Microvilli on the External Surfaces of Gastropod Tentacles and Body-Walls by NANCY J. LANE

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 neurosecretory 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 examination 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-microscopical 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 pulmonates 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 tentacles 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 hexagonal 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 indentations 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 microvilli, 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. 1, B, c; 2, B). This is about 3 or 4/1 thick, and is situated just above the normal apical cytoplasm of the epithelium. Fibres, about 70 mju. thick, run vertically upwards from the apical cytoplasm towards the microvilli. Other fibres run in the same direction from the fibrillar layer into the bases of the microvilli. It was not found possible to ascertain whether the fibres are continuous from the cytoplasm up through the fibrillar layer into the microvilli. Still other fibres, measuring about 50 mju. in diameter, run horizontally or tangentially through the fibrillar layer. The fibres are either tightly (fig. 1, B, c) or loosely (fig. 2, B) packed together. Granules, measuring from 50 to 70 m/x in diameter, are scattered between these fibrous elements. Some of the apparent granules are probably cross-sections of fibres. Amid the fibres and granules which compose the fibrillar layer beneath the microvilli, lamellar bodies were also observed. These are in the form of

FIG. 1 (plate), A, cross-section through the microvilli (mv) on an external epithelial cell of the dorsal surface of H. aspersa. Note the nucleus (n) of the cell, the gland cell (gc) which runs by it to open on the surface, the vacuolated granular cytoplasm (vgc) beneath the microvilli (mv), and the fibrillarconnexion s (fc) between their tips. The arrow indicates where the microvilli are lifting off the epithelial surface, either as a natural process or through artificial damage. B, longitudinal section through the microvilli (mv) on the surface of the tips of the optic tentacles of H. aspersa, and through the fibrillarlaye r (//) that lies under the microvilli. Note the fibres (fb) running vertically, tangentially, and horizontally to the apical cytoplasm (c), and the apparent granules (g), some of which are cross-sections of fibres. Note also the lamellar body (Ib) within the fibrillar layer and the lacuna (I) in the apical cytoplasm. C, longitudinal section through the microvilli (mv) and fibrillar layers (//) on the tip of the optic tentacle of H. aspersa. The tentacular epithelium often lies in furrows, and hence two layers of microvilli are seen in apposition. Note the fibres (fb) and granules (g) packed closely together in the fibrillar layers beneath the microvilli.

Lane—Microvilli on the external surfaces of gastropods 499 spheroids or ellipsoids. Some of them seem to have an attachment at one side to a fibre (fig. 2, B). The diameter of these lamellar bodies ranges from 0-4 to 1-i fi. They are composed of concentrically arranged lamellae, varying in number from a few to 10 or more. Each lamella averages 14 m/i in thickness. These lamellar bodies are situated in the fibrillar layer anywhere from close by the cytoplasm to the region just beneath the microvilli. In some cases the bodies are partially embedded in the cytoplasm and connected to it by fibrous strands (fig. 2, B). It is uncertain whether the fibrillar layer is intracellular or extracellular. No connexions between the bases of the microvilli can be seen in the micrographs of the tips of the tentacles, and sometimes the microvilli can be seen to extend some distance into the fibrillar layer beneath (fig. 1, c). This would suggest that the fibres in the fibrillar layer may be extensions or strands of the apical cytoplasm, which in turn form the bases of the microvilli. If so, then the microvilli on the tentacular tips are distinct from the microvilli that arise directly from the apical cytoplasm, as do those on the dorsal surfaces of the snail and slug, as well as those described by other investigators. It is note- worthy, however, that the microvilli on the tentacular tips are arrayed in precisely parallel rows, and are of constant length, which would be a remark- able occurrence if they in fact arose from loose fibres present in an extracellular layer. The apical cytoplasm, beneath