J . Zool., Lond. ( 1 983) 199,223-248

The skin of Ichfhyophis (Amphibia : Caecilia): an ultrastructural study

H A R O L D Fox* Department of Zoology, University College, London

(Accepted 13 July 1982)

(With 10 plates in the text)

The skin of the adult Ichfhyophis spp. has been investigated using light microscopy and transmission electron microscopy, and the various epidermal and dermal integral cellular components of this genus, of the order Caecilia, are compared and contrasted with those of members of the Anura and Urodela among the Amphibia. In general the epidermis of Ichthyophis is typically amphibian in appearance, though in terms ofsize the cells are large and of urodelan dimensions. Apart from the epithelial cells, in addition the epidermis includes a number of specifically different cells, some doubtless arising in sifu, possibly from the stratum germinativum, others entering the epidermis from elsewhere.

The dermis of caecilians is unique among living amphibians in possessing scales located in pockets, and for completeness their gross structure and arrangement are described and existing information on their ultrastructure is summarized. The cellular composition and arrangement of the dermal glands are described and the glandular components in the Amphibia are compared.

Contents

Introduction.. . . Materialsand methods Results . . . .

The epidermis . . Thedermis . .

The scales . . Theglands . . The granular glands The mucous glands Ducts of the glands Other dermal tissues

Discussion . . . . References . . . .

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. . . . Descriptions of Plates of adult Ichlhyophis specimens Explanation of lettering used in Plates

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Introduction

The order Caecilia (Gymnophiona or Apoda) comprises a group of living amphibians which are limb-less and long bodied worm-like animals, with no known fossil representation. The body has a series of external transverse grooves or annuli and is coloured or patterned according to species. The skin of most forms includes dermal scales. Some caecilians, however, are scale-less (i.e. Scoleomorphidae and Typhlonectidae), though Typhlonectes pressicuudu does possess minute scales in the hinder region of the body (Wake, 1975).

223 0022-5460/83/020223 + 26 $03 .00 /0 0 1983 The Zoological Society of London

224 H. FOX

Primitive characters of the caecilians are the scales and features of the skeleton, gill clefts, the hyobranchial apparatus and the urinogenital system, some of which are only recognizable in young stages during ontogeny. Other structures such as the retractile tentacles, lid-less eyes, the body elongation and in some species various fusions of the skull components, are specialized evolutionary adaptations probably for burrowing. Most genera, such as Ichthyo- phis and Hypogeophis, lay large yolky eggs on land; others like the aquatic Typhlonecfes give birth to fully developed young.

During the past hundred years or so since the classical investigations of the Sarasins (1887-1890), various workers have described the skin (and scales) of caecilians at the level of light microscopy. Only fairly recently, however, have features of the skin been examined by electron microscopy (Welsch & Storch, 1973; Fox & Whitear, 1978; Zylberberg, Castanet & Ricqles, 1980). The present work, therefore, provides further descriptions of the ultra- structure of different cellular components in the epidermis and dermis, including the archi- tecture of the two types of dermal glands, of Ichthyophis and compares and contrasts the results with previous available information on the skin of caecilians and other amphibian orders.

Materials and methods Skin of adult females of Ichthyophis orfhoplicafus and Ichthyophis kohtaoensis was prepared for

examination by light and electron microscopy. For light microscopy strips of skin from different regions of the body, including the scales, from

pithed I. orthopficatus, were fixed in formol-saline. Transverse and longitudinal serial sections 8 pm thick, from dehydrated, paraffin-embedded material, were prepared by standard methods and stained by Masson’s trichrome stain or by Weigert’s haematoxylin and aqueous eosin. Sections were photographed using a Zeiss photomicroscope. In order to examine whole mounts of skin for the gross arrangement of scales, circular strips of body skin 3 cm by 1 cm in dimensions were spread flat on glass slides. The material was macerated in 0.5% NaOH (in 70% alcohol) and thence a few drops of a solution of alizarine red S (in absolute alcohol) were added until a pale red colour appeared. Thereafter the skin was dehydrated and mounted in Canada balsam.

For electron microscopy skin from different regions of the body of I. orthoplicatus and I . kohtaoensis was fixed ice-cold for 1-3 h in a combined fixative of osmic acid and glutaraldehyde (Hirsch & Fedorko, 1968), or in 24% or 6% glutaraldehyde (1-2 h) and post-tixed in 1 % osmium tetroxide (about I h) buffered at pH 7.4. Material was dehydrated in increasing concentrations of alcohol and finally propylene oxide and embedded in Araldite epoxy resin. Silver grey sections were mounted on copper grids and stained with uranyl acetate and lead citrate. Sections were viewed under a Corinth 275 and an AEI 6B electron microscope.

Results

The epidermis The epidermis of an Ichthyophis adult from different regions of the body generally has

a similar appearance in section to that of other amphibians (Parakkal & Matolsty, 1964; Farquhar & Palade, 1965; Lavker, 1972; Welsch & Storch, 1973). In back skin of I . ortho- plicatus sectioned longitudinally (sagitally), the epidermis is about 0.03 mm thick and the dermis is up to 0.27 mm thick (Plate I(a)). The epidermis of the body of adults of both

SKIN OF ICHTHYOPHIS 225

PLATE 1. (a) Dorsal back skin of I. orthoplicatus sectioned longitudinally showing the arrangement in the dermis of the granular and mucous glands and the groups of scales in pockets. The epidermis is thin relative to the dermis. (b) Ventral (pale) body skin of I . orthoplicatus sectioned transversely showing similar dermal components to those in (a) but in a ditferent orientation. The scale sections, not shown in the Figure, are almost horizontal in position: the ducts of the glands are prominent. (Explanation ofabbreviations see p. 247).

226 H. FOX

I. kohtaoensis and I . orthoplicatus is formed from about six or seven layers of epithelial cells (Plate I(b)). In general in the two species the arrangement and ultrastructure of the cells are similar.

