Zoomorphology (1993) 113 : 47-60

Zoomorphology �9 Springer-Verlag 1993

Functional morphology of coronal and peristomeal podia in Sphaerechinus granularis (Echinodermata, Echinoida) Patrick Flammang 1,. and Michel Jangoux 1,2

1 Laboratoire de Biologic marine, Universit~ de Mons-Hainaut, 19 ave. Maistriau, B-7000 Mons, Belgium 2 Laboratoire de Biologic marine (C.P. 160/15), Universit+ Libre de Bruxelles, 50 ave. F.D. Roosevelt, B-1050 Bruxelles, Belgium

Received October 21, 1992

Summary. Coronal podia of Sphaerechinus granularis are anchoring (adhering) appendages involved in either lo- comotion or capture of drift materials. Adhesion is not due to the presumed sucker action of the disc but relies entirely on secretions of the disc epidermis. Peristomeal podia function in wrapping together food particles or food fragments in an adhesive material thus facilitating their capture by the Aristotle's lantern. In both types of podia, the disc epidermis is made up of four cell types: non-ciliated secretory cells (NCS cells) that contain gran- ules whose content is at least partly mucopolysaccharidic in nature, ciliated secretory cells (CS cells) containing granules of unknown nature, ciliated non-secretory cells (CNS cells) and support cells. The cilia of CS cells are subcuticular whereas those of CNS cells, although also short and rigid, traverse the cuticle and protrude in the outer medium. All these cells are presumably involved in an adhesive/de-adhesive process functioning as a duo- gland adhesive system. Adhesive secretion would be pro- duced by NCS cells and de-adhesive secretion by CS cells. These secretions would be controlled through stim- ulations by the two types of ciliated cells (receptor cells) which presumably interact with the secretory cells by way of the nerve plexus. This model of adhesion/de- adhesion fits well with the activities of both coronal and peristomeal podia. The secretion of NCS cells would make up a bridge of adhesive material between a podium and the substratum (coronal podia) or would coat and gather food particles (peristomeal podia), respectively. The de-adhesive material enclosed in the granules of CS cells would allow the podia (either coronal or peristo- meal) to easily become detached from the substratum and to always remain clear of any particles.

* Research Assistant, National Fund for Scientific Research (Bel- gium)

Correspondence to. P. F1ammang

A. Introduction

Regular echinoids typically have two different types of podia, coronal podia arising from the ambulacra and peristomeal podia located on the peristomeal membrane. Their importance in echinoid biology has been empha- sized by several workers (Nichols 1966; Fenner 1973; Smith 1979; De Ridder and Lawrence 1982) who consid- ered the different functions in which they are involved, i.e. locomotion, feeding, sensory perception and respira- tion. Coronal podia take part in locomotion and anchor- age (Lawrence 1987). They adhere to the substratum by means of their sucker-like discs which contain numer- ous epidermal secretory cells (Nichols 1961; Coleman 1969; Burke 1980). Peristomeal podia, on the other hand, are supposed to be sensory in function although their disc epidermis also appears to contain secretory cells (Nichols 1961; Smith 1979). According to Hermans (1983), two basic types of secretory cells (viz. cells releas- ing an adhesive secretion and cells releasing a de-adhe- sive secretion) occur in the epidermis of echinoid podia, together forming a duo-gland adhesive system. On the contrary, McKenzie (1988) reported that echinoid podia only contain one type of secretory cell and, therefore, cannot be considered duo-glandular.

The present work describes the ultrastructure of the disc of both coronal and peristomeal podia in the echin- oid Sphaerechinus granularis. It focuses especially on the epidermal secretory cells, their possible relationships to the duo-glandular model of adhesion and their role in the operation of both types of podia.

B. Materials and methods

Specimens of Sphaerechinus granularis (Lamarck, 1816) were col- lected by SCUBA diving in Brest harbour (Brittany, France) in May 1990. They were transported to the marine biology laboratory of the Mons University where they were kept in a dosed circuit marine aquarium (13 ~ C, 3.3% salinity) and fed with chicory sea lettuce (Ulva rigida C. Agardh, 1824). Podial movements were in-

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vestigated on individuals kept in small glass aquaria, adoral podia being observed by placing a mirror beneath the individuals.

For light microscopy, podia were cut off from individuals pre- viously anaesthetized with propylene phenoxetol (0.1% in sea water), fixed in Bouin's fluid, embedded in paraplast and cut into 7-gin-thick sections. Sections were stained with Masson's trichrome and Mayer's hemalum coupled with phloxine and light green. A1- cian blue pH 2.6 and the periodic acid-Schiff (PAS) techniques were used for the detection of mucopolysaccharides, and mercuric bromophenol blue and the Danielli's technique for the detection of proteins (Ganter and Joll+s 1969-70). Footprints left by echin- oids' coronal podia were obtained by allowing individuals to walk across clean microscope slides. These were stained with 0.2% tolui- dine blue in Walpole buffer pH4.2 (for the detection of mucopolysaccharides; Ganter and Joll~s 1969-70) and with 0.06% brilliant blue G in 0.6 N HC1 (for the detection of proteins; Sedmak and Grossberg 1977).

For scanning electron microscopy (SEM), three sets of coronal podia and two sets of peristomeal podia were investigated: attached coronal podia (that adhered to the aquarium wall and were cut off from the echinoid body) and both protracted and retracted unattached coronal and peristomeal podia (that did not adhere to any substratum). Podia were fixed in Bouin's fluid for 24 h. They were dehydrated in graded ethanol, dried by the critical-point method (using CO2 as transition fluid), coated with gold in a sput- ter coater and observed with a Jeol JSM-6100 scanning electron microscope. In order to observe the organization of podial tissues, some podia were embedded in Paraplast and cut into two symmet- ric parts. Paraplast was removed in toluene and the two half-podia were processed as described above. Podial spicules were cleaned in 10% (v/v) common bleach, rinsed in distilled water, air-dried, mounted on aluminum stubs and coated with gold. Footprints left by echinoids were obtained by allowing coronal podia to adhere to coverslips. After the podia became detached, the coverslips were soaked in Bouin's fluid and prepared for SEM by critical-point drying.

