Toxicon 41 (2003) 29–39 www.elsevier.com/locate/toxicon
Ultrastructural patterns of secretory activity in poison cutaneous glands of larval and juvenile Dendrobates auratus (Amphibia, Anura) R. Angela, G. Delfinob,*, G.J. Parraa b
a Instituto Colombiano de Medicina Tropical, KRA 43A n8 52S99, Sabaneta-Antioquia, Colombia Dipartimento di Biologia Animale e Genetica dell’, Universita` di Firenze, via Romana n8 17, I-50125, Firenze, Italy
Received 7 March 2002; accepted 16 July 2002
Abstract A transmission electron-microscope study has been performed on larval and juvenile skin of the Central American arrowfrog Dendrobates auratus to investigate early secretory processes and maturational changes in the serous (poison) glands. Poison biosynthesis involves the endoplasmic reticulum (both smooth and rough types), as well as Golgi stacks which release early serous product as secretory vesicles (or pre-granules). These vesicles contain fine-grained material, along with single electron-opaque bodies, spheroidal in shape, that accompany the grained product throughout its post-Gogian, maturational change. The first steps of this process involve condensation and lead to the formation of secretory granules with a glomerularlike substructure, resulting from a thick, random aggregation of rods (secretory granule subunits). Advanced maturational activity causes the loss of peculiar granule substructure: the dense bodies split into fragments, whereas the thick glomerular arrangement becomes looser, until the secretory product changes into a dispersed material. This ultrastructural study revealed biosynthesis and maturation processes in close sequence, suggesting the poison of D. auratus contains proteins and/or peptides as well as lipophilic compounds. Molecules of both these classes are known to perform several roles relevant to survival strategies in extant anurans. Furthermore, the ephemeral granules with a glomerularlike substructure detected in tadpoles and froglets exhibit the complex patterns of mature poisons in adult specimens of other anurans: Hylidae and related families. This agrees with current trends in the taxonomy of these advanced frogs and underlines the pertinence of an ontogenetic approach in investigating anuran phylogenesis. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Dendrobates auratus; Poison biosynthesis; Ultrastructure
1. Introduction Although serous (or poison) cutaneous glands in adult anurans produce secretions that are heterogeneous in composition, the transmission electron microscope (TEM) reveals that they exhibit monotonous secretory features. This consistent trait largely depends on the slow activity rate of the secretory units in adult glands, which are syncytial in * Corresponding author. Tel.: þ39-55-2288-295; fax: þ 39-552288-299. E-mail address:
[email protected] (G. Delfino).
structure (secretory syncytia), and involved in serous storage rather than the biosynthesis. The only functional performance during the storage phase is the maturational process which affects secretory granules through complex relationships with the syncytial cytoplasm holding them (Delfino et al., 2001b); however, maturation only marginally involves the biosynthesis machinery: rough endoplasmic reticulum (rer), smooth endoplasmic reticulum (ser) and Golgi stacks. Two main procedures have been developed in order to depict the role of biosynthesis organelles in anuran serous glands: (a) observation of poison neosynthesis during gland rehabilitation after secretory discharge, and (b)
0041-0101/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 0 2 ) 0 0 2 0 6 - 4
30
R. Angel et al. / Toxicon 41 (2003) 29–39
investigation into secretory differentiation and activity during gland development. A third source of information on serous biosynthesis comes from species in which glands may develop steadily in adult specimens (Flucher et al., 1986 on Xenopus laevis ). Gland discharge may be obtained by manual squeezing or compression exerted by the muscle layer (myoepithelium) ensheathing the secretory unit, under electric or pharmacological stimulations (Faraggiana, 1938a, 1939; Dockray and Hopkins, 1975; Delfino, 1980; Flucher et al., 1986; Toledo et al., 1992; Delfino et al., 2002; Nosi et al., 2002). In all cases, serous release results from mechanical activity which damages the secretory compartment, as seen by the presence of lysosomes and swollen mitochondria (Toledo et al., 1992). Gland rehabilitation involves differentiation of adenoblasts from the neck region of the gland, plus renewed activity in residual parts of the syncytium. This post-discharge activity is marked by unusual traits: accumulation of the newly synthesised product in the hyaloplasm, followed by segregation achieved by patches of membranes that fuse together (Neuwirth et al., 1979); occurrence of a set of peculiar organelles (multicored bodies, Flucher et al., 1986) and production of granules not seen in resting glands (Toledo et al., 1992). Yet another element in this functional maze is the permanence of the former product in the syncytium, which hinders recognition of a coherent secretory path (Delfino, 1991). Larval glands are the most suitable organs for investigating the dynamic aspects of poison secretion since: (a) they lack residual serous material from previous secretory cycles; and (b) the timing of their activity may be analysed along a sequence of steps of secretory differentiation and activity, consistent in all anuran species so far studied (Howes, 1947; Spannhof et al., 1954; Gillois-Chevalier, 1960; Bovbjerg, 1963; Vanable, 1964; Le Quang Trong, 1967, 1973; Delfino, 1977; Delfino and Melis, 1998; Delfino et al., 1988, 1994, 1995, 1998, 2001a; Seki et al., 1989). We recently performed several investigations into the serous glands of subtropical frogs from South America that produce granules provided with repeating substructures. Ultrastructural studies on both adult (Delfino et al., 1999; Terreni et al., 2002) and larval specimens (Delfino et al., 2001a) allowed us to depict maturational processes leading to these complex patterns. This study extends our investigations to specimens (larval as well juvenile) of Dendrobates auratus, representative of the family Dendrobatidae. We tracked the maturational pathway of early serous glands (that in adult frogs produce serous deposits containing dispersed material, Neuwirth et al., 1979), to check whether any repeating patterns occur in poison during ontogenesis. TEM investigation on serous biosynthesis and maturation could provide additional information on the composition of products which perform a wide-ranged adaptive role in survival strategies (Daly et al., 1987). Furthermore, ultrastructural features of post-Golgian, maturational processes
in larval glands can be compared in related taxa along recapitulation criteria, thus assisting phytogenetic analysis (Delfino et al., 2001a, 2002). Although the morphology of granules varies among adult anurans belonging to different groups, several larval traits of poison production are highly conservative, indicating a level of morphological canalisation during ontogenesis, as suggested by Maglia et al. (2001) for skeletal development. Accordingly, this study discusses the patterns of serous maturation in D. auratus within a phylogenetic framewok to test the viability of ultrastructural investigation into cutaneous poisons as an analysis informative on the evolutionary history of anurans.
