Z. Zellforsch. 96, 151--161 (1969)

Uhrastructure of the Tube Foot Sucker of a Regular Echinoid, Diadema antillarum Philippi,

with Especial Reference to Secretory Cells* R. COLEMAn"

Department of Zoology, Bedford College, University of London

Received January 6, 1969

Summary. Suckers of principal tube feet of a regular echinoid, Diadema antillarum, are described in an electron microscopic study. Reference is made to secretory cells and nervous elements in the sucker and their possible significance in adhesion is considered and discussed. In particular, a granule-filled secretory cell-type is described that possesses long tracts down which the granules pass to terminate on the contact edge of the sucker in secretion packets. A second cell condition is described that may represent the secretory cells in a degranulated state. The appearance and significance of mucus-producing cells in the sucker is also consid- ered.

Introduction

There is not nearly so much division of labour in tube feet in any one ambu- lacrum of regular echinoids as in irregular forms (NICHOLS, 1961). Regular echi- holds, however, show a pr imary differentiation into two distinct types of tube feet: peristomial tube feet, which are unsuckered and play no par t in locomotion, and the principal tube feet which arise from the pore-pairs of the test. These lat ter tube feet are suckered and as the name suggests, constitute the predominant type of tube foot present (Cu]~NOT, 1948; NICI~OLS, 1961). They are invoived in locomotion, anchorage, respiration and apparent ly also have subsidiary functions of sensation (NICHOLS, 1961). I n Diadema the tube feet aboral to the ambitus lack sucking discs and seem to have mainly a respiratory function (MORTENSEN, 1940). The suckers of the principal tube feet of the regular echinoids are the most highly developed of any class of echinoderms (NICHOLS, 1962) and have been the subject of several investigations at the optical microscope level (SMITH, 1937; NTCHOLS, 1961). The ul t rastructure of the echinoid sucker has been a neglected field owing to the necessity for decalcifying and at the same time preserving sub- cellular morphology. Satisfactory techniques for achieving this have only recently been developed.

The mode of act ion of tube feet in locomotion has been widely investigated (SMITH, 1937, 1950). Adhesion in tube feet has for a long time been a t t r ibuted to factors other than suction (PAIN]~, 1926), though optical microscopic and histo- chemical studies on mucus or other glandular secretions involved have been limited by the lack of resolution of optical microscopy. This work describes some ultra- s tructural observations on suckered tube feet of Diadema antillarum with special reference to secretory and nervous elements tha t are possibly involved in loco- motion.

* I am indebted to Professor N. MILLOTT for his help and encouragement during the course of this work, to Mr. RAYNOR L. JONES for his expert technical assistance, and to Dr. H. G. VEVEaS and the Zoological Society of London.

11 Z. Zellforsch., Bd. 96

152 R. COL~MA~:

Material and Methods

Suckered tube feet were dissected from fresh Diadema antillarum Philippi from Madeira and fixed immediately by immersion in ice-cold 3 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 hour prior to post-fixation in veronal acetate-buffered 1% osmium tetroxide. Tissues were then dehydrated in graded ethanols, treated with propylene oxide and embedded in Shell Epikote Resin (Epon 812), which was polymerized at 60 ~ C for 24 hours. Silver sections (ca. 60--90 nm), cut with glass knives on a Cambridge (Huxley) ultramicro- tome, were mounted on uncoated copper grids and doubly stained in uranyl acetate (WATSON, 1958) and lead citrate (REYNOLDS, 1963). Sections were examined in an AEI EM6B electron microscope containing a 25 or 50 btm objective aperture at 60 kv. Original magnifications varied from 2,500 to 10,000.

Decalci/ication After fixation in glutaraldehyde, followed by a 15 min rinse in 0.1 M sodium cacodylate

buffer (pH 7.4) containing 7.5 % sucrose, decalcification was carried out at 4 ~ C in the medium of WaRSaAWSKY and MOORE (1967) viz: 41.3 g di-sodinm EDTA (versene) A- 4.4 g NaOI-I pellets made to 1 litre with distilled water; this forms a buffered system near neutral pH. Complete decalcification was achieved in 34 hours in the above medium, which was agitated and replaced at intervals. Following decalcification the tissue was rinsed in buffered phosphate for 45 minutes prior to post-osmication and embedding as outlined above. Ultrastructural morphology was not affected by the decalcification, though occasionally a fine electron-dense deposit covered the tissue possibly due to insufficient rinsing following decalcification resulting in the formation of an EDTA-osmium compound.