Non-cornified outermost epidermal cells are somewhat flattened and have a well formed elongate nucleus with nucleopores still present. The cytoplasm includes only modest amounts of RER and there is an extensive matrix of polysomes in which bundles of tono- filaments extend throughout the cell. Mitochondria with cristae occur and numerous round, somewhat electron-translucent vesicles with smooth surfaces bound, often situated external to the nucleus. Some ofthese may be lipid droplets. Pigment granules are present (Plate II(a)).

When the outermost layer of the epidermis is keratinized (stratum corneum) it comprises extremely flattened, electron-dense keratinocytes, which have few recognizable organelles (Plate II(b)). The nucleus frequently is barely distinguishable or has disappeared. Sometimes a second layer of cells is somewhat similar in appearance. There is a dense thickened layer beneath the cell surface, about 25-30 nm thick, which is more prominent at the inner wall (Plate II(c)). The inner adjacent epidermal cells join those of the outer layer by desmosomes at short stub-like projections and the space between them contains small masses of appar- ently structureless material, some of which may well be part of the cell surface (Plate II(d)).

The intermediate cell layers (stratum spinosum) are composed of irregular shaped cells (Plate III(a)), which have large desmosomes (up to 0.8 pm long) joining them. Dense bundles of tonofilaments lead from the desmosomes into the cell (Plate III(b)). Such cells are replete with dense masses of tonofilaments extending in various directions; they are similar in appearance and thickness to the filaments arising from the desmosomes and are about 12-1 5 nm thick (Plate III(c)). Dense rounded granules, smooth vesicles, polysomes and glyco- gen granules are present but there are few mitochondria.

The roughly elongate-cuboidal-shaped basal cells (stratum basale or stratum germinati- vum) contain massive bundles of tonofilaments, leading practically perpendicularly into the cell from the rather poorly developed hemidesmosomes (Plates III(d), IV (a)). The oval-shaped nuclei tend to orientate with their long axis at right angles to the skin surface. Pinocytotic vesicles may occur within the cell margins, there is a high ribosomal content and more numerous mitochondria than in more superficial cells. Melanin granules are fre- quently present. The undulating inner surface of the basal cells is bounded by an adepidermal membrane, and fine fibrils from the cell surface traverse the adepidermal space to join the membrane (Plate IV(a)).

Flask cells occur in the body epidermis of Ichthyophis; the typical shape is best seen in sagittal sections. They are undoubtedly the “birnformige Zellen” of I. kohtaoensis skin, which are packed with mitochondria, part of whose ultrastructural profile was illustrated by Welsch & Storch (1973). In the present work it was found that the flask cells of I. koh- taoensis may be 20 pm long and 7 pm wide and the neck is 3 pm wide. They tend to orientate at right angles to the skin surface and profiles of flask cells may be found in regions of the second and third layers of epidermal cells (Plate IV(b)). A shorter stouter flask cell of I . orth- oplicatus, in profile I5 pm long, 10 pm wide (flask) and 6 pm wide (neck), terminated just beneath the stratum corneum (Plate IV(c)). A representative flask cell of I. kohtaoensis had apical microvillous-like ridges and laterally and basally similar short processes interdigitate with those of adjacent epithelial cells, to which they join by numerous desmosomes (Plate IV(d)). The cytoplasm is less dense and fibrous than that of neighbouring cells, and there is a high content of mitochondria (with well developed cristae). Flask cells contain micro-

SKIN OF I C H 7 H YOPHIS 227

PLAT^ 11. (a) Outermost layer of non-cornified epidermis of I. orthoplicafus. The somewhat flattened outer cells are similar, and joined by desmosomes, to the second layer of cells. Outer cells typically have bundles of tonofila- ments and dense granules. Other organelles are also recognizable (see text). (b) Stratum corneum of the epidermis of I. kohrao~n.si.s. The dense layer of cells has little recognizable ultrastructure. The second adjacent layer of cells is similar in ultrastructure to the non-cornified outer epidermal layer of (a). Indeed, if the stratum corneum were shed then (b) would be just like (a). Such shedding with these resulting profiles presumably occurs during life. (c) Higher magnification of a region of the stratum corneum of I. kohtaoensis showing the packed mat-like tonofila- ments. Most, if not all, of the other organelles cannot be recognized and presumably havedegenerated. (d) Desmoso- ma1 junctions between the stratum corneum and the second epidermal cell layer of I. orthoplicatus. There is a fine dense fillet below the inner surface ofthe cornified cell. (Explanation ofabbreviations see p. 247).

228 H. FOX

filaments of finer diameter and lighter electron density than the tonofilaments, and they occur in smaller looser bundles. There are also microtubules, numerous small vesicles often packed together above the nucleus, and a well developed Golgi apparatus (Plate IV(e)). Polysomes occur in abundance and there are occasional pigment granules. A sub-apical nerve terminal was seen to be closely apposed to the surface of the neck of the stout flask cell previously mentioned, though whether the nerve passes alongside or actually innervates it could not be decided (Plate IV(c)).

Merkel cells of adults of I . kohtaoensis and I . orthoplicatus, first reported in caecilians by Fox & Whitear (1978), are located above and between the basal epidermal cells but not in contact with the dermis (Plate V(a)). They occur in back and belly skin but have not been found in the tentacles and they are extremely uncommon, albeit clearly distinguishable from adjacent basal cells by their typical ultrastructure. Merkel cells of these caecilians are about 12 pm long and 9 pm wide, roughly intermediate in size between the smaller anuran and larger urodelan Merkel cells. They are usually oval or rounded in shape, with a smooth surface giving off finger-like microvillar processes about 2 pm long and up to 0.3 pm wide, which indent neighbouring cells. The processes of I . orthoplicatus appear more slender than those of I . kohtaoensis. Up to six processes were recognized in a single profile and probably up to 100 or more may originate from a single Merkel cell surface. Cell processes have microfilaments (which could possibly be actin), orientated parallel along the long axis, which insert on an apical placque. Desmosomes (up to 0.4 pm long) join Merkel cells to adjacent cells; they do not occur on the microvillar processes. Membrane-bound granules (80-120 nm in diameter) may be extremely numerous (up to 500 in a single profile), and usually they are located basally in the cell towards the basement membrane; they are absent in the cell processes (Plate V(b)); see also Fox & Whitear, 1978). The cytoplasm contains bundles of tonofilaments, some cells having only moderate fibrosity but others may have a mat-like high concentration of filaments throughout the cell. Merkel cell nuclei are variable in shape and the cytoplasm also includes mitochondria, a Golgi complex, small vesicles and occasion- ally centrioles. Nerve terminals, or their synapses of mechano-receptive nerves associated with Merkel cells, have not so far been recognized, although they are a feature of Merkel cells in anurans and urodelans. Indeed nerve profiles are difficult to distinguish in the epider- mis of Ichthyuphis though some have been seen.

