495 Microvilli on the external surfaces of gastropod tentacles and body-walls By NANCY J. LANE (From the Cytological Laboratory, Department of Zoology, University Museum, Oxford) With 2 plates (figs, i and 2) Summary In Helix aspersa the 'cuticle' on the free surface of the external epithelial cells of the optic tentacles has been shown to consist of a layer of microvilli. Microvilli are also present in the same species on the free cell borders of the body-wall, and in the slug Arion hortensis, on the outer cell surfaces of the external epithelium. In all three cases the microvilli are arranged in a hexagonal pattern. There are indications that branching may possibly occur. The microvilli have granular cores with cross- and longitudinal-striations and there are fibrillar connexions between their tips. On the tentacular and body surfaces of H. aspersa, the microvilli increase the surface area 15 and 12 times, respectively. On A. hortensis the increase in surface area is only 4 times. In H. aspersa, beneath the microvilli on the tips of the optic tentacles there is a layer, about 3 to 4 ix deep, composed of vertical, horizontal, and tangential fibres. Some of these fibres are attached to lamellar bodies, which may have a lipid content. Granules are also found among the fibres. Further, a greater depth of cuticle is found to be present on the tips of the inferior tentacles of H. aspersa than on their sides; this seems to indicate that a fibrillar layer, similar to that on the optic tentacles, may lie beneath the cuticle of microvilli on the tips of the inferior tentacles. A thicker cuticle is also found on the tips of the optic tentacles in other stylommatophoran pulmonates. It has not been found possible to ascertain whether the fibrillar layer is intracellular or extracellular, although the evidence points to the latter. Histochemical tests indicate that mucopolysaccharide is present on the surface of the cuticle. Electron micrographs show a granular precipitate caught on and between the fibrillae connecting the tips of the microvilli. It is suggested that the function of the microvilli is to hold the mucous secretions on the body-surface, which would give protection to the animals. Introduction THE free epithelial surfaces of many of the internal organs of vertebrates have been shown by electron microscopical studies to be covered with a large number of minute projections which have been termed microvilli (Dalton, 1953). Similarly, the surface membrane of various types of eggs, both vertebrate and invertebrate, has been shown to consist partially of microvilli (Rebhun, 1962). Within the phylum Mollusca, workers investigating the digestive tract (Fawcett and Porter, 1954; Lacey, 1957), the mantle (Kawaguti and Ikemoto, 1962 a, b; Emberton, 1962), the gills (Fawcett and Porter, 1954; Afzelius, 1960), and the adhesive epithelium (Hubendick, 1958) of certain lamellibranchs and gastropods, have described microvilli on the free borders [Quart. J. micr. Sci., Vol. 104, pt. 4, pp. 495-504, 1963.] 496 Lane—Microvilli on the external surfaces of gastropods of the cells. A similar structure has not previously been reported on other parts of the body, as far as is known. The first of the present observations was made during an investigation into the ultrastructure of certain neurosecretory cells which have been described in the tentacles of stylommatophoran gastropods (Lane, 1962a). These bipolar 'collar' cells have long processes, one of which terminates in the tentacular ganglion, and the other in the epithelium at the tips of the tentacles. An examination of this peripheral region of the tentacles was made in an attempt to obtain further details of the general area in which the neurosecre- tory cells terminated. During the course of these investigations, microvilli were observed on the outer cell surfaces of the optic tentacles of the snail, Helix aspersa, and further studies were carried out in order to determine whether they extended over other parts of the body-surface. Also, an examina- tion of the free surface of the body-wall of the slug, Arion hortensis, was made, in order to find out if the occurrence of microvilli was a characteristic common to other gastropod body-surfaces. Slugs were considered to be of particular interest because they are unprotected by any external shell. Histochemical tests were carried out on light-microscopical preparations in an attempt to determine the chemical contents both of granules found between and beneath the microvilli, and of the cuticle itself. Light-micro- scopical examinations were made of the inferior tentacles of H. aspersa, and of the optic tentacles of other stylommatophoran gastropods in order to study the depth of the cuticle in different parts of the tentacles. Material and methods The main source of material used in the present investigation was the garden snail, H. aspersa Miiller. The external surfaces of the optic tentacles were examined as well as pieces of the