J. Zool., Lond. (1989) 217, 213-226

Tail skin of the dipnoan Neoceratodus larva: fine structure and differentiation

HAROLD Fox Department of Biology, University College, Gower Street, London, UK

(Accepted 25 Ma-v 1988)

(With 5 plates in the text)

The fine structure of the tail skin of larval Neocerntodus forsteri, between stages 40 and 50 (Kemp. 1982). is described and where applicable specific cellular components are compared and contrasted with comparable ones in the skin of adult dipnoans, teleosts and larval and adult amphibians.

The epidermis of the early developing tail, within the range studied, differentiates a variety of different cell types. Surface epithelial lucent and vacuolated lucent cells and basal cells are distinguished, and goblet (mucous) cells, Merkel cells and macrophages appear in the epidermis towards the end of the series.

Below a poorly developed collagenous basement lamella, immature melanophores with prernelanosomes are present, and likewise there are non-myelinated nerves, some striated muscle fibres, capillaries and mesenchymal fibroblasts.

The tail epidermis is innervated by naked neurites from the beginning of the series, and the earliest recognizable Merkel cell is in synaptic association with neurites.

Contents Page

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

Introduction

Among recent works on adult dipnoan skin, there is information on its fine structure in Protopterus annectens (Kitzan & Sweeny, 1968) and P . aethiopicus (Merkel cells, Fox, Lane & Whitear, 1980), and its histochemistry in P. dolloi (Bolognani-Fantin & Porcelli, 1969) and Lepidosiren paradoxa (Singh & Singh, 1979). The detailed fine structure of the melanophores and goblet cells of skin of Protopterus sp., L. paradoxa and Neoceratodus forsteri was described by Imaki & Chavin (1975a, 6, 1984).

Among larvae, the origin and distribution of melanophores (and erythrophores) in a staged series of Neoceratodusforsteri was referred to by Kemp (1 98 I , 1982), and ciliation of the body skin was described by Whiting & Bone (1980). From the detailed descriptions of the skin of fishes in the recent extensive review by Whitear (1986), it is clear that little is known about the ultrastructure of larval dipnoan skin. Indeed, apparently the only published work is on the epidermis of early stages of Neoceratodus by Whiting & Bone (1 980), using scanning electron microscopy.

213 0952-8369/89/002213+ 14 $03.00 0 1989 The Zoological Society of London

214 HAROLD FOX

Recently, however, some tail material of a series of stages of larvae of Neoceratodusforsteri, up to about 12 weeks after hatching, became available for study. The results, described in the following account of the fine structure of the skin, show that, even during the relatively short period in terms of the animal’s life history, a barely developed tail has differentiated skin with a variety of different cell components. These cells are described and where feasible compared and contrasted, in terms of function and homology, with comparable components in the skin of adult dipnoans and teleosts, and larval and adult amphibians.

Materials and methods

Tail skin of larvae of Neoceratodus forsteri, captured in Queensland, Australia, at stages 40,42,44,46,47, 48 and 50 (Kemp, 1982), modified after the original numbered series by Semon (1893), was examined by transmission electron microscopy (TEM). Other larvae 13-16 mm total length, in serial sections, were examined by light microscopy (LM).

For LM, larvae were fixed either in neutral buffered formalin or Zenker’s fixative, dehydrated and embedded in paraffin wax, serially sectioned at 8 pm or 10 pm and stained with haematoxylin and eosin. They were photographed with a Zeiss Ultraphot 1 1.

For TEM, tail pieces were fixed in Karnovsky fixative (Karnovsky, 1965) and dehydrated and embedded in Epon-Araldite epoxy resin (in Kemp’s laboratory, Queensland) by standard methods. Silver-grey sections, prepared with a diamond knife and stained with uranyl acetate and lead citrate, were viewed under a JEM JEOL CX 11 electronmicroscope.

The following information on the larval stages of Neoceratodus of the present work is based mainly on descriptions by Kemp (1981, 1982), and, to a lesser extent, those of Whiting & Bone (1980) and Fox (1960, 1965).

