J. Zool., Lond. ( A ) (1985) 205,223-234

The tentacles of Ichthyophis (Amphibia: Caecilia) with special reference to the skin

H A R O L D Fox Department of Zoology, University College, Gower Street, London WClE 6BT

(Accepted I0 April 1984)

(With 7 plates in the text)

The skin of the paired tentacles of Ichthyophis consists of a cornified epidermis of 5-7 layers of epidermal cells, and a glandular dermis of ducted mucous glands, in association with collagen, blood vessels, fibroblasts, granulocytes, sparse melanophores and characteristic laminophores of unknown function. The epidermis is highly innervated at all levels below the stratum corneum by naked neurites, which originate as branches from large unmyelinated nerve bundles (and associated Schwann cells), located sub-epidermally, and which are part of the trigeminal cranial nerve. Myelinated nerves are also present below the epidermis, spatially associated with capillaries and glands. The study of the ultrastructure of the tentacle supports a concept of a sensory function, possibly tactile, though until further information from experimentation is available, any ideas on the specific nature of these sensory activities must remain speculative.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . The epidermis . . . . . . . . . . . . . . . . . . . . Explanation ofabbreviations used on Plates . . . . . . The dermis . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

Page . . . . . . . . . . . . . . . . . . 223 . . . . . . . . . . . . . . . . . . 224 . . . . . . . . . . . . . . . . . . 224 . . . . . . . . . . . . . . . . . . 224 . . . . . . . . . . . . . . . . . . 229 . . . . . . . . . . . . . . . . . . 232 . . . . . . . . . . . . . . . . . . 234

Introduction

The retractile tentacles of caecilians, situated in a groove behind each nostril, midway between the eye and the external nares, are unique among adult amphibians. The tentacular apparatus was first described in caecilians by Leydig (1 868). Since the classical descriptions by Wiedersheim (1 879,1880) and the Sarasins (1887-1 890) and of the innervation by Norris & Hughes ( I 91 8), the histology, innervation and homology of the tentacular apparatus were dealt with in some detail by Englehardt (1924). Afterwards, other workers contributed information on various aspects of the tentacular apparatus (see Laubmann, 1927; Marcus, 1930; de Villiers, 1938; Ramaswami, 1941) including its embryology (Badenhorst, 1978). In phylogeny, the tentacular sheath may have been derived from an epidermal sensory groove, into which the Harderian gland deposited secretions. Alternatively, the sheath represents an enlarged common duct of the Harderian gland and the tentacle is a secondary formation (Badenhorst, 1978).

Although the skin of the body of caecilians has been investigated by light and subsequently

0 The Zoological Society of London 223

0022-5460/85/020223 + 12 $03.00/0

224 H A R O L D FOX

electron microscopy (see references in Fox, 1983), the ultrastructure of tentacular skin has yet to be described. The present work aims to rectify this deficiency, and provides some information on the fine structure of both epidermis and dermis of the tentacle of Zchthyophis.

The results support a previously held view of its sensory function.

Materials and methods

For examination by electron microscopy, small paired tentacles (each one about 3-4 mm long), from 3 adult specimens each of Zchthyophis kohtaoensis and I . orthoplicatus, were removed and fixed ice-cold for 1-3 h in a combined mixture of osmic acid and glutaraldehyde buffered to pH 7.4 (Hirsch & Fedorko, 1968). After washing thoroughly with buffer, the material was dehydrated in increasing concentrations of alcohol, through absolute alcohol and finally propylene oxide, and embedded in Araldite epoxy resin. Silver grey sections were obtained by the use of a diamond knife and mounted on copper grids; they were then stained with uranyl acetate and lead citrate. Sections were viewed under an AEI 6B or JEOL JEM 100 CX I1 electron microscope.

Araldite sections of the tentacle, sectioned transversely and about 1 pm thick, were stained with toluidine blue and examined under the light microscope for gross cellular structure and topography of the skin components. Sections were photographed using a Zeiss photomicroscope.