dorsal surface of the body-wall in front of the mantle. Tissue from a similar area of the dorsal surface of the slug, A. hortensis Ferussac, was also examined. The tissues were first fixed for electron microscopy in Palade's buffered osmium at pH 7-4 (Palade, 1952), and then dehydrated and embedded in vestopal or araldite. In the case of the former medium, the material was left in phosphotungstic acid for 1 h. With the araldite-embedded tissue, final dehydration was accomplished by propylene oxide as prescribed by Luft (1961). Sections were cut on a Huxley ultramicrotome, and those showing a silver interference colour were mounted on carbon-coated, formvar-filmed grids. With araldite sections, contrast was intensified by staining for 10 to 20 min in potassium permanganate solution (Lawn, i960). The material was examined in an Akashi electron microscope (model TRS 50 El), operated at 50 kV. Photographs were taken on Uford N 50 or special lantern contrasty plates, and developed in 'universal' P.Q. developer (Ilford). Certain histochemical tests were applied to light-microscopical preparations of the optic tentacles of H. aspersa. The periodic acid / Schiff (PAS) test (McManus, 1948) for polysaccharides was performed after fixation in Zenker's Lane—Microvilli on the external surfaces of gastropods 497 fluid, embedding in paraffin, and sectioning. After fixation in formaldehyde/ calcium, post-chroming, and embedding in gelatine (Baker, 1944), sections were cut and either treated with the acid haematein (AH) test (Baker, 1946) for phospholipids, or coloured with Sudan black B. Optic tentacles were also fixed in Mann's mercuric-osmium solution and then post-osmicated (Weigl, 1910). A few araldite sections were cut at 0-25 //, on the Huxley ultramicrotome, stained in a 1% solution of toluidine blue in 1% borax, differentiated in 50% ethanol, dried, and mounted in liquid paraffin for examination (Meek, 1962). Paraffin sections of the optic tentacles of other stylommatophoran pul- monates were studied by means of the light microscope in order to make comparisons of the thickness of the cuticular border in different parts of the tentacles. The animals examined were the snails Cepaea nemoralis (Linnaeus), and H. pomatia Linnaeus, and the slugs Milax budapestensis (Hazay), A. hortensis, A. ater (Linnaeus), Agriolimax reticulatus (Miiller), and Limax maximus Linnaeus. The inferior tentacles of H. aspersa were examined for thickness of cuticle to compare with that in the optic tentacles. Observations Electron microscopy Microvilli are present on the free epithelial surface of the optic ten- tacles of H. aspersa (fig. 1, B, C). The microvilli are arranged in parallel rows; they project at right angles, or nearly at right angles, to the surface of the epithelium. Cross-sections show the microvilli to be organized in a hexa- gonal pattern. They are of nearly constant length throughout the area over which they are distributed, and, although they generally appear to be un- branched, there are indications that branching sometimes occurs near the base. The profiles of the microvilli are fairly regular, with only shallow indenta- tions and protuberances; thus their diameter is nearly constant throughout their length, except at the tips, which may be swollen to a club-like form. The core of each microvillus is granular and contains electron-dense cross- striations. The core of the tips and bases of the microvilli tends to be more electron-dense than the remainder, but this is a variable feature. Sometimes longitudinal fibrils can be seen in the core. Nearly all the club-like tips of the microvilli are connected by fibrillae. These fibrillae have a diameter of about 6 m/x or less, and a granular substance is caught in the network they form. Other similar fibrillae project from the sides of the microvilli. The measurements made of the dimensions of the microvilli are recorded in table 1. Calculations were made of the average surface-area of one microvillus. From this figure, and from the average number of microvilli per square micron, the increase in surface-area, produced by the presence of the micro- villi, was determined. These results are also recorded in table 1. 498 Lane—Microvilli on the external surfaces of gastropods TABLE I A summary of the dimensions of the microvilli in H. aspersa and A. hortensis, and calculations of their surface-area Approxi- Total mate surface- increase Average area of all in surface- Average Average Surface- number of microvilli area due Part of body length of diameter of area of 1 microvilli in 1 square to the being microvilli microvilli microvillus per square micron, presence of Animal examined iny. iny iny* micron in y? microvilli H. aspersa Surface of 0-70 0-105 0-231 65 15-02 15 the tips of the optic tentacles Dorsal 0-077 0327 38 12-43 12 surface A. hortensis Dorsal 0-42 0097 0129 305 3-92 4 surface The microvilli terminate below in a layer of cytoplasm that has an unusual, fibrillar structure (figs.