Granule-containing cells seen near the base of the epidermis are lightly textured, have an irregularly shaped nucleus filling most of the cell profile, and include mitochondria, ribo- somes and polymorphic electron-dense granules (Plate V(c)); see also Welsch & Storch, 1973). Desmosomes cannot be identified with any degree of certainty. These cells may well be leucocytes. Other cells found also in the basal epidermal region include possible lympho- cytes and macrophages, which are probably polymorphonuclear leucocytes or granulocytes, without desmosomes and of variable shape and about 12 pm by 9 pm in dimensions. Their surface is irregular and cytoplasmic extensions intrude between neighbouring cells. Numer- ous mitochondria and dense granules of variable size and shape are spread throughout the cytoplasm, which also includes a RER, polysomes, vesicles, microfilaments and a well developed Golgi complex.

Irregularly shaped immigrant melanocytes without desmosomes, are present at different levels in the epidermis (Plate V(d)). Their cytoplasm is packed with melanosomes (about 0.3 pm in diameter). Cellular extensions (dendrites) of the melanocytes, distinguishable by their pigment content, are frequently seen between the epithelial cells.

SKIN OF I C H T H Y O P H I S 229

PLATE I l l . (a) Profiles of epidermal cells of the 2nd and 3rd layers (stratum spinosum) of I . kohtaoensis, showing the large oval-shaped nuclei, granules in the cells, a high fibrosity and pronounced desmosomes joining the cells. (b) Large prominent desmosomes of epidermal cells (3rd and 4th layers). They are usually straight but frequently other large curved varieties are seen. It is possible, however, that desmosome shape appears different according to section orientation. (c) High magnification showing the heavy fibrosity of bundles of tonofilaments and dense granules in cells of the 3rd layer of the epidermis of I . orthoplicatus, typical ofother layers ofthe stratum spinosum. (d) The basal epidermal cell layer (stratum basale or stratum germinativum) of I . kohtaoensis. The large cells have an undulating margin at the epidermal-dermal interface and bundles of tonofilaments extend into the cell towards the nucleus, roughly perpendicular in direction to the epidermal surface. (Explanation ofabbrevlations see P. 247).

SKIN OF ICHTH Y O P H I S 23 I

The dermis The scales

Living anurans and urodelans have no scales in their skin at any stage in the life cycle. Nor have any vestigial elements remotely similar to them been recognized. In contrast most living members of the Caecilia retain dermal scales, either throughout the body or merely in the posterior region, according to species. Some forms thus have scale-bearing annuli only in the hinder region ofthe body (Taylor, 1972; Wake, 1975). In Ichthyophis and Hypogeophis among other caecilians, for example, scales are well developed, extremely numerous and clearly recognizable in dissected whole mounts of skin, or in microscopic sections. The number of rows of scales in some forms may reach 2000 (Taylor, 1962).

Descriptions in varied detail of scales, examined at the gross or microscopic level, have been published by a number of workers including among others P. & F. Sarasin (1887-1 890), Cockerel1 (1 9 I2), Physalix ( 1 9 I Ou, b), Datz ( 1 923), Rabl (193 I ) , Peyer ( 1 93 I) , Marcus (1934), Feuer (1962), Lawson (1963), Wake (1975) and Casey & Lawson (1980). The ultra- structure of scales of I . kohtaoensis and Hypogeophis rostratus has been described in some detail by Zylberberg, Castanet & Ricqles (1979). The following account, therefore, only briefly reviews the information on scales for completeness, in terms of their relevance structurally, topographically and functionally to other skin components.

Scales are arranged in the skin in the transverse rows encircling the body, demonstrated typically in Ichthyophis and Hypogeophis. In each row scales overlap each other laterally but the transverse rows are clearly separate. Scales are not noticeably larger dorsally or ventrally in the body, but they do vary in size. Indeed among caecilians overall size of the scales may range from 0.4 to 4.5 mm in dimensions. The flattened cycloid or ovoid scales have numerous indentations on their upper surfac-the furrows-which are easily seen in whole mounts of scales at low magnification or in microscopical sections of them.

The long axis of the scale extends transversely to the body. They occur in groups in pockets in the dermis, where each scale affixes ventrally by connective tissue. The upper end lies freely below the epidermis and the entire scale extends obliquely, roughly parallel to its neighbours. In each pocket two to six scales overlap, though up to eight may occur in some species (Wake, 1975). Six was the maximum number found in a single pocket in I. orthoplicatus and I. kohtaoensis (Plate I(a)).

Using light microscopy Wake ( 1 975) described scales to be composed of parallel-orientated collagen fibres, arranged perpendicularly to the long axis or some ofthem parallel to it. Casey & Lawson (1980) described scales of H. rostratus to comprise a basal mineralized plate of bundles of collagen fibres overlain by an ossified plate anchored to them. At the level of

PLATE IV. (a) Margin of basal epidermal cell of I . kohiaoensis showing the tonofilaments associated with poorly developed hemidesmosomes. An adepidermal membrane (basal lamina) encloses an adepidermal space across which fine fibrils extend from the cell surface to the adepidermal membrane. (b) Well developed somewhat elongated flask cell profile, of I. kohtaoensis, in the epidermis. (c) Well developed stout-shaped profile ofa flask cell, of I. orihoplicn- [us, terminating below the stratum corneum. Note the small nerve terminal alongside the neck of the flask cell. (Photograph by kind permission of Dr Mary Whitear.) (d) Neck region of the flask cell of (b), showing some indi- cation of the apical undulating membrane. Desmosomes join the flask cell to adjacent epithelial cells; there are numerous mitochondria and a Golgi complex is seen near the apex. (e) Higher magnification of the apical region of the neck of the flask cell of (d) showing the Golgi complex, microfilaments in loose bundles and coated vesicles, but there is little RER and polysomes are scarce. (Explanation ofabbreviations see p. 247).