For transmission electron microscopy (TEM), podia were fixed in 3% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.8) for 3 h at 4 ~ C. They were rinsed in cacodylate buffer and post-fixed for 1 h in 1% osmium tetroxide in the same buffer. After a final buffer wash they were decalcified according to the method of Dietrich and Fontaine (1975), dehydrated in graded ethanol and embedded in Spurr. Semithin sections (1 gm in thickness) were cut on a Rei- chert Om U2 ultramicrotome equipped with a glass knife. They were stained with a 1:1 mixture of 1% methylene blue in 1% natriumtetraborate and 1% azur II. Ultrathin sections (40-70 nm in thickness) were cut on a LKB V ultramicrotome equipped with a diamond knife. They were stained with uranyl acetate and lead citrate, and observed in a Philips EM 300 transmission electron microscope.

C. Results

L External morphology of the podia

Of the two types o f podia occurr ing in Sphaerechinus granularis, coronal podia are the mos t numerous (about 2000 for a specimen of ca. 10 cm in diameter). They are located in the five ambulacra , f rom the external edge o f the apical system to the external edge o f the peristo- meal membrane . Peristomeal podia are always ten in number (two in each ambulac rum) and are located on the peristomeal membrane a round the mouth .

Corona l podia consist o f an extensible cylindrical stem topped by a disc (Figs. 1 3). In an echinoid o f ca. 10 cm in diameter, the stem's length can vary f rom a

few m m (in retracted podia) to 3 cm (in pro t rac ted po- dia), but in bo th cases the diameter is about 350 gm. The diameter o f the disc is smaller in aboral ly located coronal podia (about 600 gm) and increases gradual ly towards the oral par t o f the test (diameter ca. 1200 gin). In the disc, a central circular area (diameter about 80% o f the disc's diameter) is separated by a groove f rom a scalloped peripheral area which is cont inuous with the stem (Figs. 1-3). W h e n a pod ium is protracted, its cen- tral area is cone-shaped and prot rudes into the external med ium (Fig. 1). W h e n retracted, the central area con- tracts inwards and sinks with respect to the peripheral area (Fig. 2). W h e n attached, the whole apical surface (i.e. bo th the central and the peripheral areas) appears to be totally flat (Fig. 3).

The central area o f the disc o f coronal podia bears cilia (about 1 gm long) (Fig. 7). These are distributed more or less uni formly but are more numerous in the middle o f the area (i.e. the par t o f the disc tha t is the more apical when a pod ium is protracted). In the periph- eral area, clusters o f cilia (about 3 gm long; Fig. 8) are a r ranged in radial rows a round the central area, each row standing on a bulge o f the peripheral area (Figs. 2, 3, 6). There are about 25 rows on a podium.

Peristomeal podia are made up of a cylindrical stem and an apical disc (Figs. 4, 5). The stem (about 300 gm in diameter in an echinoid measur ing ca. 10 cm in diame- ter) is less extensible than that o f a coronal podium, maximal length being about 0.5 cm. The disc is elliptic (700 x 500 gin), the long axis o f the ellipse being tangen- tial to the edge o f the mouth . There is no marked differ- ence between the central area o f the disc and the periph- eral area which, however, is distinguishable by its scal- loped aspect (Fig. 4). In pro t rac ted podia, the apical sur- face o f the disc is flat (Fig. 4). In the retracted podia, the central area o f the disc contracts inwards and sinks relative to the peripheral area (Fig. 5).

The central area o f the disc o f peristomeal podia bears uni formly distributed cilia (about I gm long; Fig. 7) and there are clusters o f longer cilia (about 3 gm

Figs. 1-11. Outer aspect of coronal and peristomeal podia and their ossicles in Sphaerechinus granularis. D disc; FP finger-like projec- tion; S stem

Figs. 1-3. Coronal podia (respectively protracted, retracted and attached; arrowheads show a row of ciliary clusters)

Figs. 4, 5. Peristomeal podia (protracted and retracted, respectively)

Fig. 6. Clusters of cilia arranged in a row on the disc peripheral area of a coronal podium

Fig. 7. Cilium on the disc central area (coronal podium)

Fig. 8. Cluster of cilia on the disc peripheral area (peristomeal podium)

Fig. 9. Ossicle of the rosette of a coronal podium (distal surface, inner margin downward)

Fig. 10. Spicule of the frame of a coronal podium (inner margin downward)

Fig. 11. Ossicle of the rosette of a peristomeal podium (distal sur- face, inner margin downward)

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long) on the peripheral bulges (Fig. 8). Contrary to what occurs in coronal podia, ciliary clusters form here a ci- liated belt around the central area of the disc.

II. In vivo observations

Extended coronal podia sway slowly. If they contact a substrate, they may or may not adhere to it. When an echinoid is left in the aquarium, its adoral coronal podia generally adhere to the aquarium bottom and its adoral podia are protracted, swaying slowly. As for per- istomeal podia, they seem not very active. However, when some food particles are placed on the aquarium bottom they begin a continuous movement. Each per- istomeal podium extends obliquely towards the zone in front of the mouth, presses its disc against the aquarium bottom, retracts and then starts on a new cycle. If the disc is pressed against particles, these appear to be linked together by a translucent material, but they never remain on the apical surface of the disc when the podium re- tracts.

Stimulations, consisting of a slight contact between a needle and different areas of the podia, were per- formed. Three sets of podia were investigated: pro- tracted coronal podia, attached coronal podia and pro- tracted peristomeal podia. In protracted coronal podia, contacting the stem induces the retraction of the podium, while touching the central area of the disc causes the podium to adhere to the needle and touching the periph- eral area of the disc induces the bending of the disc towards the point of stimulation. These two last stimula- tions do not lead to the retraction of the podium. On the contrary, contacting the stem of an attached coronal podia has no effect. However, touching the peripheral area of the disc induces the podium to become detached within a few seconds. In peristomeal podia, all stimula- tions we have tried always leads to the retraction of the podium.

IlL Fine structure of the disc of coronal podia

The disc of a coronal podium consists of four tissue layers: an inner mesothelium, a connective tissue layer, a nerve plexus and an outer epidermis covered by a cuti- cle.