For correlated optical microscope studies thick epon sections (ca. 1 tzm) were cut on the ultramicrotome and stained in 0.1% toluidine blue in 1% borax or in periodic acid-Schiff (PAS) reagents (LANE and EUROI"A, 1965).

Results

The sucker of the tube foot is easily dist inguished from the rest of the tube foot. I t is discoid with a depression in its contact surface. I n transverse section the periphery of the sucker is seen to have gentle ridges. Fig. 1 i l lustrates in a much simplified diagram the general construct ion of a typical sucker of a tube foot of Diadema. The distal surface of the tube foot has two morphologically dis t inct zones: the central depression, which is the ma in secretory surface and the area where suction apparen t ly occurs, and the peripheral zone. The epithelial cells of this la t ter region are cont inuous with and morphologically similar to the epithelial cells of the lateral walls of the sucker and also with the epi thel ium covering the rest of the tube foot. The free surface of the epithelial cells of the sucker is covered with arboreal microvilli, which occur in clusters with clear non-microvil lous zones between them. These microvilli t end to be longer, up to 2.5 txm, t han microvilli over the rest of the tube foot and also differ in having less pronounced electron-dense "caps" a t their apices and in lacking a fine f i lamentous outer coating. Cilia are only occasionally encountered on epithelial cells of the sucker and are extremely rare on the "secretory surface". The epithelial cells covering the sucker are columnar, and vacuolated with round to ovoid nuclei s i tuated central ly or basally in the cells. At the periphery of the sucker surrounding the "secretory surface" the epi thel ium tends to be thickened and is composed of aggregations of cells in which the nuclei are the most striking elements. I t is conceivable tha t these cells could in some way provide a seal a t the edge of the

suction zone.

Ultrastructure of Echinoid Tube Foot Sucker 153

The epithelial cells of the sucker lie on a typical basal lamina that separates them from the underlying connective tissue. The bulk of the nervous tissue in the sucker lies in the epithelial layer and is bound internally by the basal lamina (BL, Fig. 2). The nervous tissue is in the form of a neuropile tha t is swollen in one region apparently forming a "nerve ring" (Fig. 1). The neuropile consists of a network of varicose axons, in the expanded parts of which a variety of contents

- - }'::; L " : i , ~ J ! ~ N R

G SP

Fig. 1. Simplified diagram illustrating the general construction of Diadema tube foot sucker. C, dense collagen layer; Co, connective tissue layer (mainly collagen) ; CE, coelomic epithelium; D, sites of decalcified calcareous endoskeletal structures; E, epithelium; G, granular secretory

cells; L, lumen; Mu, muscle layer; MG, mucus gland; NP, neuropile; NR, nerve ring; SP, secretion packets

can be identified (Figs. 3, 4). These include neurotubules, fairly large dense granu- les (ca. 0.1 ~m diam.), and clear "synaptic" vesicles tha t are even smaller (ca. 0.06 ~m diam.). The axons, in addition to the clear vesicles, frequently have small electron-dense mitochondria in bulbous axonal endings, though there is no sign these endings constitute synapses owing to the absence of evidence of membrane specialization.

The sucker is supported by a calcareous endoskeleton (Fig. 1) but following decalcification this zone reveals a network of cells, including many pigment- containing amocbocytes and glandular secretory cells (see later). This network of cells surrounds large clear spaces, which apparently were the sites of the calcium carbonate crystals of the skeleton. This calcareous region contains consid- erable networks of collagen fibrils and more internally situated in the sucker is a very densely-packed collagen layer.

Muscle fibres are found more internal to this collagen layer, whilst the lumen of the tube foot is lined by a thin coelomic layer of cells. Some muscle cells, pos- sibly levators, seem to extend across par t of the lumen (Fig. 1).

Mucus-producing cells are extremely rarely encountered in Diadema tube foot suckers and those present are mainly in the epithelial layer, but some also appear to be present in sub-epithelial sites. These mucus-producing cells have extremely

11"

154 R. COLEMAN :

Fig. 2. Neuropile in epithelial layer; BL, basal lamina Figs. 3 and 4. T.S. and L.S. respectively of neuropile immediately adjacent to granular

secretory cells

electron-dense nuclei, whilst the cytoplasm is divided into a complex of compart- ments each filled with mucoid material of varying density (Fig. 5).