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S K I N OF ICHTH Y O P H I S 233

electron microscopy Zylberberg et al. (1980) clearly showed the scales of I. kohtaoensis and H . rostratus to be formed from deep flattened basal cells 4-5 pm thick and thence super- ficially there are bundles of parallel collagen fibres (periodicity of striation 65 nm), of varied orientation for different bundles. The scale fibres are about 100 nm in diameter, wider than that of the dermal collagen fibres, which are about 50 nm in diameter. On the upper surface of the scale and inclined towards the centre there are mineralized squamulae (separated by concentric and radial furrows), which differ and are diagnostic for different species (Taylor, 1972). A preliminary analysis by X-ray spectography identified silicon, zinc, calcium and phosphorus at the level of the squamulae (Zylberberg et al., 1980).

The glands The ultrastructure of the glands of the dermis of the adult Ichthyophis has been described

by Welsch & Storch (1973). The following account of the glands from different regions of the body, of adults of the same species I . kohtaoensis and of I. orthoplicatus, confirms some of their results, but also describes other features which are present and offers a more extensive interpretation of the topography and arrangement of the glandular architecture.

The granular glands The larger of the two types of glands, the granular or poison glands, the “Giftdrusen” or

“Kornerdrusen” of German authors, or the “Riesendrusen” (Sarasins, 1887-1 890) that tend to be located on the upper surface of the body (Physalix, 19 lob), are up to about 0.6 mm long and 0.1 5 mm wide. Like the mucous glands they have a duct traversing the epidermis to open to the exterior. Using light microscopy sagittal sections reveal an oblique orientation, probably due to their location between the individual groups of scales in their pockets-a pseudo-segmental arrangement (Plate I(a)). In transverse sections in a similar manner mucous and granular glands frequently seem to alternate, though a 1-1 relationship overall is unlikely for granular glands are less numerous than mucous glands per unit surface of skin (Physalix, 1910b), at least in some regions of the body.

Granular glands are bounded peripherally by myoepithelial cells, which join one another by desmosomes and generally form a single outer layer, though cells may overlap at their ends to form a wall more than one myoepithelial cell thick (Plate VI(a)). Skin sectioned trans- versely to the antero-posterior axis of the animal’s body frequently showed the myoepithelial cells as a narrowish band with an elongate nucleus. Other sections sagittal to the antero- posterior axis showed these cells to appear pyramidal or somewhat elongate-columnar in shape, the cells arranged side by side and having rounded nuclei (Plate VI(b)). However, this relationship of profile shape and section orientation could well be fortuitous. The flattened thin myoepithelial cells appear to be stretched, probably a result of intra-glandular tension due to the glandular content. This feature is usually particularly pronounced in the case of

PLATE V. (a) Location of a Merkel cell in the basal cell layer of the epidermis of 1. kohtaoensis. Note the well developed cell process extending apically and the concentration of membrane-bound granules in the basal region of the cell. (b) Portion of a Merkel cell of I. orthoplicarus which has a large number of membrane-bound granules and a mat-like content of microfilaments. Desmosomes join the Merkel cell to adjacent epithelial cells. (c) Granular cell in the basal epidermal region of 1. orthop/icatrcs. Probably this cell is an immigrant leucocyte. (d) Melanocyte in the basal epidermal region of I . orrhoplicatrcs. (Explanation orabbreviations see p. 247).

234 H. FOX

SKIN OF ICHTH YOPHIS 235

the mucous glands (Plate VII(d)). The outer surface of the myoepithelial cell is bounded by an extra-cellular “adepidermal space and membrane”, generally similar in appearance, arrangement and membrane thickness to that found bordering the basal epidermal cells at the epidermal-dermal interface. Myoepithelial cells have mitochondria mainly located nearer the surface and they contain enormous numbers of microfilaments and an extensive smooth endoplasmic reticulum, compared by Bock & Lertprapai (1 972) with the sarcoplas- mic reticulum of smooth muscle cells.

Internal to the myoepithelial cells there are mitochondria-rich cells, which are seen to join one another and the myoepithelial and granular cells by desmosomes (Plate VI(c)). Mito- chondria-rich cells were reported in the granular glands by Welsch & Storch (1973). The numerous mitochondria usually have a rich content of cristae, though sometimes, probably depending on the plane of section, the interior of the mitochondrion is moderately electron- dense and without cristae. The cell surface is frequently irregular or microvillous-like. There is a prominent Golgi complex (Plate VI(d)), usually with numerous small free rounded vesi- cles at the terminals of the cisternae; a smooth endoplasmic reticulum and polysomes are present, and largish round and moderately electron-dense granules are occasionally seen, usually located in the outer region of the cell.

Granule-producing cells provide the main bulk of the granular glands (Plates VI(c), VII(a)). These cells contain enormous numbers of round, membrane-bound granules, of varied appearance and electron-density in profile, whose dimensions are up to 3 pm in diameter. Granules include those with discrete electron-dense contents, others are of uniform but dif- ferent densities and others are mottled in appearance, have no membrane and seem to be in the process of dissolving their contents in the ambient medium (Plate VII(b)). Granules may well be a source of the proteinaceous poison secretions of high and varied chemical complexity described by Neuworth et al. (1 979) in other amphibians. Granule-producing cells join the mitochondria-rich cells and, when in their vicinity, the myoepithelial cells by desmosomes. Surrounding a peripherally situated nucleus the cytoplasm includes a RER, pol ysomes and mitochondria. Areas of the cell surface may be microvillous-like, though its extent may well be due to the effects of fixation. The greater part of the cell, indeed the greater part of the gland, is made up of a granulated or amorphous background substance of greater or lesser electron density, surrounding the granules. Mucus-like droplets are frequently found in some granule-producing cells, so as to suggest a mixed muco-granular type cell in the gland. The extent of this mucus-granule association, however, cannot be determined.

PLATE VI. (a) Somewhat flattened myoepithelial cells joined by desmosomes at the outer surface of a dermal granular gland of I. orthoplicatus. These cells are replete with microfilaments and there is a high content of smooth endoplasmic reticulum. The outer surface of the cells is bounded extra-cellularly by a membrane which appears similar to the adepidermal membrane ofbasal cells. (b) Myoepithelial cells surrounding a granular gland ofI. orrho- plicatiis but sectioned at a different orientation frpm those of PI. Vl.(a). Here the cells are roughly elongate-columnar in shape. There are nerves (still with sheaths) against the outer surface of the myoepithelial cells. (c) Region at the outer margin ofa granular gland of I. orthoplicatus showing profiles ofan outer myoepithelial cell and internally a mitochondria-rich cell surrounded by granule-producing cells, though the one above with a nucleus has only one granule in this section. The granules in this illustration are of uniform electron density. Note the desmosomes joining the mitochondria-rich cell to the granule-producing cells (the mitochondria-rich cells also join the myo- epithelial cells by desmosomes, though this feature is not seen in the picture), and granule producing cells also join one another by desmosomes. (d) A Golgi complex in a mitochondria-rich cell of a granular gland of I. orlhopli- catus. (Explanation of abbreviations see p. 247).

PLATE V11. (a) Granule-producing cells of a granular gland over a mitochondria-rich cell in 1. orrhoplicatus. The granules are of different electron densities. Note the microvillous-like surface of the granule-producing cell at the lower left of the picture. (b) Granules of varied sizes and profile densities of a granule-producing cell from a granular gland of 1. orthoplicatus. Some of the granules appear to be dissolving their contents in the surrounding medium: other areas are possibly remains of dissolved granules. (c) Part of a myoepithelial cell, inner mitochon- dria-rich cell and mucus-producing cell at the outer margin of a mucous gland of I. orthoplicatus. There is an extra- cellular bounding membrane ofthe myoepithelial cell, similar to that of like cells in the granular gland. (d) Profile of a region of a highly swollen mucous gland of I. kohtaoensis at the outer surface. The myoepithelial cells are extremely thin and presumably greatly stretched owing to the content of mucus in the gland. (Explanation of abbreviations seep. 247).

SKINOFICHTHYOPHIS 231

The mucous glands The rounded-oval or flask-shaped mucous glands, the “Schleimdrusen” of German

authors or “Spitzdrusen” (Sarasins, 1887-1 890), are up to 0- 13 mm long and 0.08 mm wide, the duct extending about 40 pm through the epidermis to the surface (Plate I(b)).

Mucous glands like the granular glands are enveloped by myoepithelial cells, which form a wall sometimes more than one cell thick. In general, the location, arrangement and ultra- structure of these cells appear to be similar to the comparable ones in the granular glands. Internal to the myoepithelial cells individual mitochondria-rich cells are situated at irregular but frequent intervals, and likewise they are similar in form, location and ultrastructure to those described in the granular glands (Plate VII(c)).

The inner situated mucus-producing cells contain large numbers of mucus droplets, of variable size. Many of them have coalesced to form single large droplets, which fill much of the cell and are responsible for its swollen appearance (Plate VII(d)). Owing to the enor- mous quantity of mucus in some cells, the nucleus is ultimately situated amid a reduced electron-dense cytoplasm, which has a high content of RER and polysomes; a Golgi complex may also be recognized. However, even this region of the cell can often contain mucus drop- lets (Plate VIII(a)). Mucus may often extend almost to the periphery of the gland against the myoepithelial cells (Plate VII(c)).

Nerve terminals, sometimes ensheathed and grouped together and with lucent vesicles, are found closely apposed to the outer surface of the myoepithelial cells of mucous and granular glands (Plate VI(b)). An ensheathed nerve axon (2.5 by 0.75 pm in dimensions) was seen between two outer myoepithelial cells of a mucous gland, presumably entering it (Plate VIII(b)).

Ducts of the glands The elongated duct of the granular gland is orientated roughly at right angles to the skin

through the epidermis, and formed from flattened electron-dense cells with little recognizable ultrastructure (Plate VIII(c)). Duct cells join adjacent epithelial cells of the epidermis by des- mosomes, and in transverse section likewise desmosomes are seen to join adjacent duct cells (Plates VIII(d), IX(b)). At high magnification in some profiles the lumen wall appeared to have a fine cuticular surface, and the outer wall has a dense thickened layer below the cell surface (Plate VIII(d)). Probably the duct cells are composed of closely packed tonofilaments and contain keratin, and vesicles containing a dense material may be distinguished within duct tissue. The thickness of duct cells of the two Zchthyophis species is between 0.5 and 0.7 pm in longitudinal or transverse section. The stoma cells at the skin surfice are 2.7 pm wide, but owing to their density there is little recognizable ultrastructure (Plate VIII(c)). The duct lumen may be quite broad or almost closed, and it usually contains slightly granular- translucent material, presumably derived from the granule-producing cells of the granular gland.

Other dermal tissues Nucleated profiles or their anuclear cytoplasmic extensions of melanophores are widely

scattered throughout the dermis (Plate VIII(e)), either associated spatially with the external region of the glands or apparently distributed at random. The melanophores have round,

238 H. FOX

SKIN OF ICHTH Y O P H I S 239 oval or slightly elongated melanosomes within their cytoplasm (up to 0.5 pm long in the longest dimension), which are either closely packed together or distributed peripherally in the cytoplasm. Mitochondria which are often elongated, irregularly shaped vesicles and lightly dense bodies also occur amid the granular ribosomal matrix.

In both species of Ichthyophis characteristic nucleated cellular profiles, or their cytoplas- mic extensions, are found, often located near or closely associated topographically though separate from the melanophores (Plate IX(b)). They contain parallel orientated, somewhat electron dense, disc-like or ellipsoidal bodies, some of them up to 0.8 by 0.7 pm in diameter (though edge-on the disc can reach 1.25 pm long), and 200-250 nm thick. The discs are sometimes solitary but more usually and characteristically they are found in groups of up to four or five, each group randomly orientated with respect to its neighbouring groups (Plate X(a)). A crystalline structure of the ellipsoid body is suggested, for it is formed from a series of extremely fine flat lamellae, made up of what appears to be parallel rows of macromole- cules, each row about 5 nm thick and arranged in a lattice structure (Plate X(b)). The tissue containing the ellipsoid bodies is highly granular and it includes a RER, SER, numerous smooth and coated vesicles, mitochondria and pigment granules. The nucleus is elongated and regular in shape with a smooth surface. Ellipsoid bodies may be situated close to the nucleus or peripherally in the cytoplasmic extensions. The widespread distribution in the dermis of this tissue and its highly irregular shape, suggests that the cells have numerous cytoplasmic processes. Until more is known about them, tentatively they could be called laminophores, of unknown function.

Discussion Comparison of caecilian skin with that of other amphibian orders

Epidermal structures The epidermis of the body and the external gills of the embryonic Ichthyophis kohtaoensis

(see Welsch & Storch, 1973), is two-layered, generally similar to that of larval anurans and urodelans, which is two or three layered in tail and body (Fox, 1977). The basal epidermal cells of the Ichthyophis embryo contain dense packed masses of filaments; clearly these are the Figures of Eberth, represented in similar cells in anuran (Chapman & Dawson, 1961; Singer & Salpeter, 1961) and urodelan (Kelly, 1 9 6 6 ~ ) larvae. Surface cells with vesicular inclusions and dense granules are probably mucus-producing cells. Ciliated surface cells in the caecilian external gill filaments (see also Welsch, 1981 for Ichthyophis pancisulcus), a feature of amphibian larvae (Michaels, Albright & Patt, 1971; Greven, 1980), are retained longer than those presumably occurring at the surface of the embryonic body. Caecilian embryonic epidermal cells have numerous pigment granules, a feature ofcomparable cells in

PLATE Vlll. (a) Cytoplasm of a mucus-producing cell of I . orthoplicatus containing mucus droplets of varied size. The cell abuts an outer myoepithelial cell. (b) A sheathed nerve is located between two myoepithelial cells of a mucous gland of I . orthoplicatus. Surrounding the gland are collagen fibrils and melanophore tissue. (c) Opening of a duct of a granular gland of I . orthoplicatus at the skin surface. Owing to the extreme electron density of the duct tissue, in contrast to that of adjacent epithelial cells, there is little that can be distinguished in the duct cells. Note the narrow duct cells and the wider outer stoma cells, which join neighbouring epithelial cells by desmosomes. (d) Region ofa duct of a granular gland of I. orthoplicatus with a high content oftonofilaments, a dense fillet under- neath the outer surface and the inner (luminal) ‘cuticular’ layer. (e) Protle of a melanophore in the dermis of I. or/hop/ic,u/ir.r. (Explanation ofabbreviations see p. 247).

PLATE IX. (a) Duct of dermal gland (the type could not be decided) of I. kohlaoensis in transverse section. Desmo- somes join adjacent duct cells. (b) Nucleated laminophore tissue in the dermis of I. orrhoplicarus, showing ellipsoid discs in varied orientations, either flattened face-on or partially or completely edge-on. Laminations are faintly indi- cated in some discs. (Explanation ofabbreviations see p. 247).

SKIN OFICHTHYOPHlS 24 I

PLATE X. (a) A fairly typical and common profile of laminophore tissue in the dermis ofl. orfhoplicafus, showing the ellipsoid discs arranged either singly or, more often, in groups o f up to 4 or 5 generally parallel to one another. (b) Ultrastructure of ellipsoidal discs of laminophore tissue of I. orihoplicarus sectioned edge-on, showing the disc to be made up of a series of Rattened laminae, perhaps up to 20 or so in each disc, piled on top of one another. (Explanation ofabbreviat ions see p. 247).

species of other amphibians. Other epidermal cell types present in some a t least of various amphibians, such as Leydig cells (Kelly, 19666), Merkel cells (Fox & Whitear, 1978) and "Stiftchenzellen" (Whitear, 1976), for example, or any immigrant melanocytes or poly- morphonuclear granulocytes, have not yet been reported in the embryonic epidermis. The absence of Leydig cells, which i f present would easily be recognizable in the epidermis, is a feature more typical of anuran larvae.

242 H . FOX

Using light microscopy Hetherington & Wake (1979) described lateral line organs of larval Zchthyophis spp. to include free mechanoreceptive neuromasts and electroreceptor ampull- ary organs; the latter are not present in anuran and urodelan larvae, Caecilian embryos with- in the oviduct lack the neuromast system of aquatic larvae (Wake, 1977). Bearing in mind the extensive range of different cells present in the amphibian larval epidermis (Fox, 198 11, it is clear that the embryonic and larval caecilian epidermis requires further investigation.

As is usual in amphibian larvae the embryonic basal epidermal cells of Zchthyophis are bounded at their dermal margin by an adepidermal membrane enclosing an adepidermal space (see Salpeter & Singer, 1959); thence there is an underlying collagenous basement lamella and mesenchymatous fibroblasts (Welsch & Storch, 1973, Fig. la), though a regular orthogonal pattern (Weiss & Ferris, 1954) cannot be distinguished.

