/. mar. bid. Ass. U.K. (1996), 76,451^66 451 Printed in Great Britain

THE SECRETORY SYSTEM OF THE SPINES OF NIGRA (ECHINODERMATA, OPHIUROIDEA)

BRENDAN BALL* AND MICHEL JANGOUX* *Department of Zoology, The Martin Ryan Marine Science Institute, University College Galway, Galway, Ireland. *Laboratoire de Biologie Marine (CP-160), Universite Libre de Bruxelles, B-1050 Bruxelles, Belgium

The morphology of the spines of the ophiuroid Ophiocomina nigra is described, with particular reference to the nervous system and the sensory and secretory structures of the epidermis. The nervous system is composed of two main spinal nerves, located at the centre of the spine, and their associated branches. There are three secretory cell-types described: (1) fibrillar secretory cells which produce long, javelin-shaped secretory pack- ages and, occurring exclusively in the basal two thirds of the spine, penetrate deeply with their basal regions lying close to the axial nerve running through the spine centre; (2) granular secretory cells, which also penetrate deep within the spine, contain secretory granules in the form of spherical dense vesicles (-1-3 |im in diameter); and (3) goblet secretory cells, filled with packages of loose amorphous material, are superficial in location and usually found associated with a type A ciliated sensory cell. The secretions of the fibrillar and granular secretory cells are thought to perform the functions of defence and feeding respectively. A number of different ciliated sensory cell-types have been identified. Apart from the situation with the goblet cells, no close association was found between secretory and sensory cells. It is suggested that the nervous, sensory and secre- tory cells act together to form a mucous secretion system with centralized, rather than local control. This system appears to operate when it is advantageous to produce secretion all over the body simultaneously once any portion is stimulated. Stimulation of sensory cells might result in axonal excitation of the spinal nerves and hence to the entire epineural nervous system. In this way, glands located anywhere in the body, which are controlled by the epineural system could be quickly activated to cause mucus release. This contrasts with the situation in the podia where closely associated sensory and secretory cells acting together form a secretory system with localized control.

INTRODUCTION The spines of many ophiuroids have a non-calcified central channel containing nervous tissue (Hyman, 1955). The basal portions of secretory cells usually lie close to the nervous tissue at the spine centre (Fontaine, 1964). Three types of secretory cells are generally recognized in the ophiuroid epidermis: (1) mucous cells containing an elec- tron-lucent diffuse material packaged in irregular vesicles; (2) granulated cells contain- ing an electron-dense compact material packaged in vesicles; and (3) fibrillar cells containing a moderately electron-dense material organized into parallel strands (Byrne, 1994). The secretory products of these cells are usually composed of mucopolysaccharides 452 B. BALL AND M. JANGOUX and have generally been attributed a feeding or defensive role (Buchanan, 1963; Fontaine, 1964; Pentreath, 1970). Recent work has concentrated on the sensory abilities of the spines (Cobb & Moore, 1986; Moore & Cobb, 1986) and close association has been shown between sensory and secretory cells (Whitfield & Emson, 1983). Ophiocomina nigra (Abildgaard, 1789) is a very common member of the local benthos and has been the subject of several light and electron microscopic studies in the past (Buchanan, 1962,1963; Fontaine, 1964,1965; Wilkie, 1978,1979; Ball & Jangoux, 1990a, b). Although it has been described as predominantly a 'mucus net' suspension feeder, it also utilizes deposit feeding, scavenging, and browsing (Warner, 1982). The 'startle reaction', an impressive defensive behaviour involving the release of vast quantities of a highly sulphated acid mucopolysaccharide (pH 1), is also characteristic of the (Fontaine, 1964). As such, O. nigra is an ideal model with which to examine the phenomenon of secretion and control of secretory cells in the Ophiuroidea. Secretory cells have been described recently from the tube feet of O. nigra, thought to be involved in adhesion for locomotion and feeding. These cells were found to be closely associated with sensory structures which, acting together, form a secretory system with localized control (Ball & Jangoux, 1990a). The present paper describes the ultrastructure of the spines of O. nigra and particular attention is paid to the relationship between the nervous system and the secretory and sensory epidermal structures. It is the first ultrastructural study of the spinal nerve of an ophiuroid and the first detailed study of the relationship between the spinal nerve and the associated secretory cells. The possible function of the secretory cells and the system by which secretion is controlled are discussed.