Stage 40, larva approx. 1 I mm long, operculum covers the 4 gill slits, equivalent to Semon’s stage 43: stage 42, larva approx. 11 mm long, primordia of pectoral fins appear, equivalent to Semon stage 44: stage 44, hatched probably about 1-4 weeks, 11-12 mm long, preanal fin extended forward halfway along the ventral yolk, nares visible, melanophores and erythrophores already present in the body (melanophores at stage 35), animal changes colour according to the light, equivalent to Semon stage 45: stage 46, hatched 4 weeks, larva approx. 13-14 mm long, there is a well formed alimentary canal, pronephric tubules and pronephric ducts are open into the cloaca in a 14.5 mm larva: stage 47, hatched 6 weeks, 15-1 6 mm long, spiral valve visible in gut, larva feeding, substantial development of skeletal, nervous and other morphological components of the head and pharynx, axicl skeleton appears in the pectoral fins: stage 48, hatched 8 weeks, preanal fin reached the operculum, primordia of pelvic fins appear, equivalent to Semon stage 47: stage 50, hatched 10-12 weeks, preanal fin regression, head broad, body narrow, equivalent to Semon stage 48.

According to Kemp (1981,1982), hatching of Neoceratodus in the laboratory (22 “C) occurs between stages 43 and 44, up to a month or more after laying, when larvae escape from the egg membranes through a small

PLATE I. (a) LM of proximal tail skin of a 16-mm Neocerafodus larva. Note the epidermal lucent cells (Ic), which are also present in the skin of the dorsal fin, gills, body and pectoral fins. The basal cells (bc) are flattened. Scale bar 0.02 mm. (b) LM of lucent cells (Ic) in the body epidermis, overlying the basal cells (bc) of a 13-mm Neoceratodus larva. Lucent cells also occur in the tail skin of this larva but are less numerous. Scale bar 0.02 mm. (c) SEM of region of tail epidermis of a stage 42 Neocerafodus larva. The outer cell has a high content of tonofilaments (tf) at this early stage, with mitochondria (m), granular endoplasmic reticulum (rer) and a surface packed with mucous vesicles (mv), which discharge their contents to the exterior. Scale bar 1 pm. (d) SEM of part of a basal epidermal cell (bc) of a stage 42 Neocerutodus larva. The cell is replete with tonofilaments (tf) and neurites (ne) are present between the epithelial cells. There is no basement lamella but an adepidermal membrane (am) is recognizable. Scale bar 1 pm.

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aperture in the egg albumin. However, the time during stage development of Neoceratodus is variable in different forms, some larvae proceeding through different stages quicker than others. The above information on hatching times is thus only approximate, and merely provides an indication of these features of larval development.

Results

The epidermis

Tail epidermis of Neoceratodus is about 30 pm thick (stage 42), reducing in thickness to about I7 pm (stage 46) and 10-15 pm (stage 50 and a 16-mm larva). Initially, therefore, the tail epidermis progressively becomes thinner. Body epidermis of a 13-mm and a 16-mm larva (about stages 46- 48) is about 25-30 pm thick, albeit slightly thinner in the younger stage. Plate I (a and b) shows examples of body and tail skin by LM, where the lucent cells (vide infra) are prominent.

Head epidermis (about 6 cells deep) of an 80 cm long adult Neoceratodus forsteri is much thicker, 100-150 pm thick (Pfeiffer, 1968), as might be expected.

In stages 40 and 42, the larval Neoceratodus tail is barely developed and epidermal cells of the different layers contain large lipid droplets (9-10 pm in diameter) and yolk bodies (5-6 pm in diameter). Nevertheless, there is substantial differentiation of the cells of the 2/3 layered epidermis. These are filamentous with polysomes, RER, mitochondria, SER and membrane-bounded pigment granules. Alongside the nucleus of cells of both layers, a well developed Golgi complex may reach 2-3 pm in length and 0.2 pm wide, of 5-7 stacks with terminal cisternae and numerous small, round, smooth-surfaced vesicles. The epidermal surface is lined by mucous vesicles, which appear more numerous in earlier stages of the series (Plate Ic). Microvilli and possibly microridges occur, and, in addition, there are surface blebs, up to 0.5 pm in diameter, though it is not clear whether they result from fixation. Cilia were never found on the tail surface throughout the entire series.

Highly filamentous basal cells are usually flatter than outer cells; they have mitochondria, RER and pinocytotic vesicles at the proximal surface that are particularly numerous in older stages (Plates Id, IIIc). There are no hemidesmosomes and figures of Eberth (see Fox & Whitear, 1986) in any stage of the series (Plates Id, IIIb, c).