The epidermis

In adults of Zchthyophis kohtaoensis and I. orthoplicatus the tentacular epidermis comprises 5-7 layers of epithelial cells, including the cornified surface layer (Plate I(a)). The cornified cells are

Explanation of abbreviations used on Plates As the greater part of the investigation was concerned with the tentacles of Ichthyophis orthoplicatus, all the illustrations

are of this species, though the general arrangement and ultrastructure of the tissues are the same in the tentacles of I. kohtaoensis. All the illustrations are of transverse sections and from TEM, except for Plate I(a), which is of a thick Araldite section stained with toluidine blue and viewed under the light microscope. AM BC BCN BN cc Cap c o D DM

DGr Derm Dm EB GC Gr IC IJ LC

Adepidermal membrane Basal epidermal cell Basal epidermal cell nucleus Branching nerve Cornified cell of epidermis Capillary Collagen fibrils Duct of dermal gland Dense membrane of inner wall of cornified

cell Dissolving granule Dermis Desmosome Ellipsoidal body (sideways on) of laminophore Golgi complex Granule in epidermal cell Immigrant cell in epidermis Intercellular junction Laminophore cell

Lm

MEC MG MN MPC MRC Me MUD N Ne NeB OT( 1) O W ) PNR RER RL sc TF

Laminated ellipsoidal body (edge on) of laminophore

Myoepithelial cell Mucous gland Myelinated nerve Mucus-producing cell Mitochondria-rich cell Melanophore Mucus droplet Nucleus Nerve fibre Nerve bundle in dermis Microtubule-type 1 Organelle Microtubule-type 2 Organelle Perinuclear region Rough (granular) endoplasmic reticulum Replacement layer of epidermis Stratum corneum Tonofilaments

TENTACLE SKIN OF I C H T H Y O P H I S 225

flattened, about 2.5-4 pm thick, typically electron-dense, with organelles, apart from a faint profile of a flattened nucleus, no longer recognizable (Plate I(a), (b), Plate II(a)). The region of the lateral margin between adjacent cornified cells is irregular and extremely narrow, with tight junctions at the surface. Desmosomes join outer and second layer cells: they are located on peg- like projections from the inner and outer surfaces of the cornified and replacement layer cells, respectively (Plate I(b), (c)). Tonofilaments extend inwards from the desmosomes into the second layer cells, and the intervening region, the sub-corneal space, is filled (at least in part) with an electron-lucent substance. The cornified cells are enveloped by a dense layer below the surface, about 25 nm thick. The gap between the desmosomes is between 30-40 nm thick (Plate I(c)).

PLATE I. (a) Skin section showing a multi-layered epidermis of epithelial cells with an outer stratum corneum, and a mucous gland in the dermis, whose duct is partially penetrating the epidermis. A myoepithelial cell sheath is prominent, and surrounds the mucus-producing cells. The tall columnar cells of the basal epidermal layer are clearly recognizable. (Explanation of abbreviations see p. 224); (b) Junction between the Stt'dtUm corneum and a non-cornified 2nd layer cell (replacement layer), showing projections from the inner and outer surfaces, respectively, of these cells. Intervening desmosomes are located between the cellular projections. (Explanation of abbreviations see p. 224); (c) Junction between the outer cornified and non-cornified replacement layer of the epidermis. A thickened dense layer borders the inner margin of the cornified cell which has little recognizable organelle ultrastructure. Masses of tonoflaments lead inwards from the desmosomes of the 2nd layer cell. (Explanation of abbreviations see p. 224.)