the fibrillar layer, contains granules which range in diameter from 0-18 to 1-2 /x. Vacuoles within the same size-range are also present; and sometimes irregularly shaped lacunae, up to several microns in length, lie near the junction of the fibrillar layer with the apical cytoplasm (fig. 1, B). The lateral cell membranes of the epithelial cells are very wavy, producing interdigitations of one cell into the next. Such junctions link these superficial cells closely together. The dorsal surface of the outer body-wall of H. aspersa has microvilli on the free border of the epithelial cells (fig. 1, A). These are very similar to those found on the optic tentacles, the description of which, with certain exceptions, would also apply to them. The tips and bases of the microvilli on the dorsal surface of the body are not as electron-dense as those on the optic tentacles. The longitudinal striations

FIG. 2 (plate). A, longitudinal section through the microvilli (vw) on the external dorsal surface of the slug, A. hortensis. Note the fibrils (/) and granules (gr) lying between adjacent microvilli, and rather similar granules (gr) in the cytoplasm below; note also other larger granules (g) and vacuoles (v) in the apical cytoplasm, as well as mitochondria (m). Longitu- dinal fibrils (//) can be seen within the cores of the microvilli. B, section through the fibrillar layer beneath the microvilli on the tip of the optic tentacle of H. aspersa. The section is at right angles to the plane of the surface of the cell. Note the lamellar bodies {lb) that have different diameters and different numbers of component lamellae; note also the attachments (a) of the fibres (/) to the lamellar bodies. Strands or fibres of cytoplasm (cf) can be seen coming from the cytoplasm (c) and running into fibres or lamellar bodies. Fibres (fm) can also be seen running vertically towards the bases of the microvilli. 500 Lane—Microvilli on the external surfaces of gastropods that lie within the core are sometimes so arranged, parallel to the outer membrane, that it looked as if the striation, together with the membrane, formed a double membrane, measuring about 12 m/x across. Table 1 contains a record of the dimensions of these microvilli as well as the results of calculations of their individual surface-area and total increase in surface-area per square micron. It will be seen that the microvilli on the dorsal surface are longer than those on the tentacles, but less closely packed. The outer membrane around the microvilli on the dorsal surface is a con- tinuation of the cell membrane which connects the bases of adjacent microvilli. The membrane is about 7 or 8 m/x thick. It appears tripartite, consisting of an electron-dense layer on each side of a non-electron-dense layer. The two outer layers are approximately 2 m/x thick, and the central one 4 m^x. The microvilli on the dorsal body-surface terminate in normal cytoplasm: no fibrillar layer is evident. There are a few indentations at the bases of the microvilli, and granules and vacuoles are present in the apical cytoplasm. These vacuoles range from 0-2 to 1-5 /J, in diameter. There is no definite evidence that the microvillous border undergoes a cyclic disappearance and reformation. Any indications of this (fig. 1, A) may well have been artifacts due to fixation, embedding, or sectioning. Microvilli are also present on the free surface of the external body-wall of A. hortensis (fig. 2, A). These are similar to those described on the tentacular surface of H. aspersa, with the following exceptions. The length of the microvilli is not always constant over a particular surface- area. However, it is possible that this inconstancy is due to the angle at which the sections were cut. There are indications that the microvilli branch, for sometimes a single basal projection bifurcates into two. The diameter of these microvilli is not constant throughout their length, for sometimes the tips expand into bulbous protuberances. Spheroids were observed here and there, just beyond or between the tips of the microvilli, as if the bulbous ends had been nipped off at the base, and had been caught in the mucous secretion that covers the animal. Both the protuberances and the spheroids contain electron-dense granules of a similar nature. The bases of the microvilli are not electron-dense, and the tips are only slightly so. The internal longitudinal striations, which lie parallel to the outer membrane, formed, with it, a layer measuring from 23 to 33 m/x in thickness (fig. 2, A). The