Outer cells join laterally by tight junctions and by desmosomes, which likewise join epithelial cells of both layers. Neurites are recognized from the beginning, between and within the epithelial cell layers (Plates I (d), V (c) ).

In stage 44 and restricted to the outer cells, the first indication of a lucent area around the nucleus is recognized (Plate IIa, b and vide infra). The cells of both layers of the epidermis in stage

PLATE 11. (a) SEM of outer epidermal cell of a Neoceratodus larva at stage 44, with the first indication of a lucent area (la) around the nucleus (n). A Golgi complex (9) is prominent and there are tonofilaments (to, mitochondria (m) and granular endoplasmic reticulum (rer). The round membrane-bound dense body (db), of unknown nature, is also found in outer cells of other stages (see Plate IIIa). Scale bar 1 pm. (b) SEM of outer lucent cells (Ic) of a stage 47 Neoceratodus larva. An extensive delicate fibro-granular lucent area (la) surrounds the nucleus (n), and a dense peripheral region with tonofilaments (to also contains mitochondria (m), granular endoplasmic reticulum (rer) and smooth-surfaced vesicles (sv) and mucous vesicles (mv). Golgi complexes (g) are usually found at the periphery of the lucent area. Scale bar 1 pm.

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46 are generally similar in appearance to those of the previous stage with some further differentiation in quality and number of various organelles.

Lucent cells seen by light microscopy in body and tail skin of a 13-mm larva (about stage 46), are larger and more numerous in the skin of the body, dorsal fin, gills, pectoral fins and tail of a 16-mm larva (see examples in Plate I (a, b)). The first clearly differentiated outer lucent cells, seen by electron microscopy in stage 47, show an extensive finely-granular lucent area around an irregular- shaped elongate nucleus. A dense peripheral region (3 pm thick distally) contains tonofilament bundles, mitochondria, some RER, mucous vesicles at the surface and membrane-bounded vesicles of varied size (Plate IIb). An occasional larger, round, densely granulated, membrane- bounded body is seen in outer cells of different stages (Plates IIa, IIIa). From the periphery of the lucent area, a Golgi complex, or complexes, may extend into this region (Plates IIa, IIIa, IVb). The lucent cells may also show the first signs of vacuolation (vide infra). Lucent cells are 25 pm long x 10 pm wide (nucleus up to 20 pm x 9 pm). They frequently extend right across the epidermis (as do comparable cells of the previous stage), leaving the basal cell reduced to a narrow extension alongside the basal margin.

Dense basal cells include groups of up to 4-5 elongate SER, extending parallel to the proximal margin or at random to it, in the filamentous matrix (Plate IIIb). A Golgi complex is often found distal to the nucleus (Plate IIIa).

In stages 48 and 50, there are large goblet cells, about 2-3/100 cells of tail epidermis. Plate IVc illustrates their extensive array of closely associated and irregularly-shaped, membrane-bounded mucous droplets, of lucent, finely-granular substance, up to 3 pm or more in dimensions. A well developed Golgi complex is situated proximo-laterally to the nucleus with terminal smooth vesicles, and there are occasional mitochondria, tonofilaments and RER (Plate TVd). Goblet cells are overlain by adjacent epithelial cells, leaving a short free external surface 2.5 pm long, permitting discharge of their products to the exterior.

The tail epidermis in stage 50 contains lucent cells similar to those of the previous stage. Sometimes, an extensive Golgi complex may lie close to the nucleus where the lucent area is reduced (Plate Va). In this stage, oval-shaped Merkel cells occur, situated between the outer and basal cells. They are 7.5 pm long x 5.6 pm wide (nucleus 7 pm x 5 pm) and joined to neighbouring cells by desmosomes. Around the nucleus a section of the narrow peripheral region of cytoplasm contains up to 30 or so membrane-bounded granules, within the 90-120 nm diameter range, mainly located at the proximal side of the cell. The cytoplasm also contains mitochondria, tonofilaments and a Golgi complex. Merkel cell processes were not found in the limited range of material investigated.

In addition to other naked neurites between the epithelial cells (Plate Vc), on either side against a Merkel cell proximal surface were neurites; in one case there is synaptic contact with it (Plate Vb).

