Cell Tiss. Res. 192, 489-501 (1978)
Cell and Tissue Research 9 by Springer-Verlag 1978
Paddle Cilia and D i s c o c i l i a - Genuine Structures? Observations on Cilia of Sensory Cells in Marine Turbellaria Ulrich Ehlers and Beate Ehlers* II. Zoologisches Institut und Museum der Universit~it G6ttingen, Bundesrepublik Deutschland
Summary. Kinocilia of epidermal sensory cells in fixed marine Turbellaria often terminate as flattened biconcave discs. The distal part of the ciliary axoneme curves back upon itself forming a 360 ~ loop which is enveloped by the plasmalemma. In living animals this structure can be induced by the addition of sodium cacodylate, monobasic sodium phosphate, dibasic sodium phosphate, sucrose, calcium chloride, or formaldehyde to the sea water. Specimens treated with sodium chloride, glutaraldehyde, or osmium tetroxide do not show modified cilia. In animals prepared for EM at low temperature and with a buffered hypotonic fixative less kinocilia are modified than in animals treated with a buffered iso- or hypertonic fixative and at a higher temperature. It is assumed that the unusually shaped cilia, described as "paddle cilia" or "discocilia" in other invertebrates, do not represent a genuine but an artificial structure. Key words: Paddle cilia and discocilia - Turbellaria transmission electron microscopy.
Scanning and
In recent years cilia with an uncommon structure of the ciliary membrane have been found in several marine invertebrates (e.g. Tamarin et al., 1974, 1976; Oldfield, 1975; Bergquist et al., 1977; Dilly, 1977a, b; Heimler, 1978; Storch and Alberti, 1978). The distal ends of these cilia show a flattened swelling with an annular curvature of the axoneme. The modified cilia are believed to be true features and are described as a new type of specialized organelles, called "paddle cilia", "club-footed cilia" or "discocilia". Using scanning and transmission electron microscopy we have found modified cilia resembling "paddle cilia" or "discocilia" in many species of marine Send offprint requests to." Dr. U. Ehlers, II. Zoologisches Institut und Museum der Universit~it, Berliner Str. 28, D-3400 G6ttingen, Federal Republic of Germany
* Thanks are due to Dr. C. Marschall for correcting the English of the manuscript. Financial support was provided by the Akademie der Wissenschaften und der Literatur, Mainz
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turbellarians. T h a t ciliary c l u b b i n g can be caused by external influences, e.g. high temperature ( M e c k l e n b u r g et al., 1974), v i b r a t i o n (Kharkeevich, 1977), a n d i r r a d i a t i o n (Baldetorp et al., 1977) is k n o w n . Therefore we varied the c o n d i t i o n s u n d e r which the specimens were prepared in order to determine whether the swellings o f the cilia are artefacts or genuine structures.
Materials and Methods Light microscopic and EM-studies were carried out on 29 turbellarian species, especially on 5 representativesof the family Otoplanidae (Proseriata, Neoophora): DicoelandroporaatriopapillataAx, 1956; NotocaryoplaneUa glandutosa (Ax, 1951); Parotoplana capitata Meixner, 1938; Parotoplanina geminoducta Ax, 1956; Praebursoplana steinboecki Ax, 1956. The animals were collectedat various marine localitieson the island of Sylt (North Sea). Some of the material was fixedimmediatelyafter collectionin the laboratories of the BiologischeAnstalt Helgoland, List/Sylt. Most of the turbellarians were transported live to Grttingen for fixation and further preparation including some experiments with live animals. No anesthetic agents were used. For transmission and scanning electron microscopy the animals were fixed either in (a) 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2-7.4) for 2 h, rinsed in the same buffer and postfixed in 1% osmium tetroxide in the same buffer for 11/2-2h, (b) 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2 7.4) for 2h, or (c) 1 ~o osmium tetroxide in 0.1 M sodium cacodylate buffer for 3-4h. Each fixation was carried out at 0 4 ~C, at room temperature, or initially for 10--20rain at room temperature, followed by 04~ Furthermore we used fixativesand buffers of different osmolalities(from 385 mOsm to more than 1000 mOsm)made by adding sucrose and traces ofCaCl z. The preparation for TEM followedthe steps described by Ehlers and Ehlers (1977). For SEM the fixed animals were dehydrated through graded series of ethanol or acetone. The specimenswere dried by the critical-point-method (with liquid carbon dioxide or freon) or freeze-dried. The preparations were finally sputtered with gold in a sputter coater or coated with gold-palladium in a vacuum-evaporator, and examined in a Zeiss NOVASCAN 30 at 15 kV. For light microscopy (bright-field, phase-contrast, and interference microscopy) living animals were slightlysquashed under a coverglasseither at 0 4 ~C or at room temperature. Then a small amount of sucrose (5 % or 10 %), calciumchloride (2 % or 5 %), sodium chloride (5 % or 10 %), sodium cacodylate (0.1 M, 0.2M or 0.5M), monobasic sodium phosphate (0.1 M, 0.2M or 0.5M), dibasic sodium phosphate (0.i M, 0.2 M or 0.5 M), formaldehyde (4%), glutaraldehyde (2.5%, 10% or 25%), or osmium tetroxide (2 %) was added to the squashed specimen.
Results Cilia with a modified distal tip were f o u n d in at least 16 species o f different systematical groups of m a r i n e Turbellaria. The paddle-like structures can be seen in the kinocilia o f m o n o - or multiciliary sensory cells only. These ciliary sensory cells occur frequently in the head region of the turbellarians a n d less frequently o n the other parts of the body. The perikarya of the sensory cells lie within the p a r e n c h y m a b e n e a t h the s u b e p i d e r m a l musculature. The distal parts of the cells, provided with one or several cilia, penetrate the m o n o l a y e r e d epidermis a n d reach the periphery of the b o d y (cf. Bedini et al., 1975; Ehlers a n d Ehlers, 1977). Cilia are lacking in most o f the epidermal cells in almost all T u r b e l l a r i a Proseriata studied b e l o n g i n g to the family O t o p l a n i d a e (cf. Ax, 1956). Ciliated
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epidermal cells are restricted to the ventral side of an animal, forming a ribbon-like creeping sole, which begins in the region of the head and ends near the tip of the tail. Posterior to the cephalic lobe the creeping sole shows bilateral extensions forming a small ciliary ring (Fig. 1 a) or ciliary pits. In the regions of the body with nonciliated epidermal cells (i.e. in the ventrolateral, lateral, and dorsal parts of the body as well as in the cephalic lobe) none of the cilia are locomotor organelles, but they rather belong to sensory cells. For this reason these cilia can be clearly regarded as sensory cilia in SEM pictures. The following description of the structure of modified cilia is limited to the family Otoplanidae.
