Cell Tissue Res. 203, 283-289 (1979)
Cell and Tissue Research 9 by Springer-Verlag 1979
Electron Microscopic Studies on Radular Tooth Formation in the Snails Helix pomatia L. and Limax flavus L. (Pulmonata, Stylommatophora) Klaus Kerth* Zoological Institute, Universityof Wiirzburg, Federal Republic of Germany
Summary. The radular teeth are secreted at the posterior end of the radular gland and move slowly towards the buccal cavity where they start to function. Helixpomatia and Limaxflavus were examined to determine whether the newly formed teeth already show their definite species specific shape, or whether they are gradually finished and moulded in the radular gland. Scanning electron micrographs of Helix pomatia show that teeth are secreted in the odontoblast region in their final form. Their surface is still uneven at the outset; the same is true for the newest teeth of Limax flavus. Older teeth ready for use have a smooth surface. This change seems to be brought about by secretory activity of the superior epithelium of the radular sac. Air-dried radulae, previously isolated by K O H maceration, show considerable artefacts at their posterior end. Maceration leads to shrinking of the newest teeth, but does not change their contours. The newly secreted but as yet unhardened teeth become greatly deformed during the drying process. Key words: Radula - Tooth formation - Pulmonates - Scanning electron microscopy.
The radular teeth of gastropods have a species specific shape resulting from cyclic secretory activity of the radular gland and requiring a precise "mould". It is still controversial whether or not a tooth receives its final form at the outset, or after having been transported to the buccal cavity. Most authors reported that a fully formed tooth appears at the odontoblast girdle of the radular gland (see, for example, M~irkel, 1958; Kerth and Krause, 1969; Runham, 1975; Kerth and H/insch, 1977; Wiesel and Peters, 1978). The alternative opinion, i.e., that teeth of the posterior end of the radula do not yet have their species specific shape is held, for
Sendoffprintrequeststo: Prof. Klaus Kerth, ZoologicalInstitute, Universityof Wfirzburg,R6ntgenring 10, 8700 Wtirzburg, Federal Republic of Germany * The author is verymuch indebtedto Prof. L. Schneiderfor his help on the SEM. The SEM and the Balzers equipment were kindly supplied by the Deutsche Forschungsgemeinsehaft
0302-766X/79/0203/0283/$01.40
284
K. Kerth
example, by Sick (19 58) and Menzel (1976). Light and electron microscopic as well as experimental studies prove quite clearly that the mould for the radular teeth of the pulmonates is situated in the odontoblast region (see above-mentioned references from 1969 onwards). The present study was undertaken to search for possible differences in form, between newly secreted and unworn older teeth, by means of scanning electron microscopy.
Materials and Methods
Helixpomatiaof unknown age collected near Wiirzburgduring the period fromJune to October were kept for 1-14 days at 15~ Their shell height ranged from 2.4 to 4.3 cm. Limaxflavuswere bred in 'the Zoological Institute, University of Wiirzburg. All specimens examined were adults; their length ranges from 6.5-12.0cm. For light microscopySUSA-fixedmaterial was stained with Mayer's hemalum/erythrosine and sectioned at 5-7 ~tm. The bueeal apparatus was macerated in 5 % KOH, the radula washed and embedded in glycerol gelatine. For scanning electron microscopythe buccal apparatus and a large posterior part of the radula were dissected out in the fixative(see also Kerth and H~insch, 1977).6.25% glutaraldehyde in phosphate buffer(S6rensen), pH 7.1, was used for fixation. The radula was post-fixed in the same fixativefor 3 h, washed, dehydrated, and transferred to amylacetate. Dryingby the critical point method of CO2 in a Balzers equipment was followedby 100-300.&coating with gold (Balzers sputter equ2pment). KOH maceratedradulae were air-dried and sputtered with gold.All specimenswere examined under the ISI SuperIIIA scanningelectronmicroscope.For transmissionelectron microscopy the buccal mass was fixed in 1% OsOJK2Cr2OT, pH 7.1 (Wohlfarth-Bottermann, 1957) for 1h, dehydrated in acetone, stained en bloc with 1% phosphotungstic acid and 0.5 ~ uranyl acetate, embedded in Araldite (Merck) and examined with a Siemens ElmiskopI A.
