodies were prominent and consisted of seven to nine lamellae. The cytoplasm contained numerous free ribosomes.
(5) Channel cells:
The channel cell was the large cell which was lightly stained and usually homogeneous microscopically (Fig. 6). The nucleus was also large and laid in a thin mass of cytoplasm at the basal portion of the cell. The cytoplasmic envelop was very thin except in the region of the nucleus, which contained RER, a few mitochondria, many free ribosomes, Golgi bodies and various pinocytic vesicle-like vesicles (Fig. 6B).
As illustrated in Fig. 7, the basement membrane surrounded the basal and lateral side of the cell, and very thin smooth muscles and pigment cells also surrounded the cell. The shape of the cell was cylindrical, and long U-shaped cytoplasm.
Histochemical characteristics of three secretory cells of the epidermis.
(1) Round mucous cells:
Granules in these cells were negative for acidic mucopolysaccharide and slightly positive for proteins. Lectin immunohistochemistry showed that WGA positively (Fig. 8) and SBA slightly stained the granules of round mucous cell (Table 1).
(2) Tubular mucous cells:
The staining reaction of the secretion was strongly positive with alcian blue (pH 2.5) showing the granules of acidic mucopolysaccharide and toluidine blue staining as metachromatic. Lectin immunochemistry showed that only Ricinus communis agglutinin (RCA) slightly stained the granules of the tubular mucous cells (Table 1, Fig. 8).
(3) Channel cells:
Three hours after injection of carbon particles, fluoshares (red, 0.03 μm in diameter), or FITC-dextran (MW 70Kd), these cell markers were through out the body wall (Table 2). Fig. 6C shows the basal region of the channel cell which took up the particles by pinocytosis and then released to the lumen of U-shaped cells by exocytosis. At this time, the structure of junctional complexes did not show any changes. The staining reaction of anti-hemocyanin monoclonal antibody was positive in the lumen (Fig. 9).
Estimation of the capacity of surface epithelium to transfer large molecules and particles from the hemocoelic space.
The epidermis of terrestrial slugs function not only as an integument, but it also plays a role in osmoregulation, respiration, locomotion and host defense (Furuta et al., 1995; Yuasa et al., 1998) where is covered with secreted mucus from mucous secretory cells. The external surface of the epidermis was composed of microvillous layer which increased the dorsal surface area, and most of the exposed surface consisted of microvillous-bearing epidermal cells and the mucus producing glands which consisted of simple goblet cells forming a secretion that stained for either protein or acid polysaccharide components. The epidermal cells possessed microfilaments (Fig. 3) which might function as a cytoskeleton and a contractive structure of the cells. The slug body usually could be stretched until its length is increased about 2–3 times. In Lymnaea stagnalis, Zylstra (1972) reported that the epidermal cells lining the mouth -they were submitted to a great deal of wear in the eating processcontained numerous microfilaments, and that the microfilaments appeared to function as a cytoskeleton providing a more firm matrix to resist wear by giving strength to the cells.
In the leech which possesses mucus-rich skin, two types of mucous cells are found between epidermal cells (Molinas and Huguet, 1993) and one type (more common type in the leech skin) is involved in mucus formation by their secretion of mucosubstances slightly positive for carboxylated mucopolysaccharides and for proteins. The other cell type occurs in small numbers and supplies a carboxylated mucopolysac-charide component together with neutral and sulfated fractions. However, in molluscs many investigators reported various types of mucous gland cells, for example, in L. stagnalis; Zylstra (1972a, b) described 13 different types of mucous cells, in H. aspersa: 8 types of unicellular glands (Campion, 1961), and in Pattela vulgata: 9 types of glands on the foot alone (Grenon and Walker 1978).
We reported here the existence of two major types of secretory cells. These two cells were similar to the leech-skin's secretory cells, but were different from the secretory glands and/or cells of other molluscs. In a study on nudibranchia, Onchidorus muricata, Skidmore and Rivera (1982) concluded that “the confusing variety of secretory cell types were distributed over the gastropod body and foot”. Functionally considered, the molluscan mucus contains lectins (Fountain, 1985; Fountain and Campbell, 1984; Iguchi et al., 1985; Kubota et al., 1985; Furuta et al., 1995; Yuasa et al., 1998) and antibacterial components (Iguchi et al., 1982). The water soluble fraction of the slug mucus also inhibited growth of Gram positive bacteria and fungus (in preparing for publication). The microvillous epithelial cells possessed numerous large vesicles and microvesicles (Fig. 5). This phenomenon is often probable to show endocytotic activity. Indeed, Bevelander and Nakamura (1966) reported that the endocytosed materials by mantle epithelial cells were fused with lysosomes and were thereby digested. Especially, in terrestrial molluscs, Agriolimax reticulatus, endocytosis and the associated lysosomal activity occurred on the foot epithelium (Ryder and Bowen, 1977). Multivesicular bodies were also present approximately 0.4–0.5 μm in diameter. They were enclosed by single membrane and contained small vesicles (Fig. 5). We observed the most intriguing cells which existed among columnar epithelial cells. They were lightly stained and rather homogeneous in appearance. Under electron microscope the cell was elongate Ushaped and its central lumen was filled with fluid, and surrounded by a thin layer of cytoplasm and with a large basal nucleus. Moreover, it was additionally intriguing to observe the traversal of very large molecules such as hemocyanin, dextran (MW 70Kd), carbon and latex beads (0.03 μm in diameter) 30 min after injection. Luchtel et al. (1984) described these cells as “channel cells” and suggested that fluid passed in transcellular tubules from the blood, across the cell's basal region, and into the central lumen from which the fluid might be passed onto the apical surface. Our observations supported the suggestion of Luchtel et al..
By this means the U-shaped cells (channel cells) are thought to remove (release) hemolymph accumulated during hyperhydration. Indeed, Martin and Deyrup-Olsen (1986) described the function of channel cells. Channel cells of slug's skin and the vertebrate nephron show some intriguing parallels in structure and function, and in each, a primary fluid was formed by ultrafiltration through an epithelial basement membrane (Fujita and Fujita, 1992). Moreover, they reported that slugs could exert neurohormonal control over its channel cells.
In recent years, it is said that immune system is closely associated with neuroendocrinological system, aptly termed neuroendocrinoimmunology. Further studies should be in progress to elucidate these relationships in the near future.
We thank Professor Edwin L. Cooper, Department of Neurobiology, UCLA Medical Center, for kind revision of the manuscript.