In climactic larvae of Xenopus laevis and Rana temporaria there are up to five layers of epidermal cells; in Ambystoma larvae there are five or six layers of epidermal cells (Farquhar & Palade, 1965), generally about the same number oflayers as in the adult amphibian epider- mis (Fox, 1977). Likewise the adult Zchthyophis has six or seven layers, which superficially at least bear a strong resemblance in appearance to-those of the adult Bufo bvfo (Budtz & Larsen, 1975; and pers. observ.), though the caecilian cells are larger. It is difficult to compare epithelial cell sizes in the epidermis of the three orders of amphibians, because of the varia- bility in shape, those nearer the surface are extremely flattened. Nevertheless a rough com- parison of size of cells from electronmicrographs, using the parameters of the longest axes of length and breadth, ofa random selection from larvae and adults was made. In the urodeles Proteus, Ambystoma and Salamandra cell length may reach 22 pm and width 13 pm; in anurans Rana and Xenopus comparable measurements were 10 and 9 pm and in Ichthyophis cells were up to 24 pm long and 16 pm wide. Thus caecilian epithelial epidermal cell size is more comparable to that of urodeles.

The presence in the epidermis of numerous and more superficially situated flask cells, uncommon and basally situated Merkel cells and immigrant melanocytes and macro- phages, is fairly typical of the adult amphibian skin. Again in Ichthyophis the outer stratum corneum, comprising a single layer of dense flattened keratinocytes, which (periodically) sloughs, is also an amphibian feature. The temporal cyclical programme of moulting in Ichthyophis (or for any other caecilian for that matter), is not known. Typical examples in amphibians are Bufo bufo, which at 20°C moults about once a week (Budtz & Larsen, 1973) and Rana temporaria, at room temperature every two or three days (Whitear, 1975).

Dermal structures The origin and differentiation of the dermal scales of caecilians have not yet been described

using the electron microscope. However, the ultrastructure and phylogenetic relationships, but not function, of scales of adult caecilians and of other vertebrate groups were considered by Zylberberg et al. (1980). Scale retention in living caecilians is presumably bound up in some way with a burrowing mode of life, not generally practised by anurans and urodeles. Indeed some aquatic caecilians are practically scale-less. In Zchthyophis, for example, the large number of these mineralized components arranged in specific circular and overlapping repetitive patterns along the body, associated with the musculature, permits a wide range of bodily movement and requisite rigidity when required. The great muscular strength of these animals is apparent when they are handled. The scales provide coverage and protection

S K I N OF I C ' H T H YOPHIS 243

of internal organs. Together with the poison glands they are a defence against predators. Scales overlap glands and other dermal tissues and thus may play a role in preventing desic- cation. Perhaps bodily movement influences glandular secretion owing to mechanical press- ure exerted by the scales squeezing glandular contents through the ducts to the body surface. However, until scale function is investigated with experimental precision, one can only speculate on their purpose.

Study of dermal chromatophores of adult anurans demonstrates three main types of pig- ment cells, the melanophores, iridophores and xanthophores of typical form and ultra- structure (see Bagnara, 1976). Likewise in fish, for example, there are leucophores and iridophores (Takeuchi, 1976; Menter et al., 1979), xanthophores (Takeuchi & Kajashima, 1979) and the described chromatophores (Frese, 1978; Yoshizaki & Tamura, 1979) and also melanophores in dipnoans (Imaki & Chavin, 1975a, 6). None of these chromatophores are clearly comparable in appearance with the laminophores in the dermis oflchthyophis, though the laminations could be said to show a vague similarity with the reflecting platelets of iridophores of fish (Hawkes, 1974). The laminophores with their disc-like inclusions are unlikely to be phagocytes; nor is it likely that the discs are yolk inclusions. The cells could possibly be of a pigment-containing type of unknown biochemical content and function. Or they could be blood cell derivatives akin to eosinophile-type cells, for mammalian eosino- phile leucocytes contain comparable ellipsoid bodies, either found singly or arranged in pairs within a limiting membrane, which are crystalline and lattice-like (see Fawcett, 1966: 206-2 1 I ) , and these cells have discs of comparable size to those in the Zchthyophis cells.

In amphibians, myoepithelial cells were reported in the poison (granular) glands of adult Bombina variegata (Bock & Lertprapai, 1972) and Bombina bombina (Miscalencu et al., 1973); in mucous glands of adult Rana pipiens (VoGte, 1963); and in both types of glands of Rana esculenta (Bani, 1970), of larval Xenopus laevis at climax and in the mucous glands of the adult Triturus cristatus (Fox, unpubl.). They were likewise described in a variety of amphibians including various anurans and the urodele Hynobius leechi, whose myoepithelial cells were found in the mucous and granular glands (Kim et al., 1978, 1979). Their presence, therefore, in the mucous and granular glands of Zchthyophis could well be expected.

Nerves are found leading to both types of amphibian dermal glands. Unmyelinated nerve fibres are distributed to the wall of the underside of the granular glands ofBujo bufo (Whitear, 1955), and nerve terminals with lucent granules occur between the myoepithelial and granule-producing cells of the granular glands of Bombina variegata (Bock & Lertprapai, 1972). In caecilians a plexus of nerve fibres is distributed to the undersurface of the dermal glands of Dermophis mexicanus (Ochoterena, 1932), and in the present work it is shown that nerves are distributed to the myoepithelial cells of both types of glands in Ichthyophis. The resemblance of myoepithelial cells to smooth muscle by virtue of their ultrastructure, (indeed, Neuworth et al., 1979 described granular glands of dendrobatid frogs to be sur- rounded by a layer of smooth muscle (presumably the myoepithelial cells)) and the fact that they are innervated, supports the idea of a contractile function. This role was suggested by Miscalencu et al. ( I 973) for the myoepithelial cells of the exocrine glands of mammals.

Mitochondria-rich cells have been found in the gills of larval Discoglossuspictus (Hourdry, 1974), the epidermis of larvae of Rana and Xenopus (Fox & Whitear, unpubl.), the urinary bladder of Bufo marinus (Choi, 1963), Rana catesbeiana (Strum & Danon, 1974) and Salamandra salamandra (Piceis-Polver et al., 198 1 ) and in the palatal skin of Rana (Whitear, 1975). Flask cells of amphibians are mitochondria-rich (Farquhar & Palade, 1965) and they

244 H. FOX

are found in the epidermis throughout thegroup(see Whitear, 1975; Greven, 1980). Such cells may be concerned, among other things, in moulting (Masoni & Garcia-Romeu, 1979) and with the transport of substances other than sodium (Whitear, 1975). Their high content of mitochondria indicates that they are metabolically extremely active with reference to ATP, but their exact role has yet to be determined, Mitochondria-rich cells were reported in the mucous glands of Bombina orientalis and Rana nigromaculata, and Kim et al. (1978) assumed that they were present in all amphibian mucous glands. They also occur in the granular glands of Zchthyophis kohtaoensis (Welsch & Storch, 1973); they have now been found in this species and in I . orthoplicatus in the mucous glands as well, similar in topogra- phy and ultrastructure, as might well have been expected.