General Description of the Paddle-like Cilia The kinocilia of the cranial ciliary pits (Fig. 2a, above) and of the ventral creeping sole (Fig. 3a, at left) show the normal cylindrical shape. The kinocilia of the multiciliary sensory cells of the cephalic lobe are modified forming paddle-like cilia (Figs. 1 b-e, 2a, at left). Using higher magnifications it can clearly be seen that occasionally not all the cilia of a given sensory cell show the paddle-like shape. Unmodified kinocilia also occur (Fig. 2b, arrows). Multiciliated sensory cells with paddle-like cilia are further found within the dorsal opening between the ciliated pits (Fig. 2a, arrow) and adjacent to the ventral creeping sole (Fig. 3a, arrows). Here only some or all kinocilia (Fig. 3 b, c) are paddle-like cilia. By TEM and light microscopy, the paddle-like structure of the kinocilia can be verified. T E M profiles (Fig. 4 a-d) show cilia with an axoneme forming one or more circular loops of about 1-2 ~tm in diameter. There is only one single paddle-like structure present per kinocilium. The swelling is always situated on the distal tip of the kinocilium. Generally the axoneme turns in almost piano-spiral windings, but now and then the plane of one paddle is bent in itself (Fig. 3b). The cilia measure basally about 2000 A in diameter and have the normal pattern of 9 x 2 + 2 tubules. In the region of the terminal windings the axoneme tubules start coming together and the diameter of the axoneme is somewhat reduced (Fig. 4c). The tubules can be disintegrated. In cross sections they are then seen as isolated double or single tubules which are no longer arranged in a typical ring (Fig. 4d). Normally the axoneme curves back forming a 360 ~ loop. In addition to this condition paddle-like structures showing a bent axoneme, forming a kink (Fig. 4b) can be found. When the axoneme shows two or three complete piano-spiral windings (Figs. 4d, 6), most of the plasmalemma remains partly in contact with the axoneme. Only the terminal part of the bent axoneme sometimes separates completely from the plasmalemma (Fig. 4d). The swellings surrounded by the spread plasmalemma have a content of low contrast (Fig. 4b). In the larger paddles the swelling in the ciliary membrane appears empty (Fig. 4a, c, d). In some paddles small vesicles can be seen at the concave side of the axoneme (Fig. 4a, b, arrows). These vesicles lie in regular intervals in close contact to the peripheral double tubules.
Fig. I a-e. Dicoelandropora atriopapillata. Cephalic lobe (cO with sensory bristles (sb) and part of the cephalic ciliary ring (cr), interference microscope, room temperature, a Living animal in natural sea water. I r e Treated animals with paddle cilia (pc): b In 0.1 M sodium cacodylate, e In 5 ~ sucrose, d In 2 ~o calcium chloride, e In 4 ~ formaldehyde, a--e • 830; d and e x 2080
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Fig. 2 a and b. Parotoplana capitata, a Dorsal view of the anterior end. Paddle cilia (pc) at the tip of the cephalic lobe (lower left) and the dorsal opening (arrow) between the ciliated pits. x 920. b Several multiciliated sensory cells of the cephalic lobe. Some of the cilia (arrows) are not modified to paddle cilia. x 4600
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Fig.3 a-e. Parotoplaninageminoducta,a View of the ventral creeping sole (cs)with unmodified cilia. The arrows point to 7 serially arranged multiciliated sensory cells with paddle cilia, x 1350. b and c Multiciliated sensory cells from the lateral side of the body. In c the axonemes of three paddles show more than one 360 ~ loop (arrows). b x 8000, e x 8200 Morphological Changes of the Sensory Cilia Using Different Preparatory Techniques N u m e r o u s s e n s o r y bristles o c c u r at t h e c e p h a l i c l o b e o f t h e o t o p l a n i d s d e a l t w i t h in this p a p e r (Fig. 1 a; see also A x , 1956, p. 594; B e d i n i et al., 1975, p. 257). T h e s e bristles a r e c o m p o s e d o f several cilia s t a n d i n g closely t o g e t h e r . T h e cilia a r e l o n g
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Fig. 4 a-d. Parotoplaninageminoducta (a, b, d) and Notocaryoplanellaglandulosa (e). a--ePaddle cilia of monociliary sensorycellswith one 360~loop or with a kink (b) of the axoneme. The arrows point to small vesicles,a • 14,000,b • 27,200,c x 18,000. d Paddle cilia with two (2 asterisks) or three (3 asterisks) 360~ turns of the ciliary axoneme within the enlarged plasmalemma. The axoneme has become partially separated from the ciliary membrane (arrows). • 16,000
a n d w o u n d a r o u n d each other in living a n i m a l s a n d also in some fixed specimens ( u n p u b l i s h e d S E M observations). T e r m i n a l l y each sensory bristle tapers to a sharp tip. Paddle-like cilia do n o t occur. After the a d d i t i o n o f sucrose, calcium chloride, s o d i u m cacodylate, m o n o b a s i c or dibasic s o d i u m phosphate, or formaldehyde to the sea water, the straight kinocilia of the sensory cells are modified to paddle cilia within 1-10 sec at r o o m t e m p e r a t u r e (Fig. 1 b ~ ) . A t first, only a small swelling o f the m e m b r a n e occurs at the t e r m i n a l or n e a r the distal tip o f the cilia. T h e n the swellings enlarge to form typical paddle-like structures with a curved axoneme. T h e r e b y m a n y kinocilia
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Fig. 5 a and b. Dicoelandropora atriopapillata, a and b Modified sensory bristles of the cephalic lobe. Some of the paddles have particles attached to their surfaces (arrows) or stick together (double arrow). x 5100
s h o r t e n to less t h a n 50 ~ o f their n a t u r a l length a n d b e n d d o w n to the epidermis o f the animal. L o c o m o t o r cilia n o r m a l l y d o n o t show a typical p a d d l e - l i k e structure (Fig. 1 c). O n l y f o r m a l d e h y d e causes swellings o f the p l a s m a l e m m a o f these cilia. I n sensory kinocilia t r e a t e d with the a b o v e - m e n t i o n e d solutions at 0 - 4 ~ C, the f o r m a t i o n o f p a d d l e s takes 10 sec (calcium chloride) to 10 min (sucrose); only a few or no sensory cilia are bent.