Results
Short Synopsis of Radular Gland and Tooth Formation in Pulmonates The pulmonate radular gland is an ectodermal diverticle of the buccal cavity (Fig. 1). Its superior epithelium interdigitates with the radula. At the posterior end of the radular sac, the transverse odontoblast girdle continuously secretes radular teeth and the highly flexible membrane to which they are attached. The odontoblasts are large cells arranged in groups. A n odontoblast group, by its apical profile, determines the characteristic shape of the tooth (Kerth and Krause, 1969). Runham (1975) as well as Wiesel and Peters (1978) demonstrated electron microscopically that this profile is a relief of the borders of microvilli. According to Runham (1975) the microvilli deten-nine the shape of the tooth. New teeth and the radular membrane move anteriorily towards the buccal cavity. The used area of the radula is situated outside the radular gland roughly in front of the odontophore. At its anterior end the radula becomes degraded and the oldest teeth are shed here.
Comparison Between Newly Formed and Older Teeth Already light microscopic comparison indicates that the newest teeth are largely o f final form and size (Fig. 2a b; Limaxflavus). In Fig. 2b the new tooth rises above
Radular Tooth Formation in Pulmonate Gastropods
285
l
Fig. 1. Diagrammatic sagittal section of pulmonate head showing buccal apparatus (after Runham 1963b); bc buccal cavity,iep inferiorepithelium,o odontoblast, od odontophore, oes oesophagus, rm radular membrane, sep superior epithelium, tep terminalepithelium
the surface of the odontoblast girdle. Scanning electron microscopy offers additional evidence that even the youngest tooth already has its species specific shape. The youngest teeth of Helix pomatia (Fig. 3 a, b) are not different in shape from the older except that the latter are slightly broader. However, the surfaces of older and newer teeth show striking differences. Older teeth are almost smooth, whereas newly secreted teeth in front of the main cusp look coarsely bordered. The main cusp itself reveals a granulated surface. Scanning electron micrographs of air-dried radulae, previously isolated by KOH maceration, show considerable artefacts. Older teeth are normally shaped, but new ones are heavily deformed due to shrinking (Fig. 4a, b). Such artefacts can be seen also light microscopically. The primary KOH treatment leads to limited shrinking of new teeth, but they still maintain their typical shape. Air-drying finally causes total deformation presumably due to loss of water. Transmission electron micrographs of L i m a x f l a v u s display a different internal structure of younger and older teeth. The tooth cusp in Fig. 5a (similar to Fig. 2b) rises above the odontoblast girdle and is enveloped by apical parts of terminal cells of the radular gland (Fig. 5b). Numerous long microvilli originating from the odontoblasts in front invade the tooth. Between them there is electron dense material. Such an apical bolster of microvilli is demonstrated by Wiesel and Peters (1978) in Biomphalaria glabrata. Near the surface of the tooth, coarse strands and filamentous structures lie in a fluffy matrix (Fig. 5 c). Only a few very long microvilli penetrate this region. In comparison to this, an older tooth appears largely "homogeneous" (Fig. 6a, tooth cusp) and no microvilli or individual strands can be
Fig. 2. Light micrographs of longitudinal sections of newly secreted and old teeth (Limaxflavus). SUSA, hemalum/erythrosine, a Unused old tooth outside radular gland, x 600. b Odontoblast region with new tooth; h hollow, tst top side of tooth, x 505 Fig. 3. Scanning electron micrographs of Helix pomatia (radula prepared in glutaraldehyde), a Radula region approximately at 45th transverse row. Smooth teeth with remains of superior epithelium, x 540. b Posterior end of radula. Note surface of teeth (arrows: youngest transverse row); tr transverse row. x 500 Fig. 4. Scanning electron micrographs of an air-dried radula of Helix pomatia, a Normally shaped older teeth, x 640. b Artificially deformed new teeth, x 640