Groups of cells containing very electron-dense cytoplasmic bodies bound by a unit membrane enclosing various membranous components are occasionally found between epithelial cells of the side walls of the sucker. I t is possible these cells may be pigment-carrying amoeboeytes.

Ultrastructure of Echinoid Tube Foot Sucker 155

Fig. 5. Unicellular mucus-producing gland in sub-epithelial connective tissue layer; note extremely electron-dense appearance of nucleus

Secretory Cells In the sub-epithelial zone of the sucker in the immediate vicinity of the nerve

felt and extending into the calcareous zone is a distinctive ring of cells (Fig. 1) in which the cytoplasm is granular and shows purple metachromasia after staining with alkaline toluidine blue and is weakly positive to periodic acid-Schiff reagents; thus the cytoplasm probably contains acid mucopolysaccharides. By the electron microscope the cytoplasm of these cells is seen to be packed with dense rounded granules of a fairly constant size (ca. 0.4 ~m diam.), though less rarely they may be slightly larger (ca. 0.5 ~tm) (Fig. 6). These granules are bounded by a unit membrane and contain homogenous very electron-dense material. Many of these granules have a clear region just internal to the bounding membrane giving them a dense-cored appearance. The granulated cells have regular round to ovoid nuclei, typically ca. 3 • 5 ~m. The cytoplasm rarely reveals evidence of Golgi bodies but when present these consist of short arrays of flattened saecules with terminal swellings enclosing electron-dense contents; these electron-dense swellings

Fig. 6. Granular secretory cells in region of decalcified endoskeleton Figs. 7 and 8. Secretory granules in tracts

R. COLEMAS: Ultrastructure of Echinoid Tube Foot Sucker 157

Fig. 9. Secretory packets (SP) between microvilli of adjacent epithelial cells; note fibrous elements in epithelial cells

may be the source of the dense cytoplasmic granules. Rough endoplasmic reticulum is sparse in the granulated cells and when present is located solely in the cytoplas- mic periphery in the form of simple elongated cisternae.

Bundles of collagen fibres are found in the spaces between the cell bodies of the granulated cells.

The dense granules pass from the cell bodies down long cytoplasmic extensions (Figs. 7, 8) and these tracts, which are in immediate contact with the neuropile pass into the epithelial layer. The tracts usually become distended towards the surface of the sucker and contain numerous small electron-dense mitochondria before terminating on the "secretory surface" as secretion packets (Fig. 9). Each packet contains a group of granules. These secretion packets are fairly regularly situated along the length of the "secretory surface" of the sucker. Secretion packets are separated from each other by groups of microvilli extending from neighbouring epithelial cells. These adjacent epithelial cells contain bundles of fibrous elements, which lack any discrete diagnostic banding. Fig. 10 is a composite drawing illustrating a typical glandular secretory cell in its entirety.

A second characteristic cell type is frequently found in the subepithelial granulated cell zone. In this the nucleus is less rounded and its envelope is greatly distended, with the nuclear pores becoming especially conspicuous. The cyto- plasm contains abundant vacuoles, ca. 0.2 ~m diam. devoid of content and also some larger vacuoles containing what are possibly degenerating granules. Alter- natively, this cell type may merely be a reconstitution phase of the secretory cells.

Discussion

I t is well established that suckers of tube feet of asteroids, echinoids and holo- thurians act as adhesive organs used in locomotion and where the sucker is poorly developed as, for example, in Astropecten, a sand-burrowing starfish, adhesion

158 R. COLEMAN:

ER

i

J ,

~ m

Fig. 10. Composite diagram illustrating main components of granular secretory cells; ER, rough endoplasmic rcticulum; G, granules; M, mitochondria; Mv, microvilli; N, nucleus;

SP, secretory packet

becomes of secondary importance (SMITH, 1937). PAINE (1926) analysed adhesion in tube feet of the starfish, Asterias vulgaris, which has well-developed suckers and she concluded that adhesion is due part ly to suction but also that "some sort of sticky secretion" might be involved. SMITH (1937) determined the extent to which mucus glands could be regarded as providing the means of adhesion in tube feet of the Eleuthrozoa and made observations on typical members of each

Ultrastructure of Echinoid Tube Foot Sucker 159

of the classes. He concluded tha t the adhesive mechanism in asteroids, echinoids and holothurians, all of which have tube feet with well-developed suckers, was in par t due to suction, but also to the secretion of mucus, though the ophiur- oids, whose tube feet lack well-defined suckers adhere merely by their intrinsic stickiness. The nature of glandular elements supplying this stickiness has recently been examined in several ophiuroid tube feet (BucrrA~AN, 1962).