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Fig. 6 a and h. Parotoplaninageminoducta (a) and Praebursoplanasteinboecki (b). a Multiciliatedsensory cell of the lateral body region with 3 paddle cilia; at left (arrow) more than one 360~ curvature of the axoneme. (rh) rhabdites. • 7800. b Multiciliated sensory cell with 7 paddle cilia; two paddles (arrows) with more than one 360~ loop of the axoneme. • 3600
Solutions o f s o d i u m chloride, glutaraldehyde, or o s m i u m tetroxide do n o t induce a paddle structure. I f several a n i m a l s are s i m u l t a n e o u s l y fixed for EM, paddle cilia n o r m a l l y do n o t a p p e a r in the same q u a n t i t y in all the individuals. Some o f these a n i m a l s have m a n y paddles, others have few. The p h e n o m e n o n t h a t u n d e r the influence o f identical E M processing c o n d i t i o n s the n u m b e r o f paddles per a n i m a l is i n c o n s t a n t considerably
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complicates the question of the extent to which the formation of paddle cilia is influenced by the chemical substances used for processing. Nevertheless some general statements can be made. Temperature, osmolality, and the chemical nature of the fixatives play an important role in the formation of the paddles. If the entire fixation with a buffered fixative has been performed at low temperature, some of the sensory cilia are paddle-like, whereas other sensory cilia show the typical uniform cylindrical diameter throughout their length. If the fixation has started at room temperature or if the entire fixation is carried out at room temperature, generally more sensory cilia possess a swelling, less or no unmodified sensory cilia existing in the cephalic lobe of the animals. In addition the paddles are more strongly bent (Fig. 3b); the areas of the membranes spread within the curved axonemes of the cilia are not arranged in a special manner but appear randomly orientated. Changes of the osmolality of the fixatives also influence the number and structure of the paddles. If the osmolality of the fixative is lower than the osmolality of the sea water ( < 870-930 mOsm), some single cilia always show the normal cylindrical shape. After increasing the osmolality ( > 930 mOsm) by adding more sucrose to the fixatives all the sensory cilia have paddles, which moreover often stick together. Furthermore the terminal parts of single locomotor cilia of the epidermal cells also swell. The EM fixatives mentioned under (a), (b) and (c), respectively, in Materials and Methods as well as the solutions of sucrose, calcium chloride, sodium cacodylate, monobasic or dibasic sodium phosphate, or formaldehyde apparently have the same influence on the formation of the paddles. With each of the chemicals named above the number of paddles per animal is correlated to increasing osmolality and temperature. The different dehydration substances (ethanol, acetone), drying and evaporation media are of no influence on the formation of paddles. The paddles may be reversible or irreversible structures: In living otoplanids, which for example are treated with sucrose at 0~4 ~ C for 1-2 min, the kinocilia of the bristles show terminal paddles, but the kinocilia are still straight, wound around each other and only slightly shortened. When these otoplanids are returned to natural sea water, the kinocilia of the bristles resume their normal cylindrical shape within 2-4 h. In living otoplanids treated with sucrose at room temperature, most of the modified kinocilia are disorientated, strongly bent and shortened. Such kinocilia usually detach from the animal when the otoplanids are returned to sea water.
Discussion As far as we know, paddle-like cilia in living animals have been described only by Heimler (1978) in the Aulophoralarva of Lanice. According to our observations, living, untreated marine Turbellaria do not possess comparable structures. Paddle cilia or discocilia only originate after the addition of certain chemicals to the sea water. Therefore the paddles or discs are supposedly not genuine organelles.
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In histological sections the existence of paddles has been known for 70 years (Luther, 1905, p. 27 and Plate I, Figs. 23 and 24). When Turbellaria are prepared in a fixative containing formaldehyde almost all cilia of sensory cells and some locomotor cilia of epidermal cells have distended terminal parts (unpublished light microscopical and SEM observations). In this case the solvent undoubtedly causes the swellings of the cilia. In contrast, the chemicals frequently used in electron microscopy (glutaraldehyde, osmic acid) do not regularly induce these structural changes. The formation of paddles is caused by distinct buffers and fixation additives such as sodium cacodylate, monobasic or dibasic sodium phosphate, sucrose, and calcium chloride. This interpretation is furthermore based on our investigation with fixatives of different osmolalities and different temperatures. The number of paddles of sensory cilia increases in proportion to the increasing osmolality and the increasing temperature until finally even non-sensory cilia are enlarged terminally. Obviously the plasmalemma of certain or all sensory cilia is especially permeable for certain molecules of the solutions with increasing osmolality and temperature. When several Turbellaria are fixed simultaneously in a cold buffered hypotonic solution, some animals have few paddle-like sensory cilia, other animals have many. In contrast, the locomotor cilia have blunted tips. Apparently the cytoplasm or plasmalemma of the two kinds of cilia react differently to the fixatives. The varying number of paddle cilia in simultaneously fixed individuals could be due to a different physiological condition of single sensory organelles at the moment of fixation. Thus the paddle cilia are artificial structures. Their formation seems to depend on the immediate physiological condition of the sensory cilium. The paddle-like cilia in Turbellaria correspond to the description of modified cilia in Rhabdopleura (Dilly, 1977a, Fig. 10), in Lanice (Heimler, 1978, Fig. 4ff) and especially in Mytilus (Tamarin et al., 1976, Fig. 15). It should be mentioned that Tamarin et al. fixed at room temperature and raised the osmolality of the fixative by adding 0.33 M sucrose (Tamarin et al., 1974). Lane and Nott (1975), who examined a pediveliger of Mytilus, and who used fixation media without sucrose and at a low temperature, do not describe paddle cilia. Cilia with an enlarged plasmalemma are further known in other invertebrates and vertebrates. These modified cilia belong to cells which have, or which are supposed to have, a sensory function, mostly a chemoreceptive one (Reese, 1965; Welsch and Storch, 1969; Storch and Welsch, 1969, p. 531; Barber and Wright, 1969, Fig. 5; Seifert, 1970, Fig. 94 and p. 85ff; Storch and Moritz, 1971, Fig. 3a; Moir, 1977, Fig. 7, and others). Some of these authors regard these differentiations as genuine structures, others as fixation artefacts or as a phenomenon of degeneration. In our opinion the modifications are caused by the fixation buffers used by the authors. However, whether or not the enlargement of the ciliary membrane is a genuine structure can only be determined when the specimens are prepared under different conditions. Storch (1972) observed enlargements of the ciliary plasmalemma in fixed and unfixed gastropod material, but the unfixed specimens had been stored for a short time in a hypertonic solution of glycerol. Similar conclusions may be applied to
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Cobb's observations in cilia of eulamellibranchs. The cilia with an enlarged plasmalemma described by this author (1969, Fig. 1) partly show a rectangular shape and an axonemal complex with an electron dense matrix. Kilburn et al. (1977) demonstrated that such changes in structure appear in cilia of eulamellibranchs which were transferred into hypertonic solutions. In conclusion, we do not regard the paddle-like cilia observed by us as genuine structures. Indeed, the artificial formation of paddles is instructive in indicating that cilia of sensory cells are apparently much more delicate than cilia of epidermal cells. The difference is probably due to distinct structural characteristics of the plasmalemma of the two kinds of cilia.