Fig. 5. Transmission electron micrographs of Limaxflavus. a Apical region of odontoblast group with new tooth (arrow, direction to buccal cavity) x 3500. b Apical parts of terminal cells (tep) enveloping tooth cusp. Note uneven contour of tooth cusp. x 4250. c Structure of youngest tooth near its top with coarse strands and a few very long microvilli (my); ss strands of secretion, t tooth, tc tooth cusp. • 16,000 Fig. 6. Transmission electron micrographs of tooth cusps of Limax flavus, a U n u s e d tooth outside radular gland with perfectly smooth surface, (compare Fig. 5b). x 13,000. b Smooth tooth from middle region of radular gland, arrow; gap between tooth and superior epithelium; n nucleus, x5200
288
K. Kerth
seen. Figures 5b and 6a, b reveal a considerable change of the tooth surface. The newly secreted tooth does not yet have a smooth surface. The top side contour of the cusp (Fig. 5b) unravels to form strands. Apical parts of most terminal cells of the radular gland are closely adjacent to the tooth surface. While being moved to the buccal cavity the teeth keep closest contact with the apical border of the upper epithelium. In pulmonates, the cells of this epithelium are moving anteriorily at the same speed as the teeth (Runham, 1963 b). Contact exists in a large posterior part of the radular sac. Already half-way through the length of the radular gland there is a small gap between the spongy cytoplasm and the top side of the tooth filled with a fluffy secretion (Fig. 6b). The tooth cusps in Fig. 6a and b (Fig. 6a: unused region of the radula immediately in front of the radular gland) no longer show an unraveled surface. The older tooth in Fig. 6a shows a perfectly smooth contour. It may be presumed that the newest teeth are polished by secretions of the upper epithelium. Its cells regularly show Golgi apparatus in a subapical position. Indications for a coating by an enamel-like layer, as was found in Patella by Runham et al. (1969), cannot be detected. A smoothing of the tooth surface can also be seen in Helix pomatia (Fig. 3a, b).
Discussion
Tooth formation in pulmonates has already been intensively examined (see authors mentioned in the introduction). Light and electron microscopic as well as experimental investigations show: (1) The apical profile of an odontoblast group determines the shape of the tooth, according to Runham (1975), by its apical microvilli. (2) The tooth material is largely produced by the odontoblasts and consists of secretion and shed microvilli. It has not become clear yet how the apical microvillous border originates and is shed during tooth formation and how odontoblasts discharge their secretion. Moreover, it is still controversial to what extent other epithelial cells deliver tooth material. The radula of adult pulmonates reveals a regular tooth pattern with nearly identical forms in the longitudinal row and mirror-symmetrical changes of the tooth shape in the transverse row. A mirror-symmetrical cell pattern also exists along the odontoblast girdle (Kerth and H~insch, 1977). It is possible to recognize a mouldlike contour of a tooth in the cellular arrangement of the odontoblast group. Accordingly, scanning electron micrographs of Helixpomatia demonstrate that the newest tooth receives its final form at the outset. The same is true for the prosobranchs Buccinum undatum and Pomacea bridgesi (Kraus, 1975; Mischor, 1976). However, there are some toxoglossan snails with incompletly shaped new teeth (Endean and Duchemin, 1967; Shimek, 1975). The "incomplete" new teeth found in air-dried radulae of Helix pomatia (Menzel, 1976) seem to be the result of artefactual change. Scanning electron micrographs of air-dried radulae, previously isolated by KOH treatment, show considerable artefacts. The usual 5 ~o KOH maceration for 1-2h at room temperature alone will lead to moderate shrinking of new teeth. This is probably due in part to extraction of unhardened radular proteins (Peters, 1972). Air-drying finally produces heavy deformation at the posterior end of the radula due to loss of water.