1. Mesothelium

The mesothelium surrounds the ambulacral lumen. It is a pseudostratified epithelium made up of two cell types, adluminal cells and myoepithelial cells. Adluminal cells are monociliated cells lining the ambulacral cavity. Myoepithelial cells are generally located basal to the cell bodies of adluminal cells. Myoepithelial cells enclose a bundle of myofilaments and arrange to form the muscle systems of the podia.

There are two different muscle systems in the disc of coronal podia, the retractor and the levator systems

(Fig. 13). These systems are identical to those described in the podia of other species of regular echinoids (Ni- chols 1961). The former consists of the apical end of the stem retractor muscle which anchors to the connec- tive tissue at the level of the rosette (distal part of the disc-supporting skeleton). The latter connects the frame (proximal part of the disc-supporting skeleton) to the middle of the central area of the disc. The levator system is peculiar because of the arrangement of its cells which attach to the connective tissue at their ends and extend into the ambulacral lumen, thus traversing the adluminal cell layer twice.

2. Connective tissue

The connective tissue layer occupies most of the volume of the disc. This tissue is made up of an amorphous ground substance that encloses collagen fibres, spheru- lous coelomocytes, other cells that may represent fibro- cytes or phagocytic coelomocytes and the skeletal ele- ments of the disc. The collagen fibres can be divided into two sets. In the first set, they act as ligaments be- tween the muscle systems and the skeleton (retractors and levators are linked, respectively, to the rosette and to the frame). The fibres arise from the inserting point of the muscles, extend towards the skeletal elements and invade the stereomic pores of these elements. In the sec- ond set, the collagen fibres act as epidermis-anchoring structures. They arise basally from the stereomic pores of the rosette's elements, extend towards the apex of the podium, insinuate themselves between the epidermal cells and attach apically to the support cells of the epi- dermis (Figs. 12, 15).

The disc skeleton consists of two superposed struc- tures, a distal rosette and a proximal frame. The rosette is circular with a central hole for the ambulacral lumen. It consists of five elements made of perforate stereom and which bear five to six sharp, finger-like projections extending towards the apical surface of the disc (Fig. 9). The frame consists of numerous arc-shaped spicules (Fig. 10) which form a complete ring around the ambu- lacral lumen on the proximal side of the rosette.

3. Nerve tissue

The nerve tissue of the disc chiefly consists of nerve strands. At the base of the disc lies a circular thickening of the stem nerve plexus, the nerve ring. This ring gives off radial branches (the lateral strands; Fig. 14) which go over the proximal surface of the rosette and pass through the indentations separating the finger-like pro- jections to become the nerve system of the central area of the disc. This system has two components: one in which the direction of the strands is mainly radial and a second, distal to it, in which the direction is mainly concentric (Fig. 14). The former is located about 35 gm below the surface of the epidermis and the latter about 25 gm. There are frequent connections between the two, the whole forming a network whose meshes are filled

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Fig. 12. Reconstruction of a longitudinal section through the cen- tral disc epidermis of a coronal podium of S. granularis (not to scale), BB basal body; BF bundle of filaments; BL basal lamina; CNS ciliated non-secretory cell; CPC cuticle-protruding cilium; CS ciliated secretory cell; CSG condensing secretory granule; CTP connective tissue protrusion; CU cuticle; G Golgi zone; M I mito-

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chondrion ; M P microvillar-like celt process; M T microtubule; M V microvillns; N C S non-ciliated secretory cell; N S nerve strand; R E R rough endoplasmic reticulum cisternae; SC support cell; SCC subcuticular cilium; SD septate desmosome; SG secretory granule; SR striated rootlet; VE vesicle; ZA zonula adhaerens

with connective tissue protrusions and epidermal cell processes.

The nerve tissue of the disc (Figs. 15, 23) comprises nerve cell bodies as well as numerous nerve processes which contain mitochondria, microtubules and clear and/or dense core vesicles. Beneath the central area, these processes run mainly in a plane parallel to the apical surface of the podium.

4. Epidermis

Two different epidermal organizations can be distin- guished in the disc on the basis of the cell types they enclose: the central epidermis extending over the central area of the disc and the peripheral epidermis extending over the peripheral area of the disc (Fig. 14). In both

cases, all epidermal cells are connected apically by junc- tional complexes made up of a distal zonula adhaerens and a proximal septate desmosome (Figs. 22, 25). The epidermis is covered by a cuticle which is similar all over the disc. In our preparations, this cuticle was poorly preserved (Fig. 21) possibly due to the glutaraldehyde fixation (Holland and Nealson 1978; Campbell and Crawford 1991). However, it appears to consist of two filamentous layers: a thin (about 100 nm) external layer, partly made of the fine curly filaments topping the sup- port cell microvilli, and a thicker (about 500 nm) internal layer. The cuticle is separated from the epidermis by a subcuticular space (about 3 gm thick) that is crossed by the microvilli of the epidermal cells.

a) Central epidermis. The central epidermis is made up of four cell types" non-ciliated secretory cells (NCS

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cells), ciliated secretory cells (CS cells), ciliated non-se- cretory cells (CNS cells) and support cells. All these cells are flask-shaped; their enlarged nucleus-containing bas- al parts are located at the level of or under the nerve network, sunk in the connective tissue (Figs. 12, 15-17). Each epidermal cell has a long and narrow apical neck reaching the disc surface (Fig. 15). NCS cells are the longest of all cell types (about 150 ~tm long) and their cell bodies are housed in large stereomic pores of the distal surface of the rosette (Fig. 17). The three other cell types are shorter (about 50 gm long) and their cell bodies are located just beneath or at the level of the nerve network (Fig. 16). Epidermal cell bodies occur in clusters where the four cell types are represented (NCS and support cells are however more numerous than the two ciliated cell types) and which are separated by con- nective tissue protrusions (Figs. 15, 18). At the apex, the necks of support cells enlarge so that all epidermal cells join one another to form a continuous cellular layer (Fig. 15).