References Ax, P.: Monographie der Otoplanidae (Turbellaria). Morphologie und Systematik. Akad. Wiss. Lit. Mainz, Abhandl. Math.-naturw. K1. 1955, Nr. 13, 499-796 (1956) Baldetorp, L., Mecklenburg, C.v., H~tkansson, C.H.: Ultrastructural alterations in ciliary cells exposed to ionizing radiation. A scanning and transmission electron microscopic study. Cell Tiss. Res. 180, 421-431 (1977) Barber, V.C., Wright, D.E.: The fine structure of the sense organs of the cephalopod mollusc Nautilus. Z. Zellforsch. 102, 293-312 (1969) Bedini, C., Ferrero, E., Lanfranchi, A.: Fine structural observations on the ciliary receptors in the epidermis of three otoplanid species (Turbellaria, Proseriata). Tissue &Cell 7, 253-266 (1975) Bergquist, P.R., Green, C.R., Sinclair, M.E., Roberts, H.S.: The morphology of cilia in sponge larvae. Tissue & Cell 9, 179-184 (1977) Cobb, J.L.S.: Modified cilia in the gut of the lamellibranch mollusc Tapes watlingi. J. Cell Biol. 43, 192-195 (1969) Dilly, P.N.: Material transport within specialised ciliary shafts on Rhabdopleura zooids. Cell Tiss. Res. 180, 367-381 (1977a) Dilly, P.N.: Further observations of transport within paddle cilia. Cell Tiss. Res. 185, 105-113 (1977b) Ehlers, U., Ehlers, B.: Monociliary receptors in interstitial Proseriata and Neorhabdocoela (Turbellaria Neoophora). Zoomorphologie 86, 197-222 (1977) Heimler, W.: Discocilia - a new type of kinocilia in the larvae of Lanice conchilega (Polychaeta, Terebellomorpha). Cell Tiss. Res. 187, 271-280 (1978) Kharkeevich, T.A.: Electron microscope study on the statocyst in Arenicola marina under acceleration, vibration and sound influence. Tsitologiya SSSR 19, 120-122 (1977) Kilburn, K.H., Hess, R.A., Thurston, R.J., Smith, T.J,: Ultrastructural features of osmotic shock in mussel gill cilia. J. Ultrastruct. Res. 60, 34-43 (1977) Lane, D.J.W., Nott, J.A.; A study of the morphology, fine structure and histochemistry of the foot of the pediveliger of Mytilus edulis L. J. mar. biol. Ass. U. K. 55, 477-495 (1975) Luther, A.: Zur Kenntnis der Gattung Macrostoma. In: Festschrift ftir Prof. Palm6n, Vol. 1, part 5, pp. 1-61. Helsingfors 1905 Mecklenburg, C.v., Mercke, U., H~kansson, C.H., Toremalm, N.G.: Morphological changes in ciliary cells due to heat exposure. A scanning electron microscopic study. Cell Tiss. Res. 148, 45-56 (1974) Moir, A.J.G.: On the ultrastructure of the abdominal sense organ of the giant scallop, Placopecten magellanicus (Gmelin). Cell Tiss. Res. 184, 359-366 (1977) Oldfield, S.C.: Surface fine structure of the globiferous pedicellariae of the regular echinoid, Psammechinus miliaris Gmelin. Cell Tiss. Res. 162, 377-385 (1975) Reese, T.S.: Olfactory cilia in the frog. J. Cell Biol. 25, 209-230 (1965) Seifert, K.: Die Ultrastruktur des Riechepithels beim Makrosmatiker. Eine elektronenmikroskopische Untersuchung. In: Normale und Pathologische Anatomic, Vol. 21 (W. Bargmann, V. Doerr, eds.), pp. 1-99. Stuttgart: G. Thieme 1970 Storch, V.: Elektronenmikroskopische und histochemische Untersuchungen tiber Rezeptoren yon Gastropoden (Prosobranchia, Opisthobranchia). Z. wiss. Zool. 184, 1-26 (1972)
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Storch, V., Alberti, G.: Ultrastructural observations on the gills of polychaetes. Helgol/inder wiss. Meeresunters. 31, 169-179 (1978) Storch, V., Moritz, K.: Zur Feinstruktur der Sinnesorgane von Lineus ruber O.F. Miiller (Nemertini, Heteronemertini). Z. Zellforsch. 117, 212-225 (1971) Storch, V., Welsch, U.: Uber Bau und Funktion der Nudibranchier-Rhinophoren. Z. Zellforsch. 97, 528--536 (1969) Tamarin, A., Lewis, P., Askey, J.: Specialized cilia of the byssus attachment plaque forming region in Mytilus californianus. J. Morph. 142, 321-327 (1974) Tamarin, A., Lewis, P., Askey, J.: The structure and formation of the byssus attachment plaque in Mytilus. J. Morph. 149, 199-221 (1976) Welsch, U., Storch, V.: Ober das Osphradium der prosobranchen Schnecken Buccinum undatum L. und Neptunea antiqua (L). Z. Zellforsch. 95, 317-330 (1969)
Accepted April 7, 1978
Cell and Tissue Research 9 by Springer-Verlag 1978
Paddle Cilia and D i s c o c i l i a - Genuine Structures? Observations on Cilia of Sensory Cells in Marine Turbellaria Ulrich Ehlers and Beate Ehlers* II. Zoologisches Institut und Museum der Universit~it G6ttingen, Bundesrepublik Deutschland
Summary. Kinocilia of epidermal sensory cells in fixed marine Turbellaria often terminate as flattened biconcave discs. The distal part of the ciliary axoneme curves back upon itself forming a 360 ~ loop which is enveloped by the plasmalemma. In living animals this structure can be induced by the addition of sodium cacodylate, monobasic sodium phosphate, dibasic sodium phosphate, sucrose, calcium chloride, or formaldehyde to the sea water. Specimens treated with sodium chloride, glutaraldehyde, or osmium tetroxide do not show modified cilia. In animals prepared for EM at low temperature and with a buffered hypotonic fixative less kinocilia are modified than in animals treated with a buffered iso- or hypertonic fixative and at a higher temperature. It is assumed that the unusually shaped cilia, described as "paddle cilia" or "discocilia" in other invertebrates, do not represent a genuine but an artificial structure. Key words: Paddle cilia and discocilia - Turbellaria transmission electron microscopy.