Of all the echinoderm tube feet suckers so far described at the optical micro- scope level, the general construction of that of the regular echinoid, Echinus esculcntus (SMITH, 1937; NICHOLS, 1961) corresponds most closely to tha t of Diadema antillarum and this is by no means surprising as both belong to the single order Diadematoida. Certain striking differences are, however, immediately apparent., the most striking being the lack of a cuticle over the outer epithelium in Diadema. The presence of a cuticle in Echinus may be related to the ecological distribution of the genus ; whereas it is found in the relatively cold waters around Britain, Diadema is tropical or subtropical. Features common to both species include the presence of a calcareous endoskeleton, the distribution and general arrangement of the musculature, neuropfle, connective tissue layers and also the complex distal insertion of fibrous connective tissue elements. Suggested roles for these various components in achieving locomotion have been made (SMITH, 1937).

The morphology of suckers has been the subjec~ of several recent ultrastruc- rural investigations (Cf~AET and PHILPOTT, 1964; SOUZA SANTOS, 1965, 1966; HAI~RmO~, 1966, 1968; HARRISON and PHILPOTT, 1966) and these have shed some further light on possible glandular secretion that might assist in adhesion of tube feet. Glandular cells, in much the same sites as those in Diadema, have been de- scribed in the sucker of the asteroid, Asterias/orbesi; these cells secrete "mucus granules" which are transferred in channels to be secreted at the contact surface of the sucker in packets (CrlAET and PHILPOTT, 1964). Similar "mucus granules" and secretory packets have now been identified in several other species including the asteroids, Pisaster giganteus, Acanthaster planci, Patria miniata (HARRISON and PHIZPOTT, 1966), Asterina stelli/era (SovzA SANTOS, 1966) and the echinoid, Arbacia punctulata (HARRISON, 1966). These authors found the "mucus granules" were mainly ellipsoidal in shape and contained electron-dense cores composed of fibrous hexagonal rods in a honeycomb arrangement. Small generic differences were also detected in size and structure of the granules. The granules of the secretory cells in Diadema are morphologically quite different in being smaller, more rounded and lacking the fibrous core, although the secretory packets in Diadema appear to be released in a similar manner to the other species, that is, via cellular extensions tha t terminate between clumps of microvilli from adjacent epithelial cells. The function of microvilli on the surface of the sucker and possible roles they may play in adhesion remains to be demonstrated. Recently an in- vestigation on suckered tube feet of several holothurian species has revealed secretory packets of a unique and complex ultrastructure (ttARI~mON, 1968). Although histochemically the corresponding secretion granules in the various echinoderms classes can be shown to have mucoid-like properties, they are morpho- logically quite different from mucus of other mucus-producing cells described in a wide variety of tissues (SouzA SANTOS, 1966) and are also quite different from the mucus cells in Diadema (Fig. 5). The sparsity of mucus-producing cells in the

160 R. CoLEMAN:

podia l sucker of Diadema suggests t h a t mucus m a y p l ay only a ve ry l imi ted role, if any , in adhes ion and the ma in adhesive forces p r o b a b l y come from suction, for which there are all the necessary s t ruc tu ra l components , and f rom the secre tory packets .

The neuropi le supply ing the sucker of the tube foot is v i r t ua l ly ident ical in u l t r a s t ruc tu ra l morpho logy wi th t h a t in the wall of the tube foot s tem (COLEMAN, in press) and wi th the branches of the rad ia l nerve supply ing the base of the tube foot and the " p o d i a l p i t " (MILZOTT and COLEMAN, 1969), i t will no t be consid- ered fur ther here, a p a r t f rom ment ioning i ts d i rec t con tac t wi th epi thel ia l cells of the sucker in te rna l to the basal l amina (Fig. 2). This t ends to confirm the sensory na tu re of the podia l sucker and suggests the nerve fel t is d i rec t ly invo lved in behavioura l responses to env i ronmenta l s t imul i as well as in the complex move- ments dur ing locomotion.