MATERIALS AND METHODS Adult Ophiocomina nigra were collected at 15 m, by SCUBA diving, in Killary Harbour, Ireland. The specimens were transported to the Universite Libre de Bruxelles, where they were maintained in aquaria. For light microscopy, spines were fixed and decalcified in Bouin's fluid, embedded in paraplast and sectioned (5 (xm thick). Sections were stained with Masson's trichrome, Ehrlich's haematoxylin and eosin, and alcian blue with haemalun and phloxine, for general histological observations. For histochemistry, the sections were stained with alcian blue pH 2-6, Periodic-acid-Schiff (PAS), combined alcian blue / PAS and toluidine blue pH 1 & 3, methods for the detection of mucopolysaccharides. The mercury bromophenol blue method was used for the detection of proteins. Fixation and staining methods were performed according to Ganther & Jolles (1969-1970). For transmission electron microscopy (TEM), the spines were fixed by immersion in 3% glutaraldehyde in cacodylate buffer (0-4M, pH 8,1120 mosmol.) for 1 h at 4°C. After rinsing in buffer, the tissues were post-fixed, for 1 h, in 1% osmium tetroxide in the same buffer and then rinsed in fresh buffer. Spines were then dehydrated in graded ethanol, embedded in Spurr (Spurr, 1969) and decalcified according to the double embedding technique (Holland & Grimmer, 1981). Semi-thin sections (0-5 urn) were stained with a 1:1 methylene blue-azur II mixture (Richardson et al., 1960). Ultra-thin sections were SPINES SECRETORY SYSTEM 453 stained with uranyl acetate followed by lead citrate, according to Reynolds (1963), and examined in an Philips EM 300 transmission electron microscope. For scanning electron microscopy (SEM), spines were fixed in Bouin's fluid for 12 hours (Lahaye & Jangoux, 1985) or as described for TEM. These tissues were then dehydrated in graded ethanol and dried by the critical point method, mounted on aluminium stubs and sputter coated. For examination of the skeleton, the spines were fixed in 70% ethanol and associated tissues were digested by incubating the spines for 24 h at 55°C in a neutral solution of 0-1% proteinase N (Serva). The cleaned skeleton was washed three times in deionized water and preserved in 96% ethanol prior to examina- tion (Dubois & Jangoux, 1985). Some spines were fractured, using a razor blade, to examine internal structures. The spines were air dried, mounted, coated and observed with either an ISI DS-130 or a Philips 501 scanning electron microscope.

RESULTS Gross morphology of the spine The arms of Ophiocomina nigra consist of a series of jointed vertebrae composed of a central vertebral ossicle surrounded by four arm plates, two lateral, a dorsal, and a ventral. The lateral plates, on each side of the arm, bear a single vertical row of six or seven long slender spines (~2-5 mm in length) (Figure 1A). Each spine is a single, cylindrical and blunt-tipped ossicle (Figure IB) with a non-calcified central channel. The basal articulating surface is imperforate and allows movement against the lateral arm plate (Figure 1B,C). The basal part of the ossicle stereom is labyrinthic, while the shaft is fasciculate (Figure IB). The spine is covered by an epidermis which generally appears to be smooth, although it is, in fact, pitted all over by many pores which may be the openings of glands (Figure 1D,E). Many 'hook-like' structures (Ball & Jangoux, 1990b), raised about 9 urn above the general epidermal surface, are present in the lower region of the spine (Figure 1D,E). These epidermal outgrowths bear at least one terminal cilium (Figure IF). Each spine ossicle is surrounded by a thin layer of periossicular connective tissue which is itself covered by the epidermis (Figures 2A & 3A). The ossicle stroma fills most stereomic pores as well as most of the ossicle central channel. Part of the latter, however, is occupied by two axial nerves that run along its entire length. Moreover, epidermal processes penetrate some of the stereomic pores and closely contact the nerves of the channel. Both these processes and the nerves are underlined by a single basal lamina which is in continuity with the basal lamina of the epidermis (Figure 2A,B). Conse- quently, the spine nerves are topographically basiepidermal and thus belong to the epineural plexus.