226 H A R O L D FOX

The non-cornified and clearly demarcated nucleated epithelial cells and the 2nd and 3rd layers are well differentiated with a high content of tonofilaments (Plate II(a), (b), (c)). Dense granules are present, apparently in less numbers in cells towards the surface of the epidermis. Tono- filaments frequently appear to orientate at random; in other cases they extend for some way from the desmosomes (Plate III(b)), or they are oriented dorso-ventrally from the desmosomes or circumferentially around the nucleus. The nucleus is surrounded by a homogeneous and moderately electron-dense area of finely granular substance, probably in part originating from the dispersion of material of the granules (Plate II(a), (c)). Cells of the 3rd layer are similar to those of the 2nd, though desmosomes are more irregularly distributed around the cells, and the perinuclear area includes numerous rounded granules of varied electron-density, some of which are in the process of dissolving (Plate II(b)).

All the cells of the intermediate layers-those between the stratum corneum and stratum

PLATE I I. (a) Section through the three outer layers ofepithelial cells ofthe epidermis. Both 2nd and 3rd layer cells have a high content of tonofilaments. There is a less dense agranular perinuclear area in the 2nd layer cell, though there are dense granules in the rest of the cell matrix. Granules are more numerous in cells of the 3rd layer. (Explanation of abbreviations seep. 224); (b) Cell of the 3rd layer of the epidermis showing the perinuclear region with granules of varied electron-density, some apparently in the process of dissolving. Compare this profile with that of (a), where the perinuclear region of the 2nd layer cell is practically devoid of granules. Desmosomes in different orientations join adjacent cells. (Explanation of abbreviations see p. 224); (c) Profiles of 2nd and 3rd layer epithelial cells of the epidermis showing granules presumably dissolving in the perinuclear area of the 3rd (lower) layer cell, to give rise to the condition seen in the 2nd (upper) layer cell. The intercellular junction between these cells is not prominent, but it is located just below the upper cell perinuclear region and the granules belong to the third layer cell. A small poorly-developed Golgi complex is present near the nucleus in the 3rd layer cell. (Explanation of abbreviations see p. 224.)

T E N T A C L E S K I N O F I C H T H Y O P H I S 227

germinativum-are generally similar in ultrastructure. The desmosomes are either straight or slightly curved, up to at least 540 nm in length; the thickness of the plaque is about 60 nm with the gap about 30 nm and the walls about 15 nm wide (Plate Ill(a)). Granules up to about 400 nm in

P L A T E I I I. (a) Prominent desmosomes between epidermal cells of layers 3 and 4. The desmosomes are usually straight but may often be curved, a visualized shape presumably related to the section orientation. (Explanation of abbreviations see p. 224); (b) Epidermal cell of the 3rd layer with a straight elongate bundle of tonofilaments extending from the desmosome across the cell. The cytoplasm includes a high content of tonofilaments and granules, and there are numerous polysomes but little RER, and mitochondria are rare and poorly developed. (Explanation of abbreviations see p. 224); (c) Cell of the 3rd layer of the epidermis with a pair of grouped microtubule-like organelles in the perinuclear region. Indeed, a third group is faintly recognized to the right of the lower group. (Explanation ofabbreviations seep. 224); (d) Cell of the 2nd layer of the epidermis showing a microtubule-like group of organelles similar to those of (c), but at a higher magnification. There is another more clearly delineated set of organelles on the right of the figure, which could be an inclusion of virus-type material. (Explanation of abbreviations see p. 224.)

228 H A R O L D FOX

P L A T E 1 V. (a) Cell of the 2nd layer of the epidermis with what is probably a poorly-developed Golgi complex of about five stacks. There are few, if any, terminal vesicles of the Golgi, and this apparatus is not common or substantial in the epidermal cells nearer the surface of the skin. (Explanation of abbreviations see p. 224); (b) Columnar basal epidermal cells showing an indented margin with an adepidermal membrane and space, and modest hemidesmosomes. Elongate tonofilaments extend inwards from the basal margin but there are few granules compared with more distal cells. Electron- lucent material occurs between the cells and often apparently within them as vesicles. A well developed nerve is located intercellularly in the basal cell layer. (Explanation of abbreviations seep. 224); (c) Immigrant cell, probably a leucocyte, in the basal layer of the epidermis. The polymorphic nucleus is easily distinguishable from the tall narrowish nuclei of the basal epidermal cells, directed approximately at right angles to the basal epidermal margin. (Explanation of abbreviations see p. 224); (d) Free nerve terminal in the epidermis between the 2nd and 3rd layers. Note the presence of mitochondria and glycogen granules in the nerve. The nerve profile is comparable to others in the epidermis (b) and dermis (Plate V(b)). (Explanation of abbreviations see p. 224.)

T E N T A C L E S K I N O F I C H T H Y O P H I S 229

diameter occur in the cytoplasm: others more closely surround the nucleus; these are larger and may be dissolving.

The cell matrix of the intermediate cells includes polysomes, some smooth-surface vesicles and occasionally multi-vesicular bodies. Well formed mitochondria are only rarely recognizable, nor is there a RER. In cells of the 2nd and 3rd layers, short groups of elongate microtubule-like organelles (up to about 12) are seen. Sometimes the groups are in pairs alongside the nucleus (Plate III(c), (d)). Presumably, these structures may be longer (dependent on section orientation) than recorded. An individual ‘tubule’ was > I5 nm wide and at least 350 nm long. Another small group of tubular organelles, more clearly defined and possibly not strictly comparable, included components about 20 nm thick and 200 nm long. The latter, at least, could well be virus-like inclusions (Plate IIl(d)). In other cases in cells of these layers, somewhat similar small tubular-like organelles, of about 4-5 stacks and up to 600 nm long, and often slightly curved, are probably poorly developed Golgi cisternae. Indeed, they were often slightly swollen terminally, though associated vesicles were not apparent (Plate IV(a)).

Basal epidermal cells are elongated and columnar, roughly oriented at right angles to the skin surface. The inner margin, bounded by an adepidermal membrane (50-75 nm thick) and adepi- dermal space, is irregular and indented. There are modest cell membrane thickenings, representing poorly developed hemidesmosomes, and well formed desmosomes join adjacent cells (Plate IV(b)). Basal cells have elongate nuclei, a granular matrix with abundant polysomes, poorly differentiated though frequently numerous mitochondria with few cristae, and there are masses of tonofilaments in bundles extending perpendicularly from the basal margin of the cell. Some cells have a Golgi complex and an associated array of small vesicles, and other larger dense granules are present which could be lysosomes. Occasionally a centriole is recognized.

There is a considerable quantity of electron-lucent substance located between the cells and also apparently within them, where it appears in large vesicles (Plate IV(b)). Whether some of them represent neurite substance, in some instances, which is partially degenerate, or they are glycoproteinaceous inclusions, cannot be decided.

The basal cell layer also includes occasional immigrant cells, without desmosomes, that are probably leucocytes (Plate IV(c)).

Non-myelinated nerve fibres are distributed at all levels in basal and intermediate cell layers, for the epidermis is well innervated (Plate IV(b), (d)). Nerves are located intercellularly and frequently seemingly within the cells. Actually, the nerve fibres run either between the epithelial cells or they are tightly enclosed by them in grooves, fastened in by the desmosomes at the surface (see Whitear, 1983). The epidermal nerves originate from large non-myelinated nerve bundles (with Schwann cells) seen in the dermis, which branch and supply fibres that enter the epidermis through the adepidermal membrane (Plate V(a), (b)): these are terminals either of the r. ophthalmicus profundus V, according to Norris & Hughes (191 8), or the r. maxillaris V, according to Englehardt (1924). Some of these nerves could have lost a myelin sheath before or soon after entering the tentacle. The nerve profiles in the epidermis usually include numerous mitochondria and glycogen granules, and they are of similar appearance to comparable components in the dermis (see Plate IV(b), (d), Plate V(a), (b)).