The ultrastructure of the mucus-producing and granule-producing cells of the mucous and granular glands respectively, has previously been described by Welsch & Storch (1973) and the results in the present work are broadly in agreement. In amphibians the mucus secreted by the skin may play a role in preventing desiccation and in thermoregulation and cutaneous respiration (Lillywhite & Licht, 1975). Breckenridge & Murugapillai (1974) reported that in caecilians the mucous glands produce a copioussecretion that is acidic with carboxyl and sulphate groups, has vicinal glycols and no detectable protein. The mucus is resistant to digestion by hyaluronidase and neuraminidase. They suggested that it may function by binding water and maintaining a moist surface to facilitate cutaneous respiration and also prevent desiccation; provide slipperiness to evade predators; act as a lubricant in locomotion and tunnelling by reducing friction; provide a protective covering; have a bacteriocidal and/or fungicidal action and contribute to and moisten burrows. The authors considered that if an alarm pheronome was a function of glandular secretion then it is more likely to be from the granular glandular secretions, which are predominantly proteinaceous.

Amphibian dermal glands originate in ontogeny from downgrowths of cells from the epi- dermis (Bovbjerg, 1963; Delfino, 1977). The resulting cells differentiate into the mucous and granular glands of clearly recognizable structure and function. Yet examination of the topo- graphy and ultrastructure of cellular components of these glands reveals broad similarities, shown by their myoepithelial cells which envelop the glands and the peripherally situated mitochondria-rich cells. In caecilians the two types of glands differ in size, numbers and distribution, which differences may have been present in ancestral forms from the beginning, but more likely evolved in space and time throughout phylogeny. The main difference between the two types of glands, however, relates to the secretory activity of distinct mucus- producing and granule-producing cells. Whether the glands differed morphologically in this respect from the beginning, or the secretory cells subsequently differentiated from a common progenitor cell type is not known, though as teleost fish have several distinct types of uni- cellular glands in the epidermis, the mucous, sacciform and club cells (see Mittal, Whitear & Bullock, 198 I ; Whitear, 198 l ) , the former view is more likely.

A study of glandular origin and differentiation in embryonic caecilians may possibly shed some light on this interesting problem.

I should like to thank Dr G. Vevers and the Zoological Society of London for the supply of live Zchthyophis kohtaoensis adults, and Professor C. Gans of the University of Michigan, for sending me the live Zchthyophis orthoplicatus adults, which were used in this work. Thanks are also due to D. Franklin, R. Mahoney, E. Perry, B. Pirie and Pamela Stephenson for their skilled technical assistance and to Dr Mary Whitear, for valued comments and advice during the preparation of the manuscript.

SKIN O F IC'H TH YOPHIS

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Descriptions of Plates of adult Ichthyophis specimens All the figures are of electronmicrographs except for Plate I(a) (sagittal section) and Plate

I(b) (transverse section), which are micrographs sectioned at 8 pm and stained by haema- toxylin and eosin. The sections are of body skin from various regions of the back, except for Plate I(b), which is of ventral skin and Plate VIII(e), which is of side skin. Figures on Plates VI(b)-(d), VII(c) and X(a),(b) are longitudinal (sagittal) sections and all the others are transverse to the long axis of the animal’s body.

Figures on Plates II(a), VII(c) and IX(b) were from material fixed in 6% glutaraldehyde and Figures on Plates II(d), lII(b),(c), V(b)-(d), VI(a)-(d), VII(a),(b), VIII(a)(c)(d) and X(a),(b) were fixed in 24% glutaraldehyde: post-fixation was in osmium tetroxide. All the other figures were from material fixed in a combined glutaraldehyde and osmic acid mixture (Hirsch & Fedorko, 1968). Sections were double stained in uranyl acetate and lead citrate.

Ekplanaiion oflriiering used in Plaies

AFC AM AS BC cc cv D Derm DG DL DGr Dm DM Do EP EP 1 EP 2 EP 3 Epith

Apex of neck of flask cell Adepidermal membrane Adepidermal space Basal epidermal cell Cornified outer epidermal cell Coated vesicle Duct of gland Dermals Dense granule Duct lumen Dissolving granule Desmosome Dense membrane; inner wall of cornified epidermal cell External opening of glandular duct Epidermis Epithelial cell; non-cornified outer layer of epidermis Epithelial cell; second layer of epidermis Epithelial cell: third layer of epidermis Epithelial cell of epidermis

248

FB FC GC GG GLB G PC Gr G K HDm IJ IS LB LC LM Lm MBG MeC Me1 Mf MG M PC MRC Mt Mu MUD MYC MyOS N Ne Nk Pr RER s c SN T F

H. FOX

Fibrous bundles of tonofilaments in epidermal cell Flask cell Golgi complex Granular gland Groups of ellipsoidal bodies in laminophore Granule-producing cell of granular gland Granule in granule-producing cell Granulocyte in epidermis Hemidesmosome Intercellular junction Intercellular space Laminated ellipsoidal body Laminophore Lumen margin of duct Lamination in ellipsoidal body Membrane-bound granule Merkel cell Melanophore (or Melanocyte) Microfilaments Mucous gland Mucus-producing cell Mitochondria-rich cell Mitochondrion Mucus of mucus-producing cell Mucus of droplet Myoepithelial cell Outer surface of myoepithelial cell Nucleus Nerve ending in epidermis Neck of flask cell Process of Merkel cell Granular endoplasmic reticulum Scale component of dermis Sheathed nerves Tonofil aments