Transmission microscopy of the spine epidermis and nervous system The epidermis is largely composed of T-shaped cells bordered externally by a cuticle and underlined internally by a basal lamina, which separates it from the underlying dermis (Figures 2B & 3A). 454 B. BALL AND M. JANGOUX

Figure 1. Skeletal structure of spines of Ophiocomina nigra. (A) Transverse section through arm showing arrangement of six spines on either side (arrows, spines). (B) Spine skeleton (arrow, articulating surface). (C) Section through spine showing hollow central axis. (D) Decalcified spines showing smooth surface (arrows, raised structures at base). (E) Basal region of spine (arrows, secretory pores; double arrows, sensory 'hooks'). (F) Sensory hook (arrow, cilium). c, cuticle; HCA, hollow central axis; v, arm vertebra. ECHINODERM SPINES SECRETORY SYSTEM 455

SB

Figure 2. Diagrammatic representation of the internal organization of a spine. (A) Spine sectioned transversely to show the arrangement of the different tissue types around the calcite skeleton. (B) Enlargement of a section of (A) showing the relationship between the spine nerve and the epidermal cells, bl, basal lamina; c, cover to fibrillar secretory cell; cc, ciliated sensory cell; cs, calcite skeleton; cu, cuticle; dcpt, dermal periossicular connective tissue; ep, epidermis; sb, spinal nerve branch; sn, spinal nerve; su, support cell; Tl, fibrillar secretory cell.

The cuticle (Figures 3A-C) (measuring ~0-65 um thick) is composed of three layers and closely resembles that described for the podia of O. nigra (Ball & Jangoux, 1990a). Beneath the cuticle lies a broad (~2-5-3 um thick) subcuticular space traversed by the microvilli of the epidermal cells (Figure 3A). Most epidermal cells form a superficial cover over the skeleton (Figure 3A), except in the pore spaces where they may extend all the way to the centre of the spine, terminating close to a spinal nerve (Figure 2B). At their apices, adjacent cells are joined by junctions consisting of a zonula adherens and septate desmosome (Figure 3B). The epidermis is comprised of support, ciliated and secretory cells, as described below. (1) Support cells. The support cells are the most numerous cells found in the epidermis (Figure 3A). Their cell bodies normally lie in pockets in the dermis. They extend apico- laterally as thin processes which connect with processes from adjoining cells (Figure 3A). Some of these cells run alongside secretory cells and extend through pore spaces to the spine central channel (Figure 2B). Apically the cells bear elongated microvilli, which cross the subcuticular space and pass through the cuticle, and whose tips form the outermost cuticular layer. Coupling areas occur regularly and serve to connect the cuticle and the underlying connective tissue layer via the support cells (Figure 3A). In these regions, the dermis approaches very close to the surface and anchoring filaments 456 B. BALL AND M. JANGOUX ECHINODERM SPINES SECRETORY SYSTEM 457 extend up to and connect with the basal lamina. Hemidesmosomes connect the support cells to the basal lamina and from these, bundles of tonofilaments pass vertically through the cell and extend into microvilli (Figure 3C). (2) Ciliated cells. These cells are of three different types. The first type (type A) are the most common and are found throughout the epidermis (Figure 3B). They are long, slender cells and possess a short cilium {-3-4 um in length), which projects only about 0-75 um beyond the cuticle. The cilia are club shaped and appear to have a regular 9+2 microtubular arrangement. The cells are most commonly found randomly distributed over the general spine surface but two or three are sometimes grouped together and are joined to one another and the neighbouring support cells by long septate junctions (Figure 3B). The second type of ciliated cell (type B), described in Ball & Jangoux (1990b), occurs exclusively, terminally on the 'hook-like' structures at the base of the spine (Figures 1D-F & 3D). These structures superficially resemble 'Stabchen' (Reichensperger, 1908) al- though here the cilia are raised much higher above the general spine surface (~9 \ixa). They are composed of epidermal support cells surrounding one or two ciliated sensory cells (Figure 3D,E). These ciliated cells are longer than the preceding more common type A ciliated cells. Their cilia may also project further from the cuticle (~11 um) (Figure IF). The third type of cell (type C) was only occasionally found. They bear short cilia (~0-5 um in length) which terminate in the subcuticular space well below the cuticle (Figure 3F,G). (3) Secretory cells. There are three types of secretory cells in the epidermis, fibrillar, granular and goblet cells. Fibrillar secretory cells, occur exclusively in the basal two thirds of the spine. Their cell bodies lie close to the axial nerve at the spine centre (Figures 2B & 5C), although separated from the dermis by the basal lamina. These cells then extend through the stereom pore space, opening to the exterior through cuticular pores. They produce long, javelin-shaped secretory packages, which sometimes extend beyond the cuticle and are here enclosed by a cover, probably a thickened extension of the cells plasma membrane (Figure 4A). Prior to release, there seems to be a change in osmotic pressure of the cover (Figure 4C) which then ruptures to release the secretion. Although the individual packages begin to break down prior to exposure to the environment, after exposure the secretion takes the form of an amorphous mass (Figure 4B). The secretion is formed near the nucleus, at the base of the cell, where golgi fields and endoplasmic reticulum are