Radular Tooth Formation in Pulmonate Gastropods
289
Radular teeth of Helixpomatia and Limaxflavus are secreted in their final form, but show an uneven surface. It becomes smooth probably by secretory activity of the upper epithelium whilst the teeth are moving forward in the radular gland. Thus the secretion and shaping of a pulmonate tooth is followed by smoothing of its surface and by hardening of its organic material through quinone tanning (Runham, 1963a, 1975). Not until these processes are completed is a radular tooth used.
References Endean, R., Duchemin, C.: The venom apparatus of Conus magus. Toxicon 4, 275-284 (1967) Kerth, K.: Radula-Ersatz und Z/ihnchenmuster der Weinbergschnecke im Winterhalbjahr. Zool. Jb. Anat. 88, 47-62 (1971) Kerth, K., H~insch, D.: Zellmuster und Wachstum des Odontoblastengiirtels der Weinbergschnecke Helix pomatia L. Zool. Jb. Anat. 98, 14-28 (1977) Kerth, K., Krause, G.: Untersuchungen mittels R6ntgenbestrahlung fiber den Radula-Ersatz der Nacktschnecke Limaxflavus L. Wilhelm Roux Archiv, 164, 48-82 (1969) Kraus, U.: Lichtmikroskopische Untersuchungen zur Radulabildung der Wellhornschnecke Buccinum undatum L. Zulass. Arbeit am Zool. Inst. Univ. Wfirzburg, (1975), unpublished M/irkel, K.: Bau und Funktion der Pulmonaten-Radula. Z. wiss. Zool. 160, 213-289 (1958) Menzel, R.: Autoradiographische Untersuchungen mit 35-S-Natriumsulfat fiber den Ersatz der Radula in Abh/ingigkeit yon der Temperatur bei Helixpomatia L. (Gastropoda). Zool. Jb. Anat. 97, 550-565 (1976) Mischor, B.: Bildung und Abbau der Radula von Pomacea bridgesi diffusa Blume (Gastropoda, Prosobranchia). Verh. Dtsch. Zool. Ges. Erlangen 1977, 282. Stuttgart: Fischer 1977 Peters, W.: Occurence of chitin in MoUusca. Comp. Biochem. Physiol. 41, (B) 541-550 (1972) Runham, N.W.: The histochemistry of the radula of Patella vulgata. Quart. J. Micr. Sci. 102, 371-380 (1961) Runham, N.W.: The histochemistry of the radulas of Acanthochiton communis, Lymnaea stagnalis, Helix pomatia, Scaphander lignarius and Archidoris pseudoargus. Ann. Histochim. 8, 433-442 (1963a) Runham, N.W.: A study of the replacement mechanism of the pulmonate radula. Quart. J. Micr. Sci. 104, 271-277 (1963b) Runham, N.W.: Alimentary canal. In Fretter, V., Peake, J. Pulmonates Vol. I 53-104 London, New York, San Francisco, Academic Press 1975 Runham, N.W., Thornton, P.R., Shaw, D.A., Wayte, R.C.: The mineralization and hardness of the radular teeth of the Limpet Patella vulgata. L. Z. Zellforsch. 99, 608-626 (1969) Shimek, R.L.: The morphology of the buccal apparatus of Oenopota levidensis (Gastropoda, Turridae). Z. Morph. Tiere 80, 59-96 (1975) Sick, E.: Bau und Bildung von Kiefer und Radula bei Gastropoden insbesondere auf Grund yon histochemischen Untersuchungen. Inaug. Diss. Tfibingen (1958), unpublished Wiesel, R., Peters, W.: Licht- und elektronenmikroskopische Untersuchungen am Radulakomplex und zur Radulabildung von Biomphalaria glabrata Say (= Australorbis gl.) (Gastropoda, Basommatophora) Zoomorphologie, 89, 73-92 (1978) Wohlfarth-Bottermann, K.E.: Die Kontrastierung tierischer Zellen und Gewebe im Rahmen ihrer elektronenmikroskopischen Untersuchungen an ultradfinnen Schnitten. Naturwissenschaften 44, 287-288 (1957)
Accepted September 7, 1979
Cell and Tissue Research 9 by Springer-Verlag 1979
Electron Microscopic Studies on Radular Tooth Formation in the Snails Helix pomatia L. and Limax flavus L. (Pulmonata, Stylommatophora) Klaus Kerth* Zoological Institute, Universityof Wiirzburg, Federal Republic of Germany