The cell bodies of NCS cells send several short cyto- plasmic processes near the nucleus-containing part of the cell (Fig. 17) and have one long neck reaching the surface of the disc (Fig. 15). At this level, the latter splits into numerous thick microvillar-like cell projections that radiate in between the microvilli of support ceils (Figs. 15, 24). The microvillar-like cell projections of all NCS cells together cover the whole surface of the central area of the disc. The cytoplasm of NCS cell bodies is filled with densely packed, membrane-bound secretory granules whose diameter measures from 250 to 350 nm (Fig. 19). These granules also occur in the basal cell pro- cesses, in the cell neck and in the microvillar-like cell projections (Figs. 15, 24). NCS granules contain a cy- lindrical core of parallel fibrils, which are very electron dense, surrounded by granular material of medium elec- tron density (Figs. 19, 24). These granules stain brightly with alcian blue pH 2.6 (all methods for the detection of proteins have failed). The cytoplasm also contains a well-developed Golgi apparatus near the nucleus, and cisternae of rough endoplasmic reticulum (RER) occur-

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Figs. 13-18. Fine structure of the disc of coronal podia of S. granu- laris. BL basal lamina ; CE central epidermis; CN concentric nerve strand; CO clot of coelomocytes; CS ciliated secretory cell; CTP connective tissue protrusion, FP rosette finger-like projection; L ambulacral lumen; LM levator muscle system; LN lateral nerve strand ; MP microvillar-like cell projection; M V microvillus; NCS non-ciliated secretory cell ; NS nerve strand; PE peripheral epider- mis; RN radial nerve strand; RT rosette trabeculae; SC support cell

Fig. 13. Longitudinal section through the disc (SEM)

Fig. 14. Oblique section through the disc following the x-y plane of Fig. 13 (semithin section) Figs. 15--17. Longitudinal sections through the central epidermis (apex, just beneath the nerve network, and near the distal surface of the rosette, respectively) Fig. 18. Transverse section through the central epidermis at the level of the nerve network

ring around the nucleus, at the periphery of the cell and in the basal processes (Fig. 19). RER cisternae are sometimes distended and filled with amorphous materi- al. Developing secretory granules are closely associated with Golgi membranes and RER cisternae suggesting that these organelles are involved in the synthesis of the granule content (Fig. 19). Finally, the cytoplasm in- cludes some mitochondria and a few clear vacuoles.

The cytoplasm of CS cells is filled with small, mem- brane-bound, elliptic secretory granules (150 x 250 nm) (Fig. 20). These granules show an electron-dense core surrounded by a thin clear space. The cytoplasm also contains numerous mitochondria, a Golgi apparatus, RER cisternae and longitudinally arranged microtu- bules. The apex of CS cells bears granule-containing, microvillar-like cell projections (Fig. 21) and a cilium (about 4 ~tm long) which has a regular 9 + 2 arrangement of microtubules and possesses a long striated rootlet (Fig. 22). We never observe these cilia traversing the cuti- cle and, therefore, they appear to be subcuticular. The basal part of the cell terminates within the nerve strands where granules similar in both shape and size to those occurring in CS cells can be seen in some nerve processes (Fig. 23).

The cytoplasm of CNS cells includes mitochondria, a few small clear vesicles of various shapes and sizes, and longitudinally arranged microtubules. The charac- teristic feature of these cells is a single short cilium (about 5 ~tm long) (Fig. 24) that generally traverses the cuticle, but that is sometimes observed lying above the plasma membrane between the epidermal cell microvilli. This cilium has a regular 9 + 2 arrangement of microtu- bules and possesses a long striated rootlet. It is sur- rounded by a collar of microvilli. These cilia correspond to those we observed on SEM pictures of the central area of the disc (Fig. 7).

The neck of support cells is strip-shaped in transverse section. A few microns before reaching the disc surface, it divides in two or three processes which join to the enlarged plate-shaped apical part of the cell (Fig. 15). The enlarged parts of support cells bear numerous mi- crovilli and together form a layer beneath the microvil- lar-like cell projections of NCS cells (Figs. 12, 15). Al- most the entire cytoplasm of support cells is filled with filaments arranged perpendicularly to the disc surface. In the enlarged apical part of the cell, these filaments basally connect to the collagen fibres of the connective tissue protrusions and apically extend into the microvilli (Figs. 12, 15, 22). The basal cytoplasm also encloses mi- tochondria and a small Golgi apparatus.

b) Peripheral epidermis. The peripheral epidermis is a columnar epithelium made up of three types of elongated cylindrical cells: monociliated cells, mucous cells and covering cells. This epidermis and the underlying con- nective tissue form two distinct layers whose constituents do not interpenetrate each other. Monociliated cells are located above the lateral nerve strands (those running between the finger-like projections of the rosette) where- as the two other cell types occur throughout the periph- eral area of the disc.

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processes. The sensory cilia project into the interstitium, except for their bases which are surrounded by an elec- tron-dense ring similiar to a zonula adhaerens at the opening of the depression (Fig. 5B). Structures such as a septate junction, however, have not been found. In the basal depression several microvilli are present which do not extend farther than the electron-dense ring. The cilia have a normal 9 x 2 + 2 axoneme, dynein arms are very likely lacking and their basal bodies are not con- nected to rootlets.

Phaosomous photoreceptor. In Protodrilus ciliatus one presumed phaosomous photoreceptor has been found in each tentacle (Fig. 5 C-E). It is located approximately in the middle between tip and base about 250_+ 80 gm above the base where the total length of the palps is 550-650 gin. It lies ventrally underneath the lateral mus- cle fibres opposite the blood vessel and is covered by 4.5-gin-thick epidermal cells. The sensory cell forms a small " intracel lular" cavity 3.5-5 gm in diameter. The cavity is occupied by regularly arranged, microvillus like structures which are branches of two short cilia (Fig. 5D, E). The cilia have no rootlets. Their shafts contain a regular axoneme basally, but the central mi- crotubules are reduced just above the base and dynein arms are lacking. The space between the sensory struc- tures is occupied by electron-dense fibrillar material. The cell body is lateral to the cavity, contains the nucleus 5 gm in diameter, numerous mitochondria, a well-devel- oped rough ER and a Golgi complex. The sensory cell is in contact with glial cell processes, but no such process could be found to reach the cavity. The axon of this sensory cell very probably joins the lateral palp nerve (vpn 1 in Fig. 8 A).