Figure 3. Transmission electron microscopy (TEM) of spine epidermis of Ophiocomina nigra. (A) Low power cross-section of outer spine layers showing typical arrangement of epidermal cells and dermal connective tissue separated by basal lamina (arrow, coupling areas). (B) Ciliated cell showing a cell junction, j (arrow, cilium). (C) Coupling area (arrow, anchoring filaments; double arrow, hemidesmosomes). (D) Longitudinal section through sensory 'hook'. (E) Tip of sensory 'hook' (arrows, terminal cilia). (F) Sensory cell with subcuticular cilium (arrow). (G) Enlargement of F (arrow, subcuticular cilium). bl, basal lamina; cu, cuticle; DCT, dermal connective tissue; ecp, epidermal cell cytoplasmic process; g, golgi field; j, cell junctions consisting of zonulae adherentes and septate desmosomes; mt, microtubules; mv, microvilli; sc, sensory cell; SCS, subcuticular space; su, support cell; tf, tonofilaments. 458 B. BALL AND M. JANGOUX ECHINODERM SPINES SECRETORY SYSTEM 459 found (Figure 5C). Bundles of nerve fibres that correspond to branches of the spinal nerve containing small dense and clear vesicles, run alongside the fibrillar cells at their basal extremities (Figure 5C). The secretion stains strongly with alcian blue, alcian blue and PAS, and strongly y metachromatically with toluidine blue pH 1 & 3. It does not stain with mercury bromophenol blue. These histochemical methods suggest that the secretion consists of highly sulphated acid mucopolysaccharides. Granular secretory cells (Figure 4E,F & H) also penetrate deep within the spine, with their cell bodies lying close to the axial nerve at the spine centre. The secretory granules are in the form of spherical dense vesicles (-1-3 (im in diameter) (Figure 4F). In the basal region of these cells, which lies close to nervous tissue (Figures 2B & 4H), the secretion originates as vesicles filled with a loose flocculent material. These vesicles are produced by golgi fields (Figure 4E) and as they move along the cell, they condense to form the final product (Figure 4F). The secretion stains strongly y metachromatically with tolui- dine blue pH 1 & 3, and weakly with mercury bromophenol blue. These histochemical methods suggest that the secretion consists of an acid mucopolysaccharide-protein complex, with the mucopolysaccharide appearing to be dominant. The third secretory cell-type (Figure 4D) are superficial in location. These are goblet- like cells filled with packages of loose amorphous material. They are usually found associated with a type A ciliated sensory cell (Figure 4G). The chemical nature of the secretory product is not known as no reaction occurred with any of the histological stains employed. (4) Nervous system. The nervous system is well developed and, depending on the spine area, consists of one or two axial nerves (Figure 5A) and the nerve tracks radiating from these. Two nerves co-occur in the basal two-thirds of the spine. These differ in sizes, the thicker one measures ~34 |im in diameter and the thinner one measures ~21 urn. There is no contact between these nerve chords, each of them being completely surrounded by a basal lamina which also separates the nerves from the surrounding stroma/ dermal tissue (Figure 5A). A single nerve (-15 urn in diameter) occurs in the distal third of the spine. It was not possible to determine whether the distal nerve results from the junction of the two basal nerves or corresponds to the extension of one of the two basal chords. The size of the nerves suggests that the distal chord is an extension of the thinner nerve. The spinal nerves are composed of a large number of axons ranging in diameter from 01-2-6 (im (Figure 5A). These processes contain vesicles of varying sizes, which may be divided into three main types: small dense vesicles (-0-05 (xm in diameter), dense cored vesicles (~0-l urn in diameter) and clear vesicles (-0-06 um in diameter).

Figure 4. Secretory cells of spines of Ophiocomina nigra. (A) Fibrillar secretory cell at surface (arrow, cover to secretion). (B) Exploding fibrillar secretory cell (arrow, ruptured cover). (C) Ruptured fibrillar secretory cell cover (arrow, apparent release of fluid from cover). (D) Goblet secretory cell. (E) Granular secretory cell base, note golgi field and vesicle formation. (F) Mature granular secretory cell vesicles. (G) Goblet secretory cell showing associated ciliated cell (arrow, cilium). (H) Basal region of granular secretory cell lying close to nervous tissue. CT2S, condensing granular secretory packages; G, golgi field; j, cell junctions; NT, nervous tissue; T1S, fibrillar secretion; T1SP, fibrillar secretory cell package; T2S, granular secretory cell package; T3, goblet secretory cell packages. 460 B. BALL AND M. JANGOUX

Figure 5. Nervous system of spines of Ophiocomina nigra. (A) Two axial nerves chords (arrows, continuous basal lamina). (B) Region where spinal nerve basal lamina joins with that of epidermis (double arrows ) (arrows, nerve branch). (C) Bases of secretory cells lying close to nervous tissue (arrows, nerve branches), bl, basal lamina; LSN, large spinal nerve; SSN, small spinal nerve; Tl, fibrillar secretory cell. ECHINODERM SPINES SECRETORY SYSTEM 461

At various levels along the spine, branches are given off (Figures 2B & 5B) which run close to and presumably innervate the basal parts of the deep secretory cells (Figure 5C). It should be noted, however, that although axons are juxtaposed with the base of the secretory cells, synaptic contacts were not observed. It proved impossible to follow these nerve branches for any distance to see what other structures were innervated, due to the circuitous nature of the skeletal pores, but presumably they terminate in the epineural plexus that underlies the peripheral epidermis. These peripheral branches usually contain dense cored vesicles. The basal lamina surrounding these branches merges with the basal lamina around the epidermis (Figure 5B). As a result the nervous tissue is in direct contact with the epidermis and is thus basi-epithelial, even when it is sunken deep below the level of the epidermis.

DISCUSSION Recent studies document the arrangement of the dermal and epidermal tissues in the ophiuroid calcified body wall (Markel & Roser, 1985; Hendler & Byrne, 1987). The epidermis deeply penetrates the dermal tissues of the stereom pore space and is strictly separated from the dermis by a basal lamina. The present work has shown a generally similar arrangement in the spines of Ophiocomina nigra although the organisation of the various tissues is, if anything, more complex. This complexity is exemplified in the spatial relationship between the dermal and epidermal tissues, particularly in the stereom pore spaces. Here, epidermal support and secretory cells may penetrate, through the dermal connective tissue, all the way to the spine centre with their basal lamina in continuity with that surrounding the axial nerves and their cell bodies lying close to nervous tissue. The form and function of the various epidermal components of the spine are considered below.

Nervous system Although it has long been recognized that many ophiuroid spines had a hollow central axis bearing nervous tissue (Buchanan, 1963; Fontaine, 1964), the present work shows, for O. nigra, that this tissue consists of two distinct nerves. Presumably, the spine nerves originate from the segmental nerve that penetrates the lateral arm plate and enters the spine through an aperture located on the articular surface of the spine (Byrne, 1994). It would appear that a single nerve arises from the lateral arm plate side of the articulation and then splits into the two axial nerves. The exact point at which the nerve splits in two was not, however, seen during the present study. The reason for the occurrence of two separate axial nerves in the spine is not clear. It may relate to the presence of two different secretory cell types, fibrillar cells and granular cells, each with their own function. The co-occurrence of the thicker axial nerve and the fibrillar cells only in the basal two thirds of the spine, seems to support this suggestion. The nerve axons are small and contain vesicles indicative of cholinergic (clear vesicles) and aminergic (dense cored vesicles) transmission, similar to those described for other ophiuroids (Pentreath & Cobb, 1972; Martinez, 1977; Wilkie, 1979; Markel & Roser, 1985; Hendler & Byrne, 1987). 462 B. BALL AND M. JANGOUX