layer thus formed was more electron-dense than the central part of the microvillus. The fibrillae of the microvilli arise from the tips and all along the sides down to the bases. These fibrillae interconnect to form a network as in the snail, but the interconnexions, and the granules caught in them, are more numerous between the microvilli than around the tips (fig. 2, A). Measurements of the microvilli and calculations of their surface-area are recorded in table 1. The average length is much less than that of the microvilli of H. aspersa, so that they cause far less increase in total surface-area. There are granules lying between the microvilli, close to their bases (fig. 2, A). Lane—Microvilli on the external surfaces of gastropods 501 These may have been extruded from the cytoplasm, since they have the same appearance and size as other granules lying inside the apical cytoplasm. The cytoplasm also contains larger vacuoles and granules, measuring from C15 to 0-4 fj,, and mitochondria. The wavy interdigitations of the epithelial cells measure from o-6 to 1-5 /z in length from the base to the summit of a protuberance. Light microscopy The PAS test gives a positive reaction in the form of a thin layer over the cuticle that covers the tentacular and body-surfaces. Colouring with Sudan black B shows lipid spheroids, about 1 fi in diameter, lying in the apical part of the epithelial cells as well as in the cuticle. The AH test gives a positive result, localized in particles of the same shape and distribution as those observed to exhibit sudanophilia. Mann-Kopsch preparations show osmiophil spheroids lying along the epithelial surface and cuticle of the tentacles. The osmiophil spheroids are seen as granules, although sometimes the deposition of osmium is upon a cortical border such as would be seen if a lipid droplet were impregnated with heavy metals (Baker, 1944). The araldite sections stained with toluidine blue show greater detail in the cuticle of the tentacles than do the paraffin sections. Studies of longitudinal sections of such araldite preparations suggest that there is a fibrillar layer below the microvilli only around the tip of the tentacles. The tips have a cuticle composed of two layers. The top layer is about 1-25 /x thick, and corresponds in position to the layer of microvilli shown by the electron microscope; the lower layer, about 3-5 \L thick, looks fibrillar in structure, and appears to correspond to the fibrillar basal area seen beneath the microvilli in the electron micrographs. From the tips of the tentacles towards the body, there is a gradual decrease in thickness of the cuticle, so that the sides are covered by a cuticle only 1 -25 [i deep. No fibrillar layer is evident on the sides of the tentacles. On the sides, the cuticle (or layer of microvilli, as has been shown by the electron microscope) terminates in the normal epithelial cytoplasm. Examinations of the inferior tentacles of H. aspersa show that there is a deeper layer of cuticle over the tip of the tentacles than there is over the sides, which suggests that a fibrillar basal layer is present in the tips of these tentacles as well as in the tips of the optic, or superior, tentacles. Preparations of the optic tentacles of the other stylommatophoran pulmonates were also studied in order to compare the thickness of the cuticle at the tips of their tentacles with that at the sides. A greater depth at the tip was assumed to correspond to the presence of a fibrillar layer lying beneath the normal cuticle. All the animals examined have a thicker cuticle round the tips of the tentacles than at the sides.

Discussion Within the phylum Mollusca, investigators have found microvilli on various organs in different animals. The mussel Ellipeto has microvilli on the 502 Lane—Microvilli on the external surfaces of gastropods epithelium covering the typhlosole (Fawcett and Porter, 1954) and the gastropod Patella vulgata has a brush border composed of microvilli on the intestinal epithelium (Lacey, 1957). Ellipeto and the clam Mya (Fawcett and Porter, 1954), as well as the mussel Mytilus (Afzelius, i960), have microvilli on the surface of their gills. The free surfaces of the mantle of the snail Cepaea (Emberton, 1962) and of the mantles of certain bivalved gastropods and lamellibranchs (Kawaguti and Ikemoto, 1962 a, b) are composed of microvilli. Microvilli are also present on the surface of the intact supra- muscular adhesive epithelium in gastropods, with minute depressions corre- sponding to the microvilli in the area of the muscle depression on the inner shell surface (Hubendick, 1958). Microvilli have been shown to form part of the 'cuticle' on the external surface of cestodes (Read, 1955; Threadgold, 1962). So far as is known, the present report is the first record in the phylum Mollusca of microvilli occur- ring on external epithelia which are not protected by a shell. A comparison of the structural details of the microvilli described here with those given by investigators who have studied both vertebrate and invertebrate tissues, indicate that the gastropod microvilli are very similar in size and structure to those found elsewhere. Studies of light-microscopical preparations suggest that both the optic and the inferior tentacles have a fibrillar area of cytoplasm beneath the microvilli on the tips of the tentacles. This would indicate that the fibrillar area can be in no way connected with the functioning of the eye at the tip of the optic tentacle. It may be that the fibrillar layer is a highly sensitive area present in both sets of tentacles. The sensitivity of the tips of the tentacles observed in studies of their behaviour would support such a view, for when the tips come into contact with a foreign body, the tentacles retract immediately: they are not used for tactile exploration (Moquin-Tandon, 1851). It has been observed (Lane, 1962a) that certain neurosecretory cells, which form a 'collar' around the tentacular ganglion, have their terminations in the area of the tip. It has been suggested that these cells may be modified sensory neurones (Lane, 19626), for other sensory neurones also have terminations between the epithelial cells in the tentacle tip. Clark (1956) has suggested that, in some cases, neurosecretory cells may have evolved from epidermal secretory cells, and some early investigators considered the tentacular collar cells to be primarily secretory (Jobert, 1871; Yung, 1911). The exact signifi- cance of the relationship between the terminations of the neurosecretory cells and the fibrillar layer beneath the microvilli is not clear at present, since no definite nerve-cell endings were found in the sections examined. Since all the other Stylommatophora examined have a thicker cuticle around the tips of the tentacle than along the sides, it is probable that all pulmonates with retractile tentacles have this fibrillar layer beneath the microvilli on the tentacular tips. The lamellar bodies that lie among the fibrils on the tips of the tentacles have an ultrastructural appearance somewhat similar to that of some phos- Lane—Microvilli on the external surfaces of gastropods 503 pholipid droplets. The histochemical tests indicate that there are sudanophil and osmiophil granules, within the same size-range as the lamellar bodies, lying in the same region on the tentacles. However, some osmiophil granules are also found in the cuticle on the sides of the tentacles, so that it is not certain whether the lipid granules seen in the light-microscopical preparations correspond to the lamellar bodies in the fibrillar layer of the cuticle seen under the electron microscope. It is possible that the lamellar bodies, and some of the other granules in the apical cytoplasm, may both have a lipid component. The only structures mentioned in the literature that bear any resemblance to the lamellar bodies are the spheres or granules, composed of lamellae in a spiral or concentric arrangement, which lie just beneath the microvilli in certain eggs (Afzelius, 1956). When these granules are extruded from the apical cytoplasm, the lamellar elements unfold, and sometimes the unrolled remnants remain as tangential fibres. These cortical granules have been shown to be partially lipoidal in nature (Runnstrom and Monne, 1945). Basal structures have been observed beneath microvilli in other animals. Afzelius (i960) described a layer of 'plaited' cytoplasm beneath the microvilli in the mussel gill; Kemp (1956) described a 'folded basal' cytoplasm beneath the microvilli in eggs; and Palay and Karlin (1959) found a filamentous substance that formed a 'terminal web' under the striated border in vertebrate intestinal epithelium. However, none of these structures seem to be extracellular, or to correspond exactly to the fibrillar layer described here in the tip of the gastropod tentacles. Other investigators have observed connexions between the microvilli, and mitochondria or other vesicular bodies lying in the cytoplasm just below (Kawaguti and Ikemoto, 1962a). In this study, however, the fibres running from the bases of the microvilli are in connexion with other fibres and perhaps lamellar bodies, which may in turn be connected to strands of the apical cytoplasm. The presence of granular material caught on the fibrillae connecting the microvilli, suggests that some secretory product may have been precipitated onto the fibrils. In light-microscopical preparations, the positive PAS reaction is distributed as a layer along the surface of the cuticle. This suggests that mucopolysaccharides are caught between the microvillous projections. Gastropods are noted for possessing numerous mucous cells that pour their secretion on to the body-surface to act as a lubricant or an adhesive (Campion, 1961). The microvilli of certain invertebrate eggs have been found to be embedded at their distal ends in a layer of mucopolysaccharides (Afzelius, i960). The surface-area of a single microvillus on the dorsal surface of A. hortensis is 0-13 /u.2, which is less than some of the smallest figures cited in the literature (e.g. o-2/u,2 calculated by Rhodin (1958) for the microvilli of vertebrate kidney tubules). The total surface-area of all the microvilli in 1 fj? in A. hortensis produces a relatively small increase in surface-area, in comparison with the figures calculated for the snail, and with those found elsewhere in the literature (Kemp, 1956; Rhodin, 1958; Palay and Karlin, 1959; Hubendick, 1958; Zetterqvist, 1956). 504 Lane—Microvilli on the external surfaces of gastropods I am deeply indebted to Dr. J. R. Baker, F.R.S., for his continued interest and guidance in the course of this study, as well as his permission to use the electron microscope, vacuum evaporator, and ultramicrotome granted to him. I am grateful to Professor J. W. S. Pringle, F.R.S., for accommodation in his Department. The Huxley ultramicrotome was provided by the Royal Society, and the Akashi electron microscope and Edwards vacuum evaporator by the Wellcome Trustees (grants to Dr. J. R. Baker). I wish to express my gratitude to the Canadian Federation of University Women; this work was carried out during the tenure of their Travelling Fellowship. References AFZELIUS, B. A., 1956. Exp. Cell Res., 10, 257. i960. Proc. Eur. Reg. Conf. on Electron Microscopy, Delft, z, 742. BAKER, J. R., 1944. Quart. J. micr. Sci., 85, 1. 1946. Ibid., 87, 441. CAMPION, M., 1961. Ibid., 102, 195. CLARK, R. B., 1956. Ann. Sci. Nat., Zool., 18, 199. DALTON, A. J., 1953. Int. Rev. Cytol., 2, 403. EMBERTON, L. R. B., 1962. Personal communication. FAWCETT, D. W., and PORTER, K. R., 1954. J. Morph., 94, 221. HUBENDICK, B., 1958. Ark. Zool., 11, 31. JOBERT, 1871. J. de l'Anat. et de la Phys., 2, 611. KAWAGUTI, S., and IKEMOTO, N., 1962a. Biol. J. Okayama U., 8 (nos. 1-2), 1. 19626. Ibid., 8 (nos. 1-2), 21. KEMP, N. E., 1956. J. biophys. biochem. Cytol., 2, 281. LACEY, D., 1957. Ibid., 3, 779. LANE, N. J., 1962a. Quart. J. micr. Sci., 103, 211. 19626. In Comparative neurochemistry, Proc. 5th International Neurochemical Sym- posium, Austria, edited by Richter. (In press.) LAWN, A. M., i960. J. biopbys. biochem. Cytol., 7, 197. LUFT, J. H., 1961. Ibid., 9, 409. MCMANUS, J. F. A., 1948. Stain. Tech., 23, 99. MEEK, G. A., 1962. J. roy. micr. Soc, Proc. Symposium on Cytochemical Progress in Electron Microscopy. (In press.) MOQUIN-TANDON, A., 1851. Ann. Sci. Nat., 15, 151. PALADE, G. E., 1952. J. exp. Med., 95, 285. PALAY, S., and KARLIN, L. J., 1959. J. biophys. biochem, Cytol., 5, 363. READ, C. P., 1955. In Some physiological aspects and consequences of parasitism, edited by W. H. Cole, pp. 27. New Brunswick (Rutgers Univ. Press). REBHUN, L. I., 1962. J. Ult. Res., 6, 107. RHODIN, J., 1958. Int. Rev. Cytol., 7, 485. RUNMSTROM, J., and MONNE, L., 1945. Ark. Zool., 36 A, no. 18, 1. THREADGOLD, L. T., 1962. Quart, J. micr. Sci., 103, 135. WEIGL, R., 1910. Bull. int. Acad. Sci. Cracovie B (no vol. number), 691. YUNG, E., 191 I. Rev. Suisse Zool., 19, 338. ZETTERQVIST, H., 1956. In The ultrastructural organization of the columnar absorbing cells of the mouse jejunum. Thesis, Karolinska Inst., Stockholm, Aktiebolaget Godvil. Quoted from Palay and Karlin (1959). Postscript. In an electron microscopical study of the superficial epithelium of the tentacles of Helix pomatia, Schwalbach and Lickfeld (1962) observed that the bases of the cells consist of irregular processes, with a labyrinth of extracellular spaces between them. They found that the cuticle is composed of a brush broder of microvilli. SCHWALBACH, G., and LICKFELD, K. G., 1962. Z. Zellforsch., 58, 277.