PLATE 111. (a) SEM of lateral region of an outer lucent cell of a stage 47 Neoceratodus larva. At the periphery of the lucent area (la) a Golgi complex (g) is situated near smooth-surfaced vesicles (sv), where the first signs of vacuolation (va) are recognizable. Note the membrane-bound dense body (db) (see also Plate IIa), and the Golgi complex (g) overlying the nucleus (n) of the basal cell (bc). Scale I pm. (b) SEM of basal epidermal cell (bc) of a stage 47 Neocerafodus larva, with groups of elongate smooth endoplasmic reticulum (ser) extending parallel to the proximal margin. There are no figures of Eberth or hemidesmosomes, as in all stages examined. Scale bar 1 pm. (c) SEM of pinocytotic vesicles (pv) at the proximal margin of a basal cell (bc) of a stage 48 Neoceratodus larva. Scale bar 0.5 pm.

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Other cells similar to Merkel cells in appearance, size, location and rarity, and likewise having desmosomes, occur in the epidermis. A narrowish peripheral cytoplasm contains membrane- bounded bodies, some of them larger than those in the Merkel ceils. Whether this is a Merkel cell precursor, with undifferentiated diagnostic components, cannot be decided.

A macrophage-type cell also occurs in the epidermis, without desmosomes, and containing a variety of membrane-bound inclusions of different sizes, some up to 0.3 pm in diameter.

The dermis

Below the adepidermal membrane a poorly developed basement lamella, of collagen fibrils, is barely recognizable in the tail at stages 40 and 42 (Plate Id), and even in stage 50 merely a layer of 5-6 collagen fibrils forms the lamella. In addition to striated muscle and capillaries, non- myelinated nerves extend along the tail and send off branches into the epidermis.

Melanophores are seen in stage 40; they still have lipid droplets in them at stage 50. At stage 44, melanophores contain an occasional membrane-bound melanosome, about 0.8 pm in diameter, but there are mostly premelanosomes even in stage 50, of up to 50 rod-like structures (70 nm in diameter) in various orientations but usually in parallel, within a membrane-bound vesicle. Melanocytes were not found in the epidermis in any stage examined.

Mesenchymal fibroblasts, with a high content of RER, are also present below the epidermis (Plate Vd).

Discussion

Though the tail epidermal cells of the young Neoceratodus larva are replete with lipid and yolk at stage 40, in a tail barely 2 mm long, indeed lipid is still found in epidermal cells in stage 50 3 months later, there is substantial cellular differentiation in the earliest (prehatched) stage examined. In particular, there is a high content of tonofilaments. Cilia were not seen in tail epidermis at any stage, in contrast to the ciliation of body and gill skin, described by Whiting & Bone (1980) in a comparable larval series of Neoceratodus. There is a ciliary current along the body from head to tail, from Semon stage 41 for 6 weeks after hatching, which may serve for respiration or protection from parasites or debris.

PLATE IV. (a) SEM of outer lucent cell of a stage 48 Neoceratodus larva, with vacuolation (va) at the periphery of the lucent area (la). Note the dense filamentous (tf) distal cortex of the cell, and the proximal margin with a Golgi complex (g) against the large vacuole (va). Most of the cross-section of the epidermis comprises the lucent vacuolated cell, and the basal cell (bc) in this region is flattened. Nucleus (n). Scale bar 1 pm. (b) SEM of higher magnification of an outer margin of a lucent cell of a stage 48 Neocerutodus larva. At the periphery of the lucent area (la), a Golgi complex (g). associated with smooth- surfaced (sv) and coated vesicles (cv), lies alongside a large membrane-bound vacuole, which may have a developmental relationship with the Golgi complex. Scale bar 1 pm. (c) SEM of goblet cell (gc) in the epidermis of a stage 48 Neoceratodus larva. The cell is filled with mucousdroplets (or vesicles) (mv), and is overlain by adjacent epithelial cells leaving a short free external surface. Scale bar 1 pm. (d) SEM ofextensive Golgi complex (9) in the ventrolateral region ofthe goblet cell (gc) of Plate 1Vc. Tonofilaments (tf) and granular endoplasmic reticulum (rer) are recognized at the periphery. Scale bar 0.5 pm.

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Lucent cells

The first fully developed lucent cells are free from vacuolation. This feature is only recognized in later stages 48 and 50 and originates alongside well fixed organelles in the cell; it rarely if ever occurs in basal cells. It would seem likely, therefore, that at least some vacuolation is a normal feature of lucent cells.

Lucent cells may well be exclusive to the larval stage of Neoceratodus, for they have not been reported in the adults (Imaki & Chavin, 1984: Whitear, 1986). However, swollen electron-lucent mid-epidermal cells in adult Periophthalmus kohlreuteri contain few organelles and have a peripheral region of tonofilaments (Whitear, 1986), and Rauther (1940) described by light microscopy (and illustrated) lucent cells of Periophthalmus as vesiculose Zellen, a feature similar to that found in Neoceratodus. Likewise, large electron-lucent epidermal sacciform cells occur in various species of teleosts. They have a sparse finely-granular and flocculent cytoplasm and most of them open to the exterior (Mittal, Whitear & Bullock, 198 1). In Periophthalmus lucent cells, the lucent area lies between the perinuclear cytoplasm and the cortical tonofilament region (Whitear, pers. comm.). In those of Neoceratodus, the lucent region lies within the perinuclear cytoplasm. Xenopus clear cells (vide infra) are more like the lucent cells of Neoceratodus in this respect.

Lucent cells of Neoceratodus larvae differ in fine structure from amphibian larval epidermal Leydig cells, goblet cells, the Riesenzellen of Bufo and the large clear cells of Xenopus (see Fox, 1988). Indeed, Leydig cells and Xenopus clear cells are intermediate in location and have no free external surface. Leydig cells are probably glandular; the clear cells of Xenopus are considered to be supporting cells in a delicate epidermis (Frohlich, Aurin & Kemnitz, 1977; Fox, 1988). Lucent cells of Neoceratodus and those of the amphibian larvae are similar in their electron-lucency, though this resemblance may well be superficial. There is no knowledge of the histochemistry of the Neoceratodus lucent cells, so that any comparison with the limited information on the amphibian cells is not possible.

Basal cells

Basal epidermal skein cells, comparable to basal cells with figures of Eberth in amphibian larvae, occur in Lampetra@uviatilis and L. planeri (Lane & Whitear, 1980), the leptocephalus larva of Anguilla rostrata (Leonard & Summers, 1976) and the salmonid embryo Oncorhynchus kisutch (Hawkes, 1974). Filamentous basal cells of the Neoceratodus larval tail lack figures and hemidesmosomes (see Fox & Whitear, 1986) in any stage examined. Whether figures develop in body and tail basal cells of older Neoceratodus larvae is not known. They have not been reported in

PLATE V. (a) SEM of lateral region of a lucent cell (Ic) of a stage 50 Neoceratoduslarva. There is little cellular vacuolation in this region, where the lucent area (la) is reduced and the extensive Golgi complex (g) lies close to the nucleus (n). Scale bar I pm. (b) SEM of region of a Merkel cell (mc) over a basal cell (bc), with neurites (ne) in synaptic association (sy) at the proximal margin, in a stage 50 Neocerafodus larva. Note the narrow peripheral cytoplasm containing characteristic membrane-bound granules (mg) and mitochondria (m). The nucleus (n) fills most of the cell. am, adepidermal membrane. Scale bar 0.05 pm. (c) SEM of neurites (ne) in the epidermis between outer and inner narrow basal cells (bc), of a stage 50 Neoceratodus larva. Scale bar 1 pm. (d) SEM of unmyelinated nerve bundles (ne), a melanophore (mi) with premelanophores (pm) and muscle tissue (mt) below a basal epidermal cell (bc) and poorly developed basement lamella collagen (bl). Further below there is a mesenchymal fibroblast (fb), with a high content of granular endoplasmic reticulum (rer). Scale bar 1 pm.

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the epidermis of adults of Lepidosiren paradoxa, Protopterus sp. and Neoceratodus forsteri (Kitzan & Sweeny, 1968; Imaki & Chavin, 1984; Whitear, 1986).

Goblet cells

Goblet cells appear in the tail epidermis slightly earlier than Merkel cells, and like them are rare in early stages of Neoceratodus. In appearance, larval goblet cells resemble the larger of the two types of mucous cells in the adult Pseudoohlennius cottoides (Sato, 1979), those of adult Oncorhynchus kisutch (Hawkes, 1974), and the type B goblet cells of dorso-caudal fin skin of adult Neoceratodus (Imaki & Chavin, 1984). Goblet cells of adult fishes are arranged in a single row between other epithelial cells, like some teleosts though not all, for some are arranged in several layers (Whitear, pers. comm.), to open to the exterior (Imaki & Chavin, 1984). In contrast? goblet cells of the larval Neoceratodus are relatively scarce, like those of the larval amphibians, and they are not arranged in close proximity side-by-side along the epidermis.

Goblet cells (Schleimzellen of German authors, see Whitear, 1986) are substantially larger in the adult Neoceratodus, 100 pmx25 pm (Pfeiffer, 1968) than those of the larva, and far more numerous, about 4000/sq mm of skin surface. In adult Lepidosiren paradoxa they are 50.5 pm x 38.5 pm in dimension, slightly smaller than in the adult Neoceratodus (Singh & Singh, 1979). In the salmon, goblet cells are claimed to originate from basal cells and then move towards the surface (Hawkes, 1974).

There are three types of goblet cells in Protopterus annectens, distinguished in terms of PAS staining and morphology (Kitzan & Sweeny, 1968): those of Lepidosiren paradoxa are PAS- positive and contain acid mucopolysaccharides (Singh & Singh, 1979). The cells are smaller in Protopterus after aestivation (Smith & Coates, 1937).

Protein is synthesized in the RER of the goblet cell and thence transported to the Golgi saccules, where it is linked with carbohydrate (Freeman, 1962, 1966; Henrikson & Matoltsy, 1968).

Merkel cells

Among dipnoans, large Merkel cells are present in the epidermis of adult Protopterus (Fox et al., 1980). They appear in tail epidermis of Neoceratodus 10-12 weeks after hatching, early in ontogeny, as in amphibians, and likewise as round-oval interstitial cells, apparently without processes. Membrane-bound granules (90-1 20 nm diameter) are their first reliable diagnostic characters (Fox & Whitear, 1978; Fox, 1986a, b). In larvae and adults of anurans, Merkel cells range in size from 7 pm to 9 pm across; those of urodeles are about twice as large, and in Ichthyuphis spp. among the Caecilia they are intermediate in size, about 12 pm x 9 pm (Fox & Whitear, 1978; Fox, 1983,1986a, 1987). Merkel cells of Neoceratodus larvae fit into the anuran size range, in contrast to those of adult Protoptcrus, which are more like those of urodeles (Fox et al., 1980).

In Neoceratodus, the first recognizable Merkel cell showed synaptic association with neurites. It is likely, therefore, that Merkel cell-nerve complexes are beginning to function early in larval development .

TAIL SKIN O F T H E N E O C E R A T O D U S LARVA 225

Melanophores

Adult Neoceratodus possesses melanocytes and melanophores in the epidermis and dermis, respectively, similar to those of other vertebrates. Imaki & Chavin (1 975a) showed that adult melanocytes have melanosomes and also premelanosomes in their perikarion and dendrites. Melanophores of adult Lepidosiren paradoxa, Protopterus sp. and Neoceratodus are generally similar and the premelanosomes in the dermal melanophores are best illustrated in Protopterus (Imaki & Chavin, 197%).

Eggs and cleavage blastomeres of Neoceratodus are pigmented. Later, small numbers of melanophores appear dorsally in the body and around the lens, at about stage 35 (larva 10 mm long, 37 somites). Melanophores are widely dispersed over the body by stage 43, when erythrophores first appear (Kemp, 1982).

Melanosomes occur in tail epidermal cells and melanophores below them in Neoceratodus of the present work. Nevertheless, even at stage 50, the melanophores are immature and mainly contain premelanosomes.

It is apparent, therefore that almost from its beginning there are muscles and nerves in the Neoceratodus tail. Indeed, there are nerves in tail skin at stage 40 and young Merkel cells with nerves in synapsis at stage 50. From an early age Neoceratodus larvae are equipped to feed (Kemp, 198 l), are camouflaged and presumably can respond to tactile stimuli (and to light), and can move to safer regions of their aquatic habitat, in order to escape predation or other harmful environmental features.

Dr Anne Kemp of the Queensland Museum, Brisbane, Australia, provided the fixed larval material, used for light and electron microscopy in the present work. Dr G.C. Bangma and the Hubrecht Laboratory loaned me relevant slide material of larval Neoceratodus from the J.P. Hill Collection. Their generosity is warmly acknowledged. (The author’s research collection, including the material used in this paper, is now housed in the Hubrecht Laboratory at Utrecht, Holland and will be available to workers in the field of developmental biology.) Thanks for technical assistance are due to Sylvia Carter, Elaine Clarke, Pat Fergusen and Roy Mahoney. Dr Mary Whitear gave valued advice during the preparation of the manuscript.

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