The nerves are enclosed in a basal lamina and although they are separated from the spine stroma, they have been shown to connect directly with the epidermis. This agrees with Markel & Roser's (1985) description of the situation in Ophiocomina longicauda, but differs from Hendler & Byrne's (1987) description of Ophiocomina wendti, where the nervous tissue is thought to be dermal in origin. Hendler & Byrne (1987) describe a cell type in O. wendti, which they call 'accessory cells', lying close to nervous tissue, and containing vesicles filled with loosely granular material. These cells closely resemble the early stages in the formation of the granular secretory cell granules in O. nigra. Granular secretory cells were also found in O. wendti and the 'accessory cells' might in fact represent an early stage in secretory granule formation in this species. The branches, given off at various points by the nerve chord, contain dense-cored vesicles. Vesicles of this nature have been found in the podial nerves of O. nigra (Ball & Jangoux, 1990a) and were tentatively identified as catecholamines, based on their morphology. It was suggested that they were responsible for the release of adhesive secretion from podial secretory cells. Stubbs & Cobb (1982) have provided neurophysi- ological evidence for the involvement of neurotransmittors (dopamine) in the control of secretion in , and Fontaine (1964) has suggested that the rapidity of mucus release in O. nigra indicates nervous mediation. The fact that the nerves are positioned alongside bases of the secreting cells would appear to support Fontaine's suggestion, although synaptic contacts were not observed.

Mucus secretion Mucus secretion by the spines of ophiuroids has been the subject of several studies (Buchanan, 1963; Fontaine, 1964; Pentreath, 1970). Fontaine (1964) described two types of mucous gland in the spines of O. nigra, a unicellular one which would be used for feeding and a multicellular one used for defence. More recently, three types of secretory cell have been described ultrastructurally from the dorsal arm plates of O. wendti (Hendler & Byrne, 1987). Whitfield & Emson (1983) have also described, in some detail, the structure of a fibrillar gland, from the spines of Amphipholis squamata. This study confirms the fibrillar nature of the secretion of the massive glands of the spines of O. nigra previously reported by Buchanan (1963). In this study, the glands of O. nigra have been shown to be unicellular in structure and not multicellular as suggested by Fontaine (1964). In our study, the indication of a multicellular gland may be due to the presence of epidermal support cells running alongside the fibrillar cells and penetrating the stereom pore space to varying depths. There is also often more than one fibrillar cell per pore space. The close proximity of the basal regions of the fibrillar glands to the nerves, suggests that the release of secretion may be under nervous or neurosecretory control. The exact mechanism for release is, however, elusive. Slow transport of the secretory packages by microtubules, followed by hydration causing rapid volume changes and expulsion, has been suggested for A. squamata (Whitfield & Emson, 1983). A similar system seems to occur here. The secretory packages retain their elongated shape even as they start to bulge through the external pore. At this stage they are still enclosed by a cover (cell ECHINODERM SPINES SECRETORY SYSTEM 463 membrane). Just prior to release, this cover starts to break down. Once exposed to the environment, the secretory packages seem to swell dramatically and lose their indi- viduality, causing an explosive expulsion from the cell. Fontaine (1964) suggested that these glands were responsible for the impressive mucus release associated with the 'startle reaction' in this species. The close association with the spinal nerves and the rapid release of fibrillar material supports Fontaine's suggestion. Suspension feeding by means of a mucous net, produced by the spines, has been suggested as the principal feeding type in O. nigra (Fontaine, 1965). The granular secretory cells, referred to as 'unicellular glands' by Fontaine (1964), seem the most likely candidates to produce this feeding mucus. They occur throughout the length of the spine and produce an acid mucopolysaccharide secretion. Such mucus is cohesive, elastic and strong (McKenzie, 1988). Particles impinging on the sheets of mucus, between the arm spines, would therefore distort the mucus without breaking it and become enwrapped. In this way, particles could be trapped by the mucus without actually adhering to it. Fontaine (1965) reported that shortly after assuming the feeding posture, the entire body surface of O. nigra, and especially the spines, is coated in mucus. This suggests a central control on the process of feeding-mucus secretion. The close association be- tween the basal regions of these cells and the nerves would allow rapid, co-ordinated release of mucus by a number of spines once a feeding stimulus has been received, thus greatly increasing feeding efficiency. Goblet secretory cells were only occasionally mentioned in any of the previous papers (Hendler & Byrne, 1987; Byrne, 1994), and were not highlighted by any of the stains employed in the present study. This may be due to the small size of the cells or the fact that they were often found to be empty. These cells were not closely associated with nervous tissue, but did have an accompanying sensory cell. It would appear that each of these cells operates independently venting their entire cell contents at one time.