Summary. The radular teeth are secreted at the posterior end of the radular gland and move slowly towards the buccal cavity where they start to function. Helixpomatia and Limaxflavus were examined to determine whether the newly formed teeth already show their definite species specific shape, or whether they are gradually finished and moulded in the radular gland. Scanning electron micrographs of Helix pomatia show that teeth are secreted in the odontoblast region in their final form. Their surface is still uneven at the outset; the same is true for the newest teeth of Limax flavus. Older teeth ready for use have a smooth surface. This change seems to be brought about by secretory activity of the superior epithelium of the radular sac. Air-dried radulae, previously isolated by K O H maceration, show considerable artefacts at their posterior end. Maceration leads to shrinking of the newest teeth, but does not change their contours. The newly secreted but as yet unhardened teeth become greatly deformed during the drying process. Key words: Radula - Tooth formation - Pulmonates - Scanning electron microscopy.
The radular teeth of gastropods have a species specific shape resulting from cyclic secretory activity of the radular gland and requiring a precise "mould". It is still controversial whether or not a tooth receives its final form at the outset, or after having been transported to the buccal cavity. Most authors reported that a fully formed tooth appears at the odontoblast girdle of the radular gland (see, for example, M~irkel, 1958; Kerth and Krause, 1969; Runham, 1975; Kerth and H/insch, 1977; Wiesel and Peters, 1978). The alternative opinion, i.e., that teeth of the posterior end of the radula do not yet have their species specific shape is held, for
Sendoffprintrequeststo: Prof. Klaus Kerth, ZoologicalInstitute, Universityof Wfirzburg,R6ntgenring 10, 8700 Wtirzburg, Federal Republic of Germany * The author is verymuch indebtedto Prof. L. Schneiderfor his help on the SEM. The SEM and the Balzers equipment were kindly supplied by the Deutsche Forschungsgemeinsehaft
0302-766X/79/0203/0283/$01.40
284
K. Kerth
example, by Sick (19 58) and Menzel (1976). Light and electron microscopic as well as experimental studies prove quite clearly that the mould for the radular teeth of the pulmonates is situated in the odontoblast region (see above-mentioned references from 1969 onwards). The present study was undertaken to search for possible differences in form, between newly secreted and unworn older teeth, by means of scanning electron microscopy.
Materials and Methods
Helixpomatiaof unknown age collected near Wiirzburgduring the period fromJune to October were kept for 1-14 days at 15~ Their shell height ranged from 2.4 to 4.3 cm. Limaxflavuswere bred in 'the Zoological Institute, University of Wiirzburg. All specimens examined were adults; their length ranges from 6.5-12.0cm. For light microscopySUSA-fixedmaterial was stained with Mayer's hemalum/erythrosine and sectioned at 5-7 ~tm. The bueeal apparatus was macerated in 5 % KOH, the radula washed and embedded in glycerol gelatine. For scanning electron microscopythe buccal apparatus and a large posterior part of the radula were dissected out in the fixative(see also Kerth and H~insch, 1977).6.25% glutaraldehyde in phosphate buffer(S6rensen), pH 7.1, was used for fixation. The radula was post-fixed in the same fixativefor 3 h, washed, dehydrated, and transferred to amylacetate. Dryingby the critical point method of CO2 in a Balzers equipment was followedby 100-300.&coating with gold (Balzers sputter equ2pment). KOH maceratedradulae were air-dried and sputtered with gold.All specimenswere examined under the ISI SuperIIIA scanningelectronmicroscope.For transmissionelectron microscopy the buccal mass was fixed in 1% OsOJK2Cr2OT, pH 7.1 (Wohlfarth-Bottermann, 1957) for 1h, dehydrated in acetone, stained en bloc with 1% phosphotungstic acid and 0.5 ~ uranyl acetate, embedded in Araldite (Merck) and examined with a Siemens ElmiskopI A.
Results
Short Synopsis of Radular Gland and Tooth Formation in Pulmonates The pulmonate radular gland is an ectodermal diverticle of the buccal cavity (Fig. 1). Its superior epithelium interdigitates with the radula. At the posterior end of the radular sac, the transverse odontoblast girdle continuously secretes radular teeth and the highly flexible membrane to which they are attached. The odontoblasts are large cells arranged in groups. A n odontoblast group, by its apical profile, determines the characteristic shape of the tooth (Kerth and Krause, 1969). Runham (1975) as well as Wiesel and Peters (1978) demonstrated electron microscopically that this profile is a relief of the borders of microvilli. According to Runham (1975) the microvilli deten-nine the shape of the tooth. New teeth and the radular membrane move anteriorily towards the buccal cavity. The used area of the radula is situated outside the radular gland roughly in front of the odontophore. At its anterior end the radula becomes degraded and the oldest teeth are shed here.
Comparison Between Newly Formed and Older Teeth Already light microscopic comparison indicates that the newest teeth are largely o f final form and size (Fig. 2a b; Limaxflavus). In Fig. 2b the new tooth rises above
Radular Tooth Formation in Pulmonate Gastropods
285
l
Fig. 1. Diagrammatic sagittal section of pulmonate head showing buccal apparatus (after Runham 1963b); bc buccal cavity,iep inferiorepithelium,o odontoblast, od odontophore, oes oesophagus, rm radular membrane, sep superior epithelium, tep terminalepithelium
the surface of the odontoblast girdle. Scanning electron microscopy offers additional evidence that even the youngest tooth already has its species specific shape. The youngest teeth of Helix pomatia (Fig. 3 a, b) are not different in shape from the older except that the latter are slightly broader. However, the surfaces of older and newer teeth show striking differences. Older teeth are almost smooth, whereas newly secreted teeth in front of the main cusp look coarsely bordered. The main cusp itself reveals a granulated surface. Scanning electron micrographs of air-dried radulae, previously isolated by KOH maceration, show considerable artefacts. Older teeth are normally shaped, but new ones are heavily deformed due to shrinking (Fig. 4a, b). Such artefacts can be seen also light microscopically. The primary KOH treatment leads to limited shrinking of new teeth, but they still maintain their typical shape. Air-drying finally causes total deformation presumably due to loss of water. Transmission electron micrographs of L i m a x f l a v u s display a different internal structure of younger and older teeth. The tooth cusp in Fig. 5a (similar to Fig. 2b) rises above the odontoblast girdle and is enveloped by apical parts of terminal cells of the radular gland (Fig. 5b). Numerous long microvilli originating from the odontoblasts in front invade the tooth. Between them there is electron dense material. Such an apical bolster of microvilli is demonstrated by Wiesel and Peters (1978) in Biomphalaria glabrata. Near the surface of the tooth, coarse strands and filamentous structures lie in a fluffy matrix (Fig. 5 c). Only a few very long microvilli penetrate this region. In comparison to this, an older tooth appears largely "homogeneous" (Fig. 6a, tooth cusp) and no microvilli or individual strands can be
Fig. 2. Light micrographs of longitudinal sections of newly secreted and old teeth (Limaxflavus). SUSA, hemalum/erythrosine, a Unused old tooth outside radular gland, x 600. b Odontoblast region with new tooth; h hollow, tst top side of tooth, x 505 Fig. 3. Scanning electron micrographs of Helix pomatia (radula prepared in glutaraldehyde), a Radula region approximately at 45th transverse row. Smooth teeth with remains of superior epithelium, x 540. b Posterior end of radula. Note surface of teeth (arrows: youngest transverse row); tr transverse row. x 500 Fig. 4. Scanning electron micrographs of an air-dried radula of Helix pomatia, a Normally shaped older teeth, x 640. b Artificially deformed new teeth, x 640