IV. Palp nerves

The two to five palp nerves are of different sizes depend- ing on the number of fibres included in each nerve. The

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Fig. 4A-K. Prostomial and tentacular ciliated sensory cells. A-E Sensory cells (sc) with long immobile cilia in Protodrilus. A P. haurakiensis. Apex of sensory cell and straight cilia. B P. ciliatus. Cell body of sensory cell. C P. haurakiensis. Prominent ciliary root- lets (r). D P. ciliatus. Transverse section through apices of the two sensory cells forming a ciliary tuft. E P. ciliatus. Cross-section through the mid-region of sensory cilia. Arrows point to dynein arms. F Protodriloides symbioticus. Multiciliated dendritic process. Note centriole (ce) and rootlet (arrow) between mitochondria. G P. ciliatus. Uniciliary receptor, cilium (c) associated with microvilli (my) not penetrating the cuticle. H P. ciliatus. Multiciliated dendrit- ic process with stiff cilia. I, J Sensory cells with cilia not penetrating the cuticle. P. ciliatus. I Cilium in longitudinal section. J Cross- section near base of cilium. K P. ciliatus. Transverse section through apex of uniciliary receptive ending with an electron-dense cyclinder (eric) beneath the microvilli, bb basal body; cu cuticle; dv dense vesicle; ep epidermal supportive cell; g Golgi apparatus; mb multivesicular body; n nucleus of sensory cell; sj septate junc- tion; v vesicle; za zonula adhaerens

number of nerve cell processes varies greatly between species (between 125 in ProtodriIoides symbioticus and > 1300 in Saccocirrus krusadensis). In each species the medial nerve (vpn4) is the largest one comprised of be- tween 100 fibres in Protodriloides syrnbioticus and more than 1000 in Saccocirrus krusadensis (counted near the palp bases). In the other species, the specific numbers are as follows: Protodriloides chaetifer about 180, Pro- todrilus adhaerens about 230, Protodrilus ciliatus and Protodritus haurakiensis about 600 and Saccocirrus papil- locercus about 850 (Fig. 6A, E). The other nerves are considerably smaller and only consist of 5-25 fibres in Protodriloides and Protdrilus except vpn2 in P. haura- kiensis where it is comprised of about 60 fibres. In Sacco- cirrus 10-230 nerve cell processes form the correspond- ing nerves. Where the palps are medially attached to the prostomium in Protodrilus the main palp nerve is divided by processes of epidermal cells into several bun- dles which unite again before entering the neuropile ot the brain (Fig. 6 B).

The nerves are composed of afferent and efferent fibres. Each nerve is partly enveloped by glial cell pro- cesses and in the main palp nerve they extend between the nerve fibres (Fig. 6 A, C, H). In the nerve fibres mito- chondria, neurofilaments, microtubules and vesicles are present (Fig. 6A-H) . The latter are of two types: clear vesicles with a diameter of 40 60 nm and dense-cored vesicles of 70 95 nm diameter. The nerve cell processes vary in their diameter from 0.04 by 0.11 gm to 1.0 by 1.6 gm. Synapses were recognized by accumulations of electron-lucent regular vesicles and an increased elec- tron-density and slightly thickening of the plasma mem- branes (Fig. 6C-E, G, H). Neuroneuronal and neu- romuscular junctions have frequently been observed within the nerve tracts (Figs. 6 C, D, H, 9 D). In addition, neuroepithelial synapses with the epidermal cells form- ing the field of motile cilia occur in Protodriloides (Fig. 6F, G). Neuroepithelial and neuromuscular junc- tions built up by isolated axons were encountered as well (Figs. 6E, G, 9E). Synapses are organized as but- tons or " en passant" ; the latter synapses are very likely the common type of the neuroepithelial junctions in the ventral ciliary field in Protodriloides. At the neuromuscu- lar junctions the synaptic membranes are constantly sep- arated by the extracellular lamina surrounding the palp canals or the bundles of muscle cells.

The glial cell processes associated with the nerves and basal parts of epidermal cells are characterized by nu- merous ovoid granules, 0.5-1 ~tm long and 0.!5-0.35 gm wide, and bundles of tonofilaments which often end in desmosomes with adjacent glial cells. The granules are more or less electron-dense in Protodrilus and Saccocirr- us; in Protodriloides they are somewhat larger (up to 0.5 by 1 gm) and have a fibrillar contents whose staining is considerably lighter. The glial cell processes are nor- mally 0.3-0.9 gm thick, but may be up to 2 gm across. In regions without granules the membranes are close together and lie less than 0.09 gm apart. In Protodrilus the cytoplasm stains darker than in the other two genera. The cell bodies of these ramified cells are irregularly distributed along the nerves.

Monociliated cells bear a cilium (about 7 gm long) which has a regular 9 + 2 arrangement of microtubules (Fig. 25). These cilia correspond to those we observed on SEM pictures of the peripheral area (Figs. 6, 8). The cytoplasm of these cells includes mitochondria, a few small clear vesicles (about 500 nm in diameter) of var- ious shapes and longitudinally arranged microtubules. The basal part of these cells appears to terminate within the lateral nerve strands.

Mucous cells have their cytoplasm completely filled with large vacuoles (about 2.5 gm in diameter) which enclose finely granular material of variable electron den- sity. A small number of these cells are also scattered in the epidermis of the central area.

Covering cells have a centrally or apically located nucleus. The apical cytoplasm contains mitochondria and some clear vacuoles with diameters f rom 0.5 to 1 ~tm (Fig. 25). The latter sometimes enclose amorphous mate- rial. The apical surface of support cells bears numerous microvilli that are often branched. These cells send basal processes that at tach to the basal lamina of the epider- mis. They have filaments having a 850 nm periodicity and running their entire length to contact the apical membrane at one end and the basal lamina at the other.

IV. Fine structure o f the disc o f peristomeal podia

1. Inner tissues

The mesothelium has the same structure as in coronal podia. Nevertheless, as far as the muscles are concerned, the levators do not exist, the only muscles present being the retractors (compare Figs. 13 and 27). As in coronal podia, retractors anchor to the connective tissue at the level of the rosette.