Scanning and
In recent years cilia with an uncommon structure of the ciliary membrane have been found in several marine invertebrates (e.g. Tamarin et al., 1974, 1976; Oldfield, 1975; Bergquist et al., 1977; Dilly, 1977a, b; Heimler, 1978; Storch and Alberti, 1978). The distal ends of these cilia show a flattened swelling with an annular curvature of the axoneme. The modified cilia are believed to be true features and are described as a new type of specialized organelles, called "paddle cilia", "club-footed cilia" or "discocilia". Using scanning and transmission electron microscopy we have found modified cilia resembling "paddle cilia" or "discocilia" in many species of marine Send offprint requests to." Dr. U. Ehlers, II. Zoologisches Institut und Museum der Universit~it, Berliner Str. 28, D-3400 G6ttingen, Federal Republic of Germany
* Thanks are due to Dr. C. Marschall for correcting the English of the manuscript. Financial support was provided by the Akademie der Wissenschaften und der Literatur, Mainz
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turbellarians. T h a t ciliary c l u b b i n g can be caused by external influences, e.g. high temperature ( M e c k l e n b u r g et al., 1974), v i b r a t i o n (Kharkeevich, 1977), a n d i r r a d i a t i o n (Baldetorp et al., 1977) is k n o w n . Therefore we varied the c o n d i t i o n s u n d e r which the specimens were prepared in order to determine whether the swellings o f the cilia are artefacts or genuine structures.
Materials and Methods Light microscopic and EM-studies were carried out on 29 turbellarian species, especially on 5 representativesof the family Otoplanidae (Proseriata, Neoophora): DicoelandroporaatriopapillataAx, 1956; NotocaryoplaneUa glandutosa (Ax, 1951); Parotoplana capitata Meixner, 1938; Parotoplanina geminoducta Ax, 1956; Praebursoplana steinboecki Ax, 1956. The animals were collectedat various marine localitieson the island of Sylt (North Sea). Some of the material was fixedimmediatelyafter collectionin the laboratories of the BiologischeAnstalt Helgoland, List/Sylt. Most of the turbellarians were transported live to Grttingen for fixation and further preparation including some experiments with live animals. No anesthetic agents were used. For transmission and scanning electron microscopy the animals were fixed either in (a) 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2-7.4) for 2 h, rinsed in the same buffer and postfixed in 1% osmium tetroxide in the same buffer for 11/2-2h, (b) 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2 7.4) for 2h, or (c) 1 ~o osmium tetroxide in 0.1 M sodium cacodylate buffer for 3-4h. Each fixation was carried out at 0 4 ~C, at room temperature, or initially for 10--20rain at room temperature, followed by 04~ Furthermore we used fixativesand buffers of different osmolalities(from 385 mOsm to more than 1000 mOsm)made by adding sucrose and traces ofCaCl z. The preparation for TEM followedthe steps described by Ehlers and Ehlers (1977). For SEM the fixed animals were dehydrated through graded series of ethanol or acetone. The specimenswere dried by the critical-point-method (with liquid carbon dioxide or freon) or freeze-dried. The preparations were finally sputtered with gold in a sputter coater or coated with gold-palladium in a vacuum-evaporator, and examined in a Zeiss NOVASCAN 30 at 15 kV. For light microscopy (bright-field, phase-contrast, and interference microscopy) living animals were slightlysquashed under a coverglasseither at 0 4 ~C or at room temperature. Then a small amount of sucrose (5 % or 10 %), calciumchloride (2 % or 5 %), sodium chloride (5 % or 10 %), sodium cacodylate (0.1 M, 0.2M or 0.5M), monobasic sodium phosphate (0.1 M, 0.2M or 0.5M), dibasic sodium phosphate (0.i M, 0.2 M or 0.5 M), formaldehyde (4%), glutaraldehyde (2.5%, 10% or 25%), or osmium tetroxide (2 %) was added to the squashed specimen.
Results Cilia with a modified distal tip were f o u n d in at least 16 species o f different systematical groups of m a r i n e Turbellaria. The paddle-like structures can be seen in the kinocilia o f m o n o - or multiciliary sensory cells only. These ciliary sensory cells occur frequently in the head region of the turbellarians a n d less frequently o n the other parts of the body. The perikarya of the sensory cells lie within the p a r e n c h y m a b e n e a t h the s u b e p i d e r m a l musculature. The distal parts of the cells, provided with one or several cilia, penetrate the m o n o l a y e r e d epidermis a n d reach the periphery of the b o d y (cf. Bedini et al., 1975; Ehlers a n d Ehlers, 1977). Cilia are lacking in most o f the epidermal cells in almost all T u r b e l l a r i a Proseriata studied b e l o n g i n g to the family O t o p l a n i d a e (cf. Ax, 1956). Ciliated
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epidermal cells are restricted to the ventral side of an animal, forming a ribbon-like creeping sole, which begins in the region of the head and ends near the tip of the tail. Posterior to the cephalic lobe the creeping sole shows bilateral extensions forming a small ciliary ring (Fig. 1 a) or ciliary pits. In the regions of the body with nonciliated epidermal cells (i.e. in the ventrolateral, lateral, and dorsal parts of the body as well as in the cephalic lobe) none of the cilia are locomotor organelles, but they rather belong to sensory cells. For this reason these cilia can be clearly regarded as sensory cilia in SEM pictures. The following description of the structure of modified cilia is limited to the family Otoplanidae.