Cilia and related structures Cilia are a very common and obviously important constituent of the spines of O. nigra. Most of the ciliated cells have specialised cilia (short with a club shaped tip) and appear to be sensory (Byrne, 1994). A close association was not found between the sensory cells and the fibrillar and granular secretory cells, although fibrillar cells and the sensory 'hook-like' structures (type B) are found in the same basal region of the spines only. This is unlike the situation in A. squamata where a close association between sensory and secretory cells is found (Whitfield & Emson, 1983). Ophiuroid ciliated sensory cells do not appear to give rise to sensory axons. As suggested by Cobb & Moore (1986), the ciliated cells may act as mechanoreceptors or chemoreceptors and a stimulus picked up by these could be quickly transferred to the mucous cells, through the well developed spinal nervous system which is juxtaposed with the base of the secretory cells. Such a system would not require sensory cells to be located close to mucous cells, as mucus release is not normally localized but rather stimulation of any area results in copious release of mucus over the entire body surface. Presumably, 464 B. BALL AND M. JANGOUX however, two different sets of sensory cells would be necessary, one to initiate feeding mucus release and the other defensive mucus release, otherwise both secretions would be released in response to any kind of stimulation. This is counter to the situation in the goblet cells and podial adhesive cells (Ball & Jangoux, 1990a), where mucus release is local. As described in Ball & Jangoux (1990b), the sensory 'hook-like' structures appear to be unique to O. nigra. They resemble 'Stabchen' but differ both in their size and the fact that they are multicellular, formed by an extension of the epidermis containing both support and ciliated cells. In A. squamata (Whitfield & Emson, 1983), the 'Stabchen' are simply outpushings of the cuticle containing the cilium of a sensory cell and its associated microvilli. It is unclear why such large structures should be positioned near the base of the spine. These 'sensory-hooks' and the fibrillar secretory cells co-occur in the basal region, suggesting that they may function as a sensory-secretory pair in the release of defensive mucus. However, Ball & Jangoux (1990b) suggested another com- plimentary or alternative role for the sensory hooks, i.e. to recognize when mucus- laden spines are ready for cleaning and to initiate podial cleaning of this mucus. Small subcuticular cilia, similar to those seen in the present study, were described by Cobb & Moore (1986) from the arms of Ophiura ophiura. These were identified as possible photoreceptors and considered responsible for light sensitivity in this species. Wilson et al. (1977) have remarked on the extreme sensitivity to light exhibited by O. nigra. The subcuticular cilia described here may function in photoreception.

The spine secretory system Considering that mucus secretion in O. nigra is often a whole-body response, it must involve some central control following appropriate peripheral stimulation. Excitation of any sensory cell may cause a message to be sent from peripheral axons to the main spinal nerves and hence to the ectoneural nervous system. Glands, anywhere in the body, lying in close proximity to nervous tissue would then be quickly activated. Both the fibrillar secretion for defence and the granular secretion for mucous-net feeding operate under this system. Although this type of system might appear to be energy wasteful, as vast quantities of mucus must be produced, the gains outweigh the losses. Preservation of life is of paramount importance and may override all other functions. The production of vast quantities of mucus during the defensive 'startle reaction' would be worthwhile if it facilitated escape from predation. During mucous-net feed- ing, large quantities of mucus are needed to produce the mucous net between the spines. For maximum efficiency, it is necessary to coat as many spines as possible quickly once a feeding stimulation has been received. The presence of a centrally controlled mucus secreting system in the spines of O. nigra, working in conjunction with the localized system of 'sensory-secretory com- plexes' in the podia (Ball & Jangoux, 1990a), may help to explain the apparent success of this species and the Ophiuroidea at large. ECHINODERM SPINES SECRETORY SYSTEM 465

The authors would like to thank Dr B.F. Keegan for reading and commenting on the manu- script; Dr L. Devos and Dr K. Wauters for SEM facilities and assistance, E. Bricourt and J. Harray for TEM assistance. Work supported by an ECC Grant (Stimulation Programme, ref. ST- 2J.0105.2) to Dr M. Jangoux and Dr B.F. Keegan.

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Submitted 19 January 1995. Accepted 3 August 1995.