The dermis

The dermis and more central regions of the tentacle include masses of collagen fibrils, well formed capillaries, mesenchymal cells (probably fibroblasts), granulocytes, occasional

230 H A R O L D FOX

PLATE V. (a) Nerve bundle (a continuation of the nerve of (b)), extends below the epidermis and supplies a branch that enters the epidermis through the adepidermal membrane. Presumably, the nerve terminals seen in Plate IV(b), (d) originated from such a tentacular sub-epidermal branch of the trigeminal nerve. (Explanation of abbreviations see p. 224); (b) Unmyelinated nerve bundle and Schwann cells in the dermis. The epidermis is in the upper right of the picture. (Explanation of abbreviations see p. 224); (c) Capillary in the sub-epidermal region of the tentacle with associated mesenchymal cell, melanophore process and unmyelinated nerve bundle. (Explanation of abbreviations see p. 224); (d) Capillary and associated myelinated nerve. (Explanation of abbreviations see p. 224.)

melanophores, and laminophores that are also present in the body dermis (Fox, 1983) and membranous labyrinth (Jorgensen, 198 1) of Zchthyophis (Plate V(a)-(d), Plate VII(a), (b)).

In addition to the large unmyelinated nerve bundles previously mentioned, there are well developed myelinated nerves, up to 8 p m in maximum cross-section and whose sheath is 200 nm thick. They are located, like the non-myelinated nerves, alongside capillaries and near glands (Plate V(c), (d)).

Mucous glands with ducts of flattened epithelial cells are present, though an actual opening at the skin surface was not recorded (Plate I(a)).

Each gland consists of a sheath of myoepithelial cells surrounding peripheral mitochondria-rich cells and the large swollen mucus-producing cells with a prominent RER (Plate VI(a), (b)). The

T E N T A C L E S K I N O F I C H T H Y O P H I S 23 1

P L A T E VI. (a) Margin of a mucous gland in the dermis. A peripheral sheath of myoepithelial cells surrounds large swollen mucus-producing cells, which have a high content of RER at their periphery. Collagen fibrils surround the myoepithelial sheath of the gland. (Explanation of abbreviations see p. 224); (b) Region of a mucous gland showing a mitochondria-rich cell at the periphery, enveloped by a myoepithelial cell. (Explanation of abbreviations see p. 224.)

roundish-shaped mucous glands are about 0.04 mm in diameter, somewhat smaller than the comparable glands in the body.

Scales were not seen in the dermis but at the base of the tentacle, near the sac, masses of collagen fibrils closely packed together and oriented in three dimensions may possibly represent incipient dermal scales and define the region where the tentacle merges into the sac.

232 H A R O L D F O X

PLATE V 11. (a) Laminophore tissue and nerve bundles in the sub-epidermal (and more central) region of the tentacle. The area includes masses of collagen fibrils, and other similar regions may also include fibroblast tissue, sparse melanophore tissue and capillaries. (Explanation of abbreviations seep. 224); (b) Higher magnification of a laminated disc of laminophore tissue in the tentacle. The views are sideways and edge-on. The profiles are similar to comparable tissue in the dermis of the body of Ichthyophis. (Explanation of abbreviations see p. 224.)

Discussion The ultrastructure of epidermal epithelial cells of the tentacles of Zchthyophis kohtaoensis and I . orthoplicatus is similar to that of their comparable body epidermal cells (Welsch & Storch, 1973; Fox, 1983). However, the absence of Merkel cells in the epidermis of the tentacles, when they occur in the rest of the body (Fox & Whitear, 1978; Fox, 1983), and the fact that the tentacles are probably sensory organs (vide infra), is somewhat unexpected. Merkel cells occur in tentacles of the larval Xenopus luevis, synapsing with nerve fibres (Fox & Whitear, 1978; Ovalle, 1979). Caecilian tentacles develop at metamorphosis when there is epidermal keratinization and loss of