General Description of the Paddle-like Cilia The kinocilia of the cranial ciliary pits (Fig. 2a, above) and of the ventral creeping sole (Fig. 3a, at left) show the normal cylindrical shape. The kinocilia of the multiciliary sensory cells of the cephalic lobe are modified forming paddle-like cilia (Figs. 1 b-e, 2a, at left). Using higher magnifications it can clearly be seen that occasionally not all the cilia of a given sensory cell show the paddle-like shape. Unmodified kinocilia also occur (Fig. 2b, arrows). Multiciliated sensory cells with paddle-like cilia are further found within the dorsal opening between the ciliated pits (Fig. 2a, arrow) and adjacent to the ventral creeping sole (Fig. 3a, arrows). Here only some or all kinocilia (Fig. 3 b, c) are paddle-like cilia. By TEM and light microscopy, the paddle-like structure of the kinocilia can be verified. T E M profiles (Fig. 4 a-d) show cilia with an axoneme forming one or more circular loops of about 1-2 ~tm in diameter. There is only one single paddle-like structure present per kinocilium. The swelling is always situated on the distal tip of the kinocilium. Generally the axoneme turns in almost piano-spiral windings, but now and then the plane of one paddle is bent in itself (Fig. 3b). The cilia measure basally about 2000 A in diameter and have the normal pattern of 9 x 2 + 2 tubules. In the region of the terminal windings the axoneme tubules start coming together and the diameter of the axoneme is somewhat reduced (Fig. 4c). The tubules can be disintegrated. In cross sections they are then seen as isolated double or single tubules which are no longer arranged in a typical ring (Fig. 4d). Normally the axoneme curves back forming a 360 ~ loop. In addition to this condition paddle-like structures showing a bent axoneme, forming a kink (Fig. 4b) can be found. When the axoneme shows two or three complete piano-spiral windings (Figs. 4d, 6), most of the plasmalemma remains partly in contact with the axoneme. Only the terminal part of the bent axoneme sometimes separates completely from the plasmalemma (Fig. 4d). The swellings surrounded by the spread plasmalemma have a content of low contrast (Fig. 4b). In the larger paddles the swelling in the ciliary membrane appears empty (Fig. 4a, c, d). In some paddles small vesicles can be seen at the concave side of the axoneme (Fig. 4a, b, arrows). These vesicles lie in regular intervals in close contact to the peripheral double tubules.
Fig. I a-e. Dicoelandropora atriopapillata. Cephalic lobe (cO with sensory bristles (sb) and part of the cephalic ciliary ring (cr), interference microscope, room temperature, a Living animal in natural sea water. I r e Treated animals with paddle cilia (pc): b In 0.1 M sodium cacodylate, e In 5 ~ sucrose, d In 2 ~o calcium chloride, e In 4 ~ formaldehyde, a--e • 830; d and e x 2080
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Fig. 2 a and b. Parotoplana capitata, a Dorsal view of the anterior end. Paddle cilia (pc) at the tip of the cephalic lobe (lower left) and the dorsal opening (arrow) between the ciliated pits. x 920. b Several multiciliated sensory cells of the cephalic lobe. Some of the cilia (arrows) are not modified to paddle cilia. x 4600
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Fig.3 a-e. Parotoplaninageminoducta,a View of the ventral creeping sole (cs)with unmodified cilia. The arrows point to 7 serially arranged multiciliated sensory cells with paddle cilia, x 1350. b and c Multiciliated sensory cells from the lateral side of the body. In c the axonemes of three paddles show more than one 360 ~ loop (arrows). b x 8000, e x 8200 Morphological Changes of the Sensory Cilia Using Different Preparatory Techniques N u m e r o u s s e n s o r y bristles o c c u r at t h e c e p h a l i c l o b e o f t h e o t o p l a n i d s d e a l t w i t h in this p a p e r (Fig. 1 a; see also A x , 1956, p. 594; B e d i n i et al., 1975, p. 257). T h e s e bristles a r e c o m p o s e d o f several cilia s t a n d i n g closely t o g e t h e r . T h e cilia a r e l o n g
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Fig. 4 a-d. Parotoplaninageminoducta (a, b, d) and Notocaryoplanellaglandulosa (e). a--ePaddle cilia of monociliary sensorycellswith one 360~loop or with a kink (b) of the axoneme. The arrows point to small vesicles,a • 14,000,b • 27,200,c x 18,000. d Paddle cilia with two (2 asterisks) or three (3 asterisks) 360~ turns of the ciliary axoneme within the enlarged plasmalemma. The axoneme has become partially separated from the ciliary membrane (arrows). • 16,000
a n d w o u n d a r o u n d each other in living a n i m a l s a n d also in some fixed specimens ( u n p u b l i s h e d S E M observations). T e r m i n a l l y each sensory bristle tapers to a sharp tip. Paddle-like cilia do n o t occur. After the a d d i t i o n o f sucrose, calcium chloride, s o d i u m cacodylate, m o n o b a s i c or dibasic s o d i u m phosphate, or formaldehyde to the sea water, the straight kinocilia of the sensory cells are modified to paddle cilia within 1-10 sec at r o o m t e m p e r a t u r e (Fig. 1 b ~ ) . A t first, only a small swelling o f the m e m b r a n e occurs at the t e r m i n a l or n e a r the distal tip o f the cilia. T h e n the swellings enlarge to form typical paddle-like structures with a curved axoneme. T h e r e b y m a n y kinocilia
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Fig. 5 a and b. Dicoelandropora atriopapillata, a and b Modified sensory bristles of the cephalic lobe. Some of the paddles have particles attached to their surfaces (arrows) or stick together (double arrow). x 5100
s h o r t e n to less t h a n 50 ~ o f their n a t u r a l length a n d b e n d d o w n to the epidermis o f the animal. L o c o m o t o r cilia n o r m a l l y d o n o t show a typical p a d d l e - l i k e structure (Fig. 1 c). O n l y f o r m a l d e h y d e causes swellings o f the p l a s m a l e m m a o f these cilia. I n sensory kinocilia t r e a t e d with the a b o v e - m e n t i o n e d solutions at 0 - 4 ~ C, the f o r m a t i o n o f p a d d l e s takes 10 sec (calcium chloride) to 10 min (sucrose); only a few or no sensory cilia are bent.
Paddle Cilia and Discocilia in Turbellaria
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Fig. 6 a and h. Parotoplaninageminoducta (a) and Praebursoplanasteinboecki (b). a Multiciliatedsensory cell of the lateral body region with 3 paddle cilia; at left (arrow) more than one 360~ curvature of the axoneme. (rh) rhabdites. • 7800. b Multiciliated sensory cell with 7 paddle cilia; two paddles (arrows) with more than one 360~ loop of the axoneme. • 3600
Solutions o f s o d i u m chloride, glutaraldehyde, or o s m i u m tetroxide do n o t induce a paddle structure. I f several a n i m a l s are s i m u l t a n e o u s l y fixed for EM, paddle cilia n o r m a l l y do n o t a p p e a r in the same q u a n t i t y in all the individuals. Some o f these a n i m a l s have m a n y paddles, others have few. The p h e n o m e n o n t h a t u n d e r the influence o f identical E M processing c o n d i t i o n s the n u m b e r o f paddles per a n i m a l is i n c o n s t a n t considerably
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complicates the question of the extent to which the formation of paddle cilia is influenced by the chemical substances used for processing. Nevertheless some general statements can be made. Temperature, osmolality, and the chemical nature of the fixatives play an important role in the formation of the paddles. If the entire fixation with a buffered fixative has been performed at low temperature, some of the sensory cilia are paddle-like, whereas other sensory cilia show the typical uniform cylindrical diameter throughout their length. If the fixation has started at room temperature or if the entire fixation is carried out at room temperature, generally more sensory cilia possess a swelling, less or no unmodified sensory cilia existing in the cephalic lobe of the animals. In addition the paddles are more strongly bent (Fig. 3b); the areas of the membranes spread within the curved axonemes of the cilia are not arranged in a special manner but appear randomly orientated. Changes of the osmolality of the fixatives also influence the number and structure of the paddles. If the osmolality of the fixative is lower than the osmolality of the sea water ( < 870-930 mOsm), some single cilia always show the normal cylindrical shape. After increasing the osmolality ( > 930 mOsm) by adding more sucrose to the fixatives all the sensory cilia have paddles, which moreover often stick together. Furthermore the terminal parts of single locomotor cilia of the epidermal cells also swell. The EM fixatives mentioned under (a), (b) and (c), respectively, in Materials and Methods as well as the solutions of sucrose, calcium chloride, sodium cacodylate, monobasic or dibasic sodium phosphate, or formaldehyde apparently have the same influence on the formation of the paddles. With each of the chemicals named above the number of paddles per animal is correlated to increasing osmolality and temperature. The different dehydration substances (ethanol, acetone), drying and evaporation media are of no influence on the formation of paddles. The paddles may be reversible or irreversible structures: In living otoplanids, which for example are treated with sucrose at 0~4 ~ C for 1-2 min, the kinocilia of the bristles show terminal paddles, but the kinocilia are still straight, wound around each other and only slightly shortened. When these otoplanids are returned to natural sea water, the kinocilia of the bristles resume their normal cylindrical shape within 2-4 h. In living otoplanids treated with sucrose at room temperature, most of the modified kinocilia are disorientated, strongly bent and shortened. Such kinocilia usually detach from the animal when the otoplanids are returned to sea water.
Discussion As far as we know, paddle-like cilia in living animals have been described only by Heimler (1978) in the Aulophoralarva of Lanice. According to our observations, living, untreated marine Turbellaria do not possess comparable structures. Paddle cilia or discocilia only originate after the addition of certain chemicals to the sea water. Therefore the paddles or discs are supposedly not genuine organelles.
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499
In histological sections the existence of paddles has been known for 70 years (Luther, 1905, p. 27 and Plate I, Figs. 23 and 24). When Turbellaria are prepared in a fixative containing formaldehyde almost all cilia of sensory cells and some locomotor cilia of epidermal cells have distended terminal parts (unpublished light microscopical and SEM observations). In this case the solvent undoubtedly causes the swellings of the cilia. In contrast, the chemicals frequently used in electron microscopy (glutaraldehyde, osmic acid) do not regularly induce these structural changes. The formation of paddles is caused by distinct buffers and fixation additives such as sodium cacodylate, monobasic or dibasic sodium phosphate, sucrose, and calcium chloride. This interpretation is furthermore based on our investigation with fixatives of different osmolalities and different temperatures. The number of paddles of sensory cilia increases in proportion to the increasing osmolality and the increasing temperature until finally even non-sensory cilia are enlarged terminally. Obviously the plasmalemma of certain or all sensory cilia is especially permeable for certain molecules of the solutions with increasing osmolality and temperature. When several Turbellaria are fixed simultaneously in a cold buffered hypotonic solution, some animals have few paddle-like sensory cilia, other animals have many. In contrast, the locomotor cilia have blunted tips. Apparently the cytoplasm or plasmalemma of the two kinds of cilia react differently to the fixatives. The varying number of paddle cilia in simultaneously fixed individuals could be due to a different physiological condition of single sensory organelles at the moment of fixation. Thus the paddle cilia are artificial structures. Their formation seems to depend on the immediate physiological condition of the sensory cilium. The paddle-like cilia in Turbellaria correspond to the description of modified cilia in Rhabdopleura (Dilly, 1977a, Fig. 10), in Lanice (Heimler, 1978, Fig. 4ff) and especially in Mytilus (Tamarin et al., 1976, Fig. 15). It should be mentioned that Tamarin et al. fixed at room temperature and raised the osmolality of the fixative by adding 0.33 M sucrose (Tamarin et al., 1974). Lane and Nott (1975), who examined a pediveliger of Mytilus, and who used fixation media without sucrose and at a low temperature, do not describe paddle cilia. Cilia with an enlarged plasmalemma are further known in other invertebrates and vertebrates. These modified cilia belong to cells which have, or which are supposed to have, a sensory function, mostly a chemoreceptive one (Reese, 1965; Welsch and Storch, 1969; Storch and Welsch, 1969, p. 531; Barber and Wright, 1969, Fig. 5; Seifert, 1970, Fig. 94 and p. 85ff; Storch and Moritz, 1971, Fig. 3a; Moir, 1977, Fig. 7, and others). Some of these authors regard these differentiations as genuine structures, others as fixation artefacts or as a phenomenon of degeneration. In our opinion the modifications are caused by the fixation buffers used by the authors. However, whether or not the enlargement of the ciliary membrane is a genuine structure can only be determined when the specimens are prepared under different conditions. Storch (1972) observed enlargements of the ciliary plasmalemma in fixed and unfixed gastropod material, but the unfixed specimens had been stored for a short time in a hypertonic solution of glycerol. Similar conclusions may be applied to
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Cobb's observations in cilia of eulamellibranchs. The cilia with an enlarged plasmalemma described by this author (1969, Fig. 1) partly show a rectangular shape and an axonemal complex with an electron dense matrix. Kilburn et al. (1977) demonstrated that such changes in structure appear in cilia of eulamellibranchs which were transferred into hypertonic solutions. In conclusion, we do not regard the paddle-like cilia observed by us as genuine structures. Indeed, the artificial formation of paddles is instructive in indicating that cilia of sensory cells are apparently much more delicate than cilia of epidermal cells. The difference is probably due to distinct structural characteristics of the plasmalemma of the two kinds of cilia.