The connective tissue layer is well developed and con- sists of a ground substance enclosing collagen fibres, different cell types and the skeletal elements. The con- nective tissue and the epidermis of peristomeal podia

Figs. 19--25. Fine structure of the disc epidermis of coronal podia of S. granularis. BB basal body; C cilium; CC covering cell; CNS ciliated non-secretory cell; CS ciliated secretory cell; CSG condens- ing secretory granule; CU cuticle; G Golgi zone; MC monociliated cell; MP microvillar-like cell projection; N nucleus; NCS non- ciliated secretory cell; RER rough endoplasmic reticulum cisternae; SC support cell; SD septate desmosome; SG secretory granule; SR striated rootlet; V vacuole; ZA zonula adhaerens

Fig. 19. Aspect and formation of secretory granules in a non-ciliat- ed secretory cell

F i g s . 211-22. Ciliated secretory cell (nucleus and basal cytoplasm, microvillar-like cell projection, and apex, respectively)

Fig. 23. Detailed view of a nerve strand (arrowheads show vesicles similar to the secretory granules of ciliated secretory cells, compare with Figs. 20 and 22)

Fig. 24. Apex of a ciliated non-secretory cell

Fig. 25. Longitudinal section through the peripheral epidermis (apex)

SC NCS CS CNS SC

55

SCC CPC

�9 ' _ c I �9 " ' - - , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " '

. . . . . . . . . . . i , . " ' " ' , . . . . . . . . . . . . . . .

Fig. 26. Reconstruction of a longitudinal section through the cen- tral disc epidermis of a peristomeal podium of S. granularis (not to scale). BF bundle of filaments ; BL basal lamina; BNP basiepith- elial nerve plexus; CNS ciliated non-secretory cell; CS ciliated se- cretory cell; CPC cuticle-protruding cilium; CSG condensing secre- tory granule; CT connective tissue; CU cuticle; G Golgi zone; MI mitochondrion; MP microvillar-like cell projection; MT micro- tubule; M V microvilli; NCS non-ciliated secretory cell; RER rough endoplasmic reticulum cisternae; SC support cell; SCC subcuticu- lar cilium; SD septate desmosome; SG secretory granule; SR striat- ed rootlet; V vacuole; ZA zonula adhaerens

form two distinct layers whose constituents do not inter- penetrate each other (Figs. 26, 27, 29). There is no frame and the skeleton consists only of a rosette. This rosette is made up of five trapezoid elements that are more irregular in shape and size than their equivalents in cor- onal podia and that bear blunt finger-like projections (Fig. 11).

The organization of the nerve tissue follows the same basic plan as in coronal podia except beneath the central area of the disc where it consists of a thick basiepidermic plexus instead of radial and concentric strands. This plexus encloses neurone bodies as well as numerous nerve processes which contain mitochondria, microtu- bules and clear and/or dense core vesicles.

2. Epidermis

The central epidermis is made up of four cell types: non-ciliated secretory cells (NCS cells), ciliated secretory cells (CS cells), ciliated non-secretory cells (CNS cells) and support cells. These cells share many common fea- tures with their equivalents in coronal podia, principally

56

Figs. 27-30. Fine structure of the disc of peristomeal podia of S. granularis. BE bundle of filaments; BL basal lamina; C cilium; CArS ciliated non-secretory cell; CO clot of coelomocytes; CS ciliated secretory cell; CT connective tissue; CU cuticle; E epidermis; L ambulacral lumen; M P microvillar-like cell process; N C S non-ciliated secretory cell; R E R rough endoplasmic reticulum cisternae; SC support cell; SG secretory granule; V vacuole

Fig. 27. Longitudinal section through the disc (SEM)

Figs. 28, 29. Longitudinal sections through the central epidermis (apex and base, arrowheads show membrane thickenings)

Fig. 30. Transverse section through the central epidermis

as far as the cytoplasmic content is concerned. However, the shape differs in that all the epidermal cells of peristo- meal podia are cyclindrical cells together forming a col- umnar epithelium about 60 pm thick (Figs. 26, 28, 29). Support cells are the most numerous. They form a sup- portive meshwork into which groups of the other cell types fit (Fig. 30). In these groups (which enclose one to three cells), NCS and CS cells are almost always asso- ciated (Fig. 30). CNS cells are proportionally less numer- ous than the two aforesaid cell types and they may occur together with NCS and CS cells or isolated.

NCS cells have a basally located nucleus. Their cyto-

plasmic content is very similar to that of the NCS cells of coronal podia, viz. a well-developed Golgi apparatus, RER cisternae and numerous secretory granules. These secretory granules measure about 250 pm in diameter and contain rather homogeneous material (Figs. 28, 30), different from that of coronal podia which enclose a complex fibrillar core. As in coronal podia, they stain brightly with alcian blue pH 2.6. The apical surface of NCS cells bears microvillar-like cell projections contain- ing secretory granules (Fig. 28) and their basal part atta- ches to the basal lamina of the epidermis by way of membrane thickenings (Fig. 29).

57

Except for their shape, both types of ciliated cells (CS and CNS) are almost identical to their equivalents of coronal podia (Fig. 28). CS cells, however, appear to be proportionally more numerous in the central epi- dermis of peristomeal podia and are larger, thus contain- ing more secretory granules.

Support cells have an apically located nucleus. The apical cytoplasm contains numberous clear vacuoles with diameters from 100 to 400 nm. The apical surface of support cells bears numerous microvilli that are often branched (Fig. 28). These cells attach to the epidermis basal lamina (Fig. 29). They enclose a bundle of fila- ments (about 500 nm in diameter; Fig. 30) having a 350 nm periodicity and running their entire length to contact the apical membrane at one end and the basal lamina at the other.

The peripheral epidermis is identical to that of coron- al podia for the cell types encountered, their location and their morphology. The cuticle is also similar to that of coronal podia (Fig. 28).

V. Characterization of the footprints

The prints left by coronal podia of S. granularis are disc-shaped, and, for an echinoid of about 10 cm in di- ameter, measure between 600 and 900 lain in diameter (this corresponds to the diameter of the central area of the disc of adoral podia). The whole footprint is made up of a rather homogeneous material which, however, appears to be less abundant in the middle of the print. Other smaller footprints (about 100 gm in diameter) were also observed. Under the light microscope, these prints have the same aspect as the middle area of the large footprints. All footprints stain brightly with tolui- dine blue but do not stain with brilliant blue, indicating that they are at least partly mucopolysaccharidic in na- ture.