Fig. 5. Transmission electron micrographs of Limaxflavus. a Apical region of odontoblast group with new tooth (arrow, direction to buccal cavity) x 3500. b Apical parts of terminal cells (tep) enveloping tooth cusp. Note uneven contour of tooth cusp. x 4250. c Structure of youngest tooth near its top with coarse strands and a few very long microvilli (my); ss strands of secretion, t tooth, tc tooth cusp. • 16,000 Fig. 6. Transmission electron micrographs of tooth cusps of Limax flavus, a U n u s e d tooth outside radular gland with perfectly smooth surface, (compare Fig. 5b). x 13,000. b Smooth tooth from middle region of radular gland, arrow; gap between tooth and superior epithelium; n nucleus, x5200
288
K. Kerth
seen. Figures 5b and 6a, b reveal a considerable change of the tooth surface. The newly secreted tooth does not yet have a smooth surface. The top side contour of the cusp (Fig. 5b) unravels to form strands. Apical parts of most terminal cells of the radular gland are closely adjacent to the tooth surface. While being moved to the buccal cavity the teeth keep closest contact with the apical border of the upper epithelium. In pulmonates, the cells of this epithelium are moving anteriorily at the same speed as the teeth (Runham, 1963 b). Contact exists in a large posterior part of the radular sac. Already half-way through the length of the radular gland there is a small gap between the spongy cytoplasm and the top side of the tooth filled with a fluffy secretion (Fig. 6b). The tooth cusps in Fig. 6a and b (Fig. 6a: unused region of the radula immediately in front of the radular gland) no longer show an unraveled surface. The older tooth in Fig. 6a shows a perfectly smooth contour. It may be presumed that the newest teeth are polished by secretions of the upper epithelium. Its cells regularly show Golgi apparatus in a subapical position. Indications for a coating by an enamel-like layer, as was found in Patella by Runham et al. (1969), cannot be detected. A smoothing of the tooth surface can also be seen in Helix pomatia (Fig. 3a, b).
Discussion
Tooth formation in pulmonates has already been intensively examined (see authors mentioned in the introduction). Light and electron microscopic as well as experimental investigations show: (1) The apical profile of an odontoblast group determines the shape of the tooth, according to Runham (1975), by its apical microvilli. (2) The tooth material is largely produced by the odontoblasts and consists of secretion and shed microvilli. It has not become clear yet how the apical microvillous border originates and is shed during tooth formation and how odontoblasts discharge their secretion. Moreover, it is still controversial to what extent other epithelial cells deliver tooth material. The radula of adult pulmonates reveals a regular tooth pattern with nearly identical forms in the longitudinal row and mirror-symmetrical changes of the tooth shape in the transverse row. A mirror-symmetrical cell pattern also exists along the odontoblast girdle (Kerth and H~insch, 1977). It is possible to recognize a mouldlike contour of a tooth in the cellular arrangement of the odontoblast group. Accordingly, scanning electron micrographs of Helixpomatia demonstrate that the newest tooth receives its final form at the outset. The same is true for the prosobranchs Buccinum undatum and Pomacea bridgesi (Kraus, 1975; Mischor, 1976). However, there are some toxoglossan snails with incompletly shaped new teeth (Endean and Duchemin, 1967; Shimek, 1975). The "incomplete" new teeth found in air-dried radulae of Helix pomatia (Menzel, 1976) seem to be the result of artefactual change. Scanning electron micrographs of air-dried radulae, previously isolated by KOH treatment, show considerable artefacts. The usual 5 ~o KOH maceration for 1-2h at room temperature alone will lead to moderate shrinking of new teeth. This is probably due in part to extraction of unhardened radular proteins (Peters, 1972). Air-drying finally produces heavy deformation at the posterior end of the radula due to loss of water.
Radular Tooth Formation in Pulmonate Gastropods
289
Radular teeth of Helixpomatia and Limaxflavus are secreted in their final form, but show an uneven surface. It becomes smooth probably by secretory activity of the upper epithelium whilst the teeth are moving forward in the radular gland. Thus the secretion and shaping of a pulmonate tooth is followed by smoothing of its surface and by hardening of its organic material through quinone tanning (Runham, 1963a, 1975). Not until these processes are completed is a radular tooth used.
References Endean, R., Duchemin, C.: The venom apparatus of Conus magus. Toxicon 4, 275-284 (1967) Kerth, K.: Radula-Ersatz und Z/ihnchenmuster der Weinbergschnecke im Winterhalbjahr. Zool. Jb. Anat. 88, 47-62 (1971) Kerth, K., H~insch, D.: Zellmuster und Wachstum des Odontoblastengiirtels der Weinbergschnecke Helix pomatia L. Zool. Jb. Anat. 98, 14-28 (1977) Kerth, K., Krause, G.: Untersuchungen mittels R6ntgenbestrahlung fiber den Radula-Ersatz der Nacktschnecke Limaxflavus L. Wilhelm Roux Archiv, 164, 48-82 (1969) Kraus, U.: Lichtmikroskopische Untersuchungen zur Radulabildung der Wellhornschnecke Buccinum undatum L. Zulass. Arbeit am Zool. Inst. Univ. Wfirzburg, (1975), unpublished M/irkel, K.: Bau und Funktion der Pulmonaten-Radula. Z. wiss. Zool. 160, 213-289 (1958) Menzel, R.: Autoradiographische Untersuchungen mit 35-S-Natriumsulfat fiber den Ersatz der Radula in Abh/ingigkeit yon der Temperatur bei Helixpomatia L. (Gastropoda). Zool. Jb. Anat. 97, 550-565 (1976) Mischor, B.: Bildung und Abbau der Radula von Pomacea bridgesi diffusa Blume (Gastropoda, Prosobranchia). Verh. Dtsch. Zool. Ges. Erlangen 1977, 282. Stuttgart: Fischer 1977 Peters, W.: Occurence of chitin in MoUusca. Comp. Biochem. Physiol. 41, (B) 541-550 (1972) Runham, N.W.: The histochemistry of the radula of Patella vulgata. Quart. J. Micr. Sci. 102, 371-380 (1961) Runham, N.W.: The histochemistry of the radulas of Acanthochiton communis, Lymnaea stagnalis, Helix pomatia, Scaphander lignarius and Archidoris pseudoargus. Ann. Histochim. 8, 433-442 (1963a) Runham, N.W.: A study of the replacement mechanism of the pulmonate radula. Quart. J. Micr. Sci. 104, 271-277 (1963b) Runham, N.W.: Alimentary canal. In Fretter, V., Peake, J. Pulmonates Vol. I 53-104 London, New York, San Francisco, Academic Press 1975 Runham, N.W., Thornton, P.R., Shaw, D.A., Wayte, R.C.: The mineralization and hardness of the radular teeth of the Limpet Patella vulgata. L. Z. Zellforsch. 99, 608-626 (1969) Shimek, R.L.: The morphology of the buccal apparatus of Oenopota levidensis (Gastropoda, Turridae). Z. Morph. Tiere 80, 59-96 (1975) Sick, E.: Bau und Bildung von Kiefer und Radula bei Gastropoden insbesondere auf Grund yon histochemischen Untersuchungen. Inaug. Diss. Tfibingen (1958), unpublished Wiesel, R., Peters, W.: Licht- und elektronenmikroskopische Untersuchungen am Radulakomplex und zur Radulabildung von Biomphalaria glabrata Say (= Australorbis gl.) (Gastropoda, Basommatophora) Zoomorphologie, 89, 73-92 (1978) Wohlfarth-Bottermann, K.E.: Die Kontrastierung tierischer Zellen und Gewebe im Rahmen ihrer elektronenmikroskopischen Untersuchungen an ultradfinnen Schnitten. Naturwissenschaften 44, 287-288 (1957)
Accepted September 7, 1979