TENTACLE S K I N O F I C H T H Y O P H I S 233

the lateral line organs (Wake, 1977). Perhaps their post-larval origin is inhibitory or precludes in some way the differentiation of Merkel cells in the tentacle. It is possible that Merkel cells are extremely scarce or absent in the head region, though specific details of their overall distribution in caecilian skin are unknown. Nevertheless, Merkel cells are not uncommon in the head of fishes and anuran larvae, and in lampreys they are more plentiful in this region than elsewhere (Whitear, pers. comm.). However, if Merkel cells are not a feature of head skin of Zchthyophis, then their absence in the tentacles would not be unexpected. Nor were flask cells, or any other cell type comparable to them, found in tentacular skin, in contrast to the report by Englehardt (1924) of ‘Flaschenzellen’ in the tentacles of Zchthyophis glutinosus. They were first described in caecilian body skin by the Sarasins (1887-1890), later confirmed by Datz (1923) and Gabe (1971), who reported flask cells only in ventral body skin after metamorphosis. Though not common, flask cells were found in back epidermis of Zchthyophis (Fox, 1983). Whether the tentacles of Ichthyophis glutinosus (Englehardt, 1924) differ from those of Z. kohtaoensis and I . orthoplicatus in respect of flask cells, or these are extremely scarce in the latter species and thus are difficult to locate, or they were misinterpreted by light microscopy, is not clear.

The dermal mucous glands of the tentacles, though smaller, are generally similar to the comparable mucous glands in the rest of the body. Mucus presumably reaches the surface of the tentacular skin via the ducts, and may be used to lubricate the sheath during movement of the tentacle. It is possible that the tentacular glands are peripheral components of the orbital Harderian glands, known to surround the eye region and also the hinder region of the tentacle and part of the tentacular muscle, at the margin where the tentaculai sac is found (see Badenhorst, 1978). Specific demarcation areas of the tentacular base and sac are difficult to differentiate where they merge. Indeed, a few communal ducts of the orbital glands open into the sheath. Nevertheless, as, for practical purposes, only the tentacles were removed from the head of Ichthyophis during preparation, it would seem likely that small dermal mucous glands and their ducts are normal components of at least the hinder tentacle region.

Tentacles are probably vascularized by the stapedial artery or another branch of the internal carotid artery, and are drained by a branch of the jugular vein (de Villiers, 1938). They are retracted into the tentacular sac by retractor tentaculi muscles, homologues of the retractor bulbi of other amphibians (Englehardt, 1924) and derived from the extrinsic eye musculature. Innervation of the muscle is by the r. abducens VI cranial nerve (Norris & Hughes, 1918). Retraction is by muscles acting at the base of the tentacle and no muscle-type tissue was recognized within the tentacle at any level.

The tentacle itself is supplied by branches of the trigeminal cranial nerve (vide supra). The epidermis below the cornified layer is well innervated throughout by free nerve fibres. It seems likely, therefore, that the tentacle function is a sensory one, the ‘Tastorgan’ of Englehardt (1 924). A tactile function has been assumed by most workers since the Sarasins (1 887- 1890), though Marcus (1930) and Badenhorst (1978) considered the tentacles to have an olfactory (presumably chemosensory) function also.

In very young amphibian larvae, nerve fibres have already penetrated the larval epidermis before Merkel cells are recognized (Fox & Whitear, 1978), and such nerves are functional in Xenopus luevis (Roberts & Hayes, 1977). They are probably mechanoreceptive, and deformation of the neurite membrane should be an adequate stimulus (Whitear, 1983). The free nerve endings could nevertheless be chemoreceptors (see Fox et ul., 1980). However, until experimental evidence is available so as to be clearer about the properties of the tentacular component nerves, hypotheses on the specific function of the caecilian tentacle must remain speculative.

234 H A R O L D FOX

Skilled technical assistance was provided by David Franklin, Roy Mahoney and Brian Pirie. Dr Mary

Dr H. G. Vevers of the Zoological Society of London and Prof C. Cans of the University of Michigan Whitear kindly gave valued advice during the preparation of the manuscript.

generously supplied me with the adults of Zchthyophis.

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