References Ax, P.: Monographie der Otoplanidae (Turbellaria). Morphologie und Systematik. Akad. Wiss. Lit. Mainz, Abhandl. Math.-naturw. K1. 1955, Nr. 13, 499-796 (1956) Baldetorp, L., Mecklenburg, C.v., H~tkansson, C.H.: Ultrastructural alterations in ciliary cells exposed to ionizing radiation. A scanning and transmission electron microscopic study. Cell Tiss. Res. 180, 421-431 (1977) Barber, V.C., Wright, D.E.: The fine structure of the sense organs of the cephalopod mollusc Nautilus. Z. Zellforsch. 102, 293-312 (1969) Bedini, C., Ferrero, E., Lanfranchi, A.: Fine structural observations on the ciliary receptors in the epidermis of three otoplanid species (Turbellaria, Proseriata). Tissue &Cell 7, 253-266 (1975) Bergquist, P.R., Green, C.R., Sinclair, M.E., Roberts, H.S.: The morphology of cilia in sponge larvae. Tissue & Cell 9, 179-184 (1977) Cobb, J.L.S.: Modified cilia in the gut of the lamellibranch mollusc Tapes watlingi. J. Cell Biol. 43, 192-195 (1969) Dilly, P.N.: Material transport within specialised ciliary shafts on Rhabdopleura zooids. Cell Tiss. Res. 180, 367-381 (1977a) Dilly, P.N.: Further observations of transport within paddle cilia. Cell Tiss. Res. 185, 105-113 (1977b) Ehlers, U., Ehlers, B.: Monociliary receptors in interstitial Proseriata and Neorhabdocoela (Turbellaria Neoophora). Zoomorphologie 86, 197-222 (1977) Heimler, W.: Discocilia - a new type of kinocilia in the larvae of Lanice conchilega (Polychaeta, Terebellomorpha). Cell Tiss. Res. 187, 271-280 (1978) Kharkeevich, T.A.: Electron microscope study on the statocyst in Arenicola marina under acceleration, vibration and sound influence. Tsitologiya SSSR 19, 120-122 (1977) Kilburn, K.H., Hess, R.A., Thurston, R.J., Smith, T.J,: Ultrastructural features of osmotic shock in mussel gill cilia. J. Ultrastruct. Res. 60, 34-43 (1977) Lane, D.J.W., Nott, J.A.; A study of the morphology, fine structure and histochemistry of the foot of the pediveliger of Mytilus edulis L. J. mar. biol. Ass. U. K. 55, 477-495 (1975) Luther, A.: Zur Kenntnis der Gattung Macrostoma. In: Festschrift ftir Prof. Palm6n, Vol. 1, part 5, pp. 1-61. Helsingfors 1905 Mecklenburg, C.v., Mercke, U., H~kansson, C.H., Toremalm, N.G.: Morphological changes in ciliary cells due to heat exposure. A scanning electron microscopic study. Cell Tiss. Res. 148, 45-56 (1974) Moir, A.J.G.: On the ultrastructure of the abdominal sense organ of the giant scallop, Placopecten magellanicus (Gmelin). Cell Tiss. Res. 184, 359-366 (1977) Oldfield, S.C.: Surface fine structure of the globiferous pedicellariae of the regular echinoid, Psammechinus miliaris Gmelin. Cell Tiss. Res. 162, 377-385 (1975) Reese, T.S.: Olfactory cilia in the frog. J. Cell Biol. 25, 209-230 (1965) Seifert, K.: Die Ultrastruktur des Riechepithels beim Makrosmatiker. Eine elektronenmikroskopische Untersuchung. In: Normale und Pathologische Anatomic, Vol. 21 (W. Bargmann, V. Doerr, eds.), pp. 1-99. Stuttgart: G. Thieme 1970 Storch, V.: Elektronenmikroskopische und histochemische Untersuchungen tiber Rezeptoren yon Gastropoden (Prosobranchia, Opisthobranchia). Z. wiss. Zool. 184, 1-26 (1972)
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Storch, V., Alberti, G.: Ultrastructural observations on the gills of polychaetes. Helgol/inder wiss. Meeresunters. 31, 169-179 (1978) Storch, V., Moritz, K.: Zur Feinstruktur der Sinnesorgane von Lineus ruber O.F. Miiller (Nemertini, Heteronemertini). Z. Zellforsch. 117, 212-225 (1971) Storch, V., Welsch, U.: Uber Bau und Funktion der Nudibranchier-Rhinophoren. Z. Zellforsch. 97, 528--536 (1969) Tamarin, A., Lewis, P., Askey, J.: Specialized cilia of the byssus attachment plaque forming region in Mytilus californianus. J. Morph. 142, 321-327 (1974) Tamarin, A., Lewis, P., Askey, J.: The structure and formation of the byssus attachment plaque in Mytilus. J. Morph. 149, 199-221 (1976) Welsch, U., Storch, V.: Ober das Osphradium der prosobranchen Schnecken Buccinum undatum L. und Neptunea antiqua (L). Z. Zellforsch. 95, 317-330 (1969)
Accepted April 7, 1978