The second g landula r cell t ype found in Diadema m a y represent a d is t inc t t ype of cell b u t i t is more l ikely to represent a p a r t l y deg ranu la t ed cell a f te r the release of i ts secre tory granules. The clear vacuoles are p r o b a b l y the former sites of the granules and the larger dense bodies m a y be inclusions re la ted to lysosomes involved in the degrada t ion of cell organelles t h a t are exhaus ted or no longer funct ional .

The chemical na tu re of the secre tory granules in Diadema is no t y e t cer ta in and is stil l being elucidated. However , the secre tory cells resemble in the i r fine s t ruc ture the ve r t eb ra t e secre tory cells producing po lypep t ide hormones (PwARS~, 1968).

R e f e r e n c e s

BUCHANAN, J . B . : A re-examination of the glandular elements in the tube feet of some common British Ophiuroids. Proc. zool. Soc. Lond. 138, 645--650 (1962).

CHAET, A. B., and D. E. PHILPOTT: A new subcellular particle secreted by the starfish. J. Ultrastruct. Res. 11, 354~362 (1964).

CU~NOT, L. : Echinodermes. In: Trait6 de Zoologic (ed. P-P. GRASSY). Paris: Masson & Cie. 1948.

HARRISON, G. A. : Light and electron microscopic studies of secretion packets having adhesive qualities in the tube feet of echinoderms. 6th Int. Cong. Electr. Micr., Kyoto, Japan 2, 411~412 (1966).

- - Subcellular particles in Echinoderm tube feet. II . Class Holothuroidea. J. Ultrastruct. Res. 23, 124--133 (1968).

- - , and D. P~rOTT: Subcellular particles in Echinoderm tube feet. I. Class Asteroidea. J. Ultrastruct. Res. 16, 537--547 (1966).

LA~E, B. P., and D. L. EUROPA: Differential staining of ultrathin sections of epon-embedded tissues for light microscopy. J. Histochem. Cytochem. 13, 579--582 (1965).

MILLOTT, N., and R. COLEMAn: The podial pit - - a new structure in the Echinoid Diadema antillarum Philippi. Z. Zellforsch. 95, 187--197 (1969).

MORTE~SEN, T. : In: A monograph of the Echinoidea. I I I (i), 245. Copenhagen: Reitzel 1940. NICHOLS, D. : A comparative histological study of the tube feet of two regular Echinoids.

Quart. J. micr. Sci. 162, 157--180 (1961). - - In: Echinoderms. London: Hutchinson 1962. PAINE, V . L . : Adhesion of the tube feet in starfishes. J. exp. Zool. 45, 361--366 (1926). PEARSE, A. G. E. : Common cytochemical and ultrastructural characteristics of cells producing

polypeptide hormones (the APUD series) and their relevance to thyroid and ultimobranchial C cells and calcitonin. Proc. roy. Soc. B 170, 71--80 (1968).

REYNOLDS, E. S. : The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol. 17, 208--212 (1963).

Ultrastructure of Echinoid Tube Foot Sucker 161

SMITH, J. E. : The structure and function of the tube feet in certain Echinoderms. J . Mar. Biol. Ass. 22, 345--357 (1937).

- - The motor nervous system of the starfish, Astropecten irregularis (Pennant) with special reference to the innervation of the tube feet and ampullae. Phil. Trans. B 234, 521--558 (1950).

SovzA SANTOS, H. DE: Estudo da ultraestrutura dos p~s ambulacrs de Asterina stelli/era (Hup6)-Echinodermata-Ast~roidea. Bolm. Fac. Filos. Ci~nc. Univ. S. Paulo 25, 175--229 (1965).

- - The ultrastructure of the mucous granules from starfish tube feet. J . Ultrastruct. Res. 16, 259--268 (1966).

WARSHAWSKY, H., and G. MOORE: A technique for the fixation and decalcification of rat incisors for electron microscopy. J. Histochem. Cytochem. 15, 542--549 (1967).

WATSOn, M. L. : Staining of tissue sections for electron microscopy with heavy metals. J . biophys, biochem. Cytol. 4, 4 7 5 ~ 7 8 (1958).

Dr. RAYMOND COLEMAN Department of Zoology Bedford College (University of London) Regent's Park London, N.W. 1., England