D. Discussion

L Functions of coronal and peristomeal podia

Both the coronal and peristomeal podia of Sphaerechin- us granularis are built according to the same basic plan; they consist of a cylindrical stem topped by a specialized disc. The stem and the disc together form a functional unit with the stem allowing the podium to lengthen, flex and retract (antagonistic action of the hydrostatic pressure of the ambulacral fluid and the retractor mus- cles of the podium), and the disc allowing it to adhere onto the substratum.

Whatever their location (aboral or adoral), coronal podia end in a well-developed disc although this last is smaller in those of the aboral hemisphere. All coronal podia are anchoring appendages, the adorals being rath- er more involved in locomotion and the aborals in the capture of drift material which they hold on the test surface (a behaviour called 'covering reaction' and al- ready reported for S. granularis by Dambach and Hent-

schel 1970). The adhesive strength of coronal podia is brought about by the operation of their disc. Until now, two means of adhesion were thought to operate simulta- neously: the mechanical action of the sucker and the chemical action of the disc epidermal secretions (Nichols 1961 ; Coleman 1969; Burke 1980). The former was re- garded as a result of the operation of the levator muscles of the disc which, when contracting, would create a suc- tion cavity (Nichols 1961). However, adhesion of coron- al podia in S. granularis appears to rely entirely on disc epidermal secretions. Indeed, the occurrence of numer- ous epidermal secretory cells (whose secretory, granule- filled, apical projections cover the whole surface of the central area of the disc), the fact that the apical surface of attached podia is almost totally flat and the complete- ness of the footprints (they do not have areas devoid of adhesive material), all plead for an adhesive process only mediated by secretion.

Peristomeal podia lack the levator muscles and their associated skeletal frame (which was regarded as a lack of sucker; Nichols 1961) and were generally considered not very adhesive. This, together with their localization around the mouth, led the authors to suggest that they would be solely sensory in function, the secretory cells of the disc only ensuring that the podium adheres to an object long enough to test it (Nichols 1961; Smith 1979). In S. granularis, however, the secretory equipment of peristomeal podia is quite developed - it is at least comparable to that of coronal podia - and their adhesi- vity must consequently be quite important, suggesting an additional function for these podia. It has been shown that the buccal podia of clypeasteroid echinoids (which, like peristomeal podia of S. granularis, do not possess a levator muscle system and rely entirely on their apical epidermis secretions for adhesion) and the penicillate buccal podia of spatangoid echinoids are able to handle particles and to bring them to the mouth (Mooi 1986; De Ridder etal. 1987, respectively). Nevertheless, in S. granularis, the peristomeal podia do not need to handle plant or algal materials as the lantern can protract and pick them up by itself (Buchanan 1969). In accordance with their behaviour, we suggest rather that the function of peristomeal podia would be to wrap particles or small food fragments located beneath the mouth in an adhe- sive material in order to make the lantern's work easier. Such a technique is also used by the penicillate buccal podia of the spatangoid Echinocardium cordaturn (Pen- nant, 1777) which wrap sand grains in an adhesive mate- rial before trapping them in between their digitations (Flammang et al. 1991).

On the other hand, peristomeal podia are certainly also sensory, being presumably able to test the ' edibility' of detritic materials. These sensory abilities are possibly due to the clusters of monociliated cells located on the peripheral area of the disc (the fact that these ceils termi- nate within the lateral nerve strands is indicative of their sensory function). This sensory equipment is very similar to that of coronal podia and these last may also have important sensory abilities. Indeed, a coronal podium does not attach with every contact made, suggesting that certain criteria must be met before adhesion occurs. As

58

in peristomeal podia, the radial rows of monociliated cells located on the peripheral area of the disc could be the site of sensory perception (i.e. the site of initiation of the attachment behaviour).

II. Comparison with other echinoderms and other taxa

It is obvious that both coronal and peristomeal podia have important adhesive abilities based on the secretory activity of their disc epidermis. The major difference be- tween the epidermis of the central area of the disc of coronal and peristomeal podia lies in the shape of the epidermal cells, flask-shaped in the former and cylindri- cal in the latter. As for the rest, the epidermis consists of similar cell types in both podial types: non-ciliated secretory cells, ciliated secretory cells, ciliated non-secre- tory cells and support cells.

Non-ciliated secretory cells (NCS cells) contain gran- ules whose content is at least partly mucopolysaccharidic in nature. The aspect of these secretory granules is differ- ent according to whether they belong to NCS ceils of coronal or peristomeal podia. In the former, the granule content consists of a central core of fibrils surrounded by granular material while, in the latter, it consists of a rather homogeneous material. In asteroids, Engster and Brown (1972) have pointed out a relationship be- tween the internal organization of adhesive cell secretory granules and the possible adhesive strength of the podia: asteroids confined to hard rocky substratum have com- plex granules enclosing a highly organized core whereas soft substratum dwelling species have granules of consid- erably simpler ultrastructure. Similarly, the different as- pect of NCS cell secretory granules from coronal and peristomeal podia of S. granularis could be related to the adhesive strength the podia have to produce: a stronger adhesive strength is required to anchor to a substrate (coronal podia) than to wrap particles (peristo- meal podia). NCS cells of S. granularis are typically api- cal tuft cells (according to the terminology of McKenzie, 1988), i.e. their secretory granules are released at the tip of microvillar-like cell projections (see also Flam- mang and Jangoux 1992). Such cells have already been observed in the coronal podia of other species of regular echinoids (Coleman 1969; Burke 1980), in the penicillate podia of the spatangoid echinoid E. cordatum (Flam- mang et al. 1991) and in the locomotory podia and buc- cal tentacles of several holothuroid species (Cameron and Fankboner 1984; McKenzie 1987; Flammang and Jangoux 1992). In the podia of all these species, NCS cells are adhesive in function.

Both ciliated secretory cells (CS cells) and ciliated non-secretory cells (CNS cells) terminate within the nerve plexus with is indicative of their sensory function (CS cells thus may be considered neurosecretory-like). Ciliated cells morphologically similar to these two cell types occur in the podial adhesive areas of most echino- derm species. In S. granularis, CS cells appear to be more developed in peristomeal podia than in coronal podia. Nevertheless, in both types of podia, they contain granules of unknown nature and bear a cilium which

is subcuticular. CS cells are remarkably similar in all echinoderm species whose podia have been examined. In all cases, they are neurosecretory-like cells containing small electron-dense granules and often presenting a subcuticular cilium (Engster and Brown 1972; McKenzie 1987; Ball and Jangoux 1990; Flammang et al. 1991; Flammang and Jangoux 1992). Some workers have con- sidered them as de-adhesive cells although the way in which they operate remains enigmatic. Ball and Jangoux (1990) proposed that their secretions could act as neu- rotransmitters controlling or terminating the release of the adhesive secretion. On the other hand, Flammang et al. (1991) suggested that their granule content would be released into the outer medium, acting directly as a de-adhesive. (McKenzie, 1988, observed CS cells in podia of several species of regular echinoids but he did not recognize their secretory function, hence his asser- tion that echinoid podia cannot be considered duo-glan- dular.) CNS cells are monociliated cells whose cilium traverses the cuticle and protrudes into the outer medi- um. These cells, like CS cells, are almost identical in the podia of all the echinoderm species so far studied (Engster and Brown 1972; Burke 1980; McKenzie 1987; Ball and Jangoux 1990; Flammang et al. 1991; Flam- mang and Jangoux 1992).

Adhesive organs involved in temporary adhesion and resembling the podial adhesive systems of echinoderms have been found in numerous species from other marine invertebrate taxa (e.g. in Turbellaria, Tyler 1976; in Gas- trotricha, Tyler and Rieger 1980; or in Polychaeta, Gelder and Tyler 1986). In all species studied, these or- gans consists of two types of closely associated secretory cells. Cells of the first type are modified epidermal cells similar to echinoderm NCS cells. They release an adhe- sive material. Cells of the second type are derived from sensory nerve cells and resemble echinoderm CS cells. They are de-adhesive in function.

IlL A model for the adhesion mechanism of coronal and peristomeal podia

Epidermal cells of the central area of the disc of the podia of S. granularis are presumably involved in an adhesive/de-adhesive process functioning as a duo-gland adhesive system as proposed by Hermans (1983). In the duo-gland adhesive model we propose the adhesive and de-adhesive secretions would be respectively produced by NCS and CS cells. The secretion of NCS cells would make a bridge of adhesive material between the coronal podia and a substrate (this bridge being the footprint left by the podium after it has become detached from the substrate) or would coat and gather food particles (peristomeal podia). The de-adhesion would be due to the material enclosed in the granules of CS cells. Their action would allow the podium to easily become de- tached from the substrate and to always remain clear of any particles. All these secretions would be controlled by stimulations of the two types of ciliated cells (receptor cells) which presumably interact with the secretory cells by way of the nerve plexus. Tyler (1988) reviewed that

59

most adhesives in marine organisms involve an associa- tion of protein and glycans and that the nature of the de-adhesive material generally remains unknown. The latter was, however, proposed to be mucopolysaccharid- ic in echinoderms (Thomas and Hermans 1985). In S. granularis, we only succeed in showing the glycan com- ponent of the presumed adhesive substance.

This functional model of adhesion/de-adhesion fits well the activities of both coronal and peristomeal podia of S. granularis. When a coronal podium extends, the central part of its disc forms a conical projection due to the increased internal hydrostatic pressure exerted by the ambulacral fluid. As the podium sways slowly, the long cilia of the peripheral area of the disc are generally the first to contact a substrate (drift material or sea- bottom). I f the substrate is suitable, their stimulation induces the reorientation of the disc perpendicular to the substrate. The tip of the conical projection of the disc is then brought in contact with the substra tum and it is necessarily at this site that the release of an adhesive secretion by NCS cells starts the a t tachment process (cu- ticle-protruding cilia of CNS cells being the first to con- tact the substratum, it is likely that they trigger NCS cell secretion). At this stage, coronal podia may some- times break adhesion (release of CS cell granules) and retract, which explains the small footprints observed on the glass slides. (These footprints would be the areas of contact between the central protrusive par t of the podia and the substrate.) I f the podium carries on its action, its disc squeezes further against the substrate. The levator muscles would then contract to flatten the apical surface of the disc thus allowing all NCS cells to make contact with the substrate and to release their adhesive material. This flattening is made possible by the rigidity of the peripheral area and of the external part of the central area of the disc; rigidity which is itself due to the presence of the rosette and to the com- plex interpenetration of the connective tissue layer and the epidermis in the central area. Detachment of the podium f rom the substrate is presumably initiated by the secretion of CS cell secretory granules. This, together with the pulling action of the retractor muscles, allows the podium to retract f rom the substrate.

As suggested above, peristomeal podia could wrap food particles. Indeed, the close association in their disc epidermis of one or two NCS cells with one CS cell is reminiscent o f the sensory-secretory complexes of the penicillate buccal podia of E. cordatum (F lammang et al. 1991). As in coronal podia, the contact between a parti- cle and the cuticle-protruding cilia of CNS cells would lead to the release of the adhesive granules of NCS cells whose content would coat and gather the food frag- ments. The large elliptic apical surface of the disc pro- vides the max imum contact area with the substrate. Thanks to the exocytosis of CS cell granules, the podium would no longer stick to the particles and could then retract without carrying them along. The great develop- ment of CS cells (large cells with very numerous secreto- ry granules) may be related to the repetitive behaviour of peristomeal podia which requires an important de- adhesive potential. This also makes us think that CS

cell granule content would be a real de-adhesive instead of a neurotransmitter which, by extension, would also be true for coronal podia.

Acknowledgements. We thank Professor P. Lassere for providing facilities at the Observatoire Oc6anologique de Roscoff (Brittany, France) and E. Bricourt, J. Harray and M. Klinkert for technical assistance. The work was supported by FRFC contract n ~ 2.4549.91. Contribution of the "Centre Interuniversitaire de Biolo- gie Marine" (CIBIM).

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