2. Materials and methods 2.1. Animals and larval stages D. auratus belongs to the large family Dendrobatidae, confined to Southern Central America and South America. Dendrobatid frogs have gained celebrity on account of the toxic skin secretion, used by Indian hunters for poisoning blowgun darts (Myers and Daly, 1983). Tadpoles were reared in the Laboratorio de Zoologı`a, Instituto Colombiano de Medicina, Medelin Colombia—where pre-fixation was also performed—until they reached ontogenetic ranges relevant to gland organogenesis, namely 38 – 40, 42 – 43 and 44 – 45, according to Gosner (1960). In these stages several generations of glands and advance gland Anlagen coexist in the same specimen, allowing observation of a wide range of secretory patterns (Delfino et al., 2001a). 2.2. Tissue processing Skin strips (4 – 9 mm2 in surface area) were removed from the dorsal surface of the animals (both trunk and hind limb areas) previously sacrificed with 0.2% chlorobutanol. They were treated (4 h, 4 8C) with glutaraldehyde in 0.1 M, pH 7.0 phosphate buffer (Sabatini et al., 1963). After prefixation the tissue fragments were washed with phosphate buffer and despatched in 2 – 4 ml of this solution (with the addition of a drop of glutaraldehyde) to the Dipartimento di Biologia Animale e Genetica (Universita` di Firenze, Italy), to be processed according to previous investigations (Delfino et al., 1998, 2001a). The skin specimens were rinsed once more in 0.1 M, pH 7.0 cacodylate buffer, reduced in size and postfixed in 1% OsO4 (90 min), again using the cacodylate buffer. After rinsing in the buffer, the skin samples were dehydrated in graded ethanol, soaked in propyleneoxide and infiltrated with Eponw 812 (purchased from Fluka), to obtain flat, prismatic blocks. These were cut with a NOVA LKB ultramicrotome into semithin (1– 2 mm) and ultrathin (yellow –white interference colour) sections. Semithin sections were stained with buffered tolouidine blue for preliminary LM observations. Ultrathin sections were
R. Angel et al. / Toxicon 41 (2003) 29–39
collected on 300 mesh, uncoated copper grids, then electron dense stained in sequence with saturated, hydroalcoholic uranyl acetate and 2 mg/ml alkaline lead citrate solutions. Finally, these samples were observed (80 kV) using a Siemens 101 electron microscope. 2.3. Skin specimen sampling through semithin sections As well as on the back skin of the trunk, investigations were performed on the skin of the leg (or zeugopodium ). In anurans, remarkable collections of serous glands usually occur in this hind limb region and eventually only differ from trunk glands in size (Delfino et al., 1982). Therefore, observation of semithin sections from leg skin in the appropriate larval stages provides the opportunity of localising several glands in various developmental and functional phases even in small section areas.
31
(adenomere) displays an obvious centripetal polarisation, with peripheral nuclei (ovoidal to kidney-shaped) and inner poison aggregates (Fig. 1(B) and (D)). The gland neck represents the stem compartment of the organ and consists of undifferentiated cells (adenoblasts as well as myoblasts), that are stratified in a few layers at the dermal –epidermal boundary. Both the terms ‘neck’ and ‘intercalary tract’ describe this gland region, on account of its position, between the large secretory unit and the elongate duct. In tangential semithin sections it is not possible to see the duct along its axis, but in transverse sections its narrow, threeradiated lumen is obvious as it crosses the cell layers of the epidermis (Fig. 1(A) and (B)). In describing ultrastructural patterns of secretory activity in the Section 3.2, we will respect the morpho-functional polarisation of serous glands. 3.2. Transmission electron microscope
3. Results 3.1. Light microscope Tangential sections of the skin provide relatively wide surface areas for investigation and reveal several cutaneous glands (Fig. 1(A)). As an effect of tangential section, the cutaneous structures are arranged concentrically, with a peripheral epidermis, intermediate loose dermis and central dense dermis. Cutaneous glands occupy the intermediate layer, profiting from its rich blood supply; the two fundamental types of anuran cutaneous glands can easily be recognised namely serous and mucous. Mucous glands exhibit epithelial secretory units with obvious interstices between component cells (Fig. 1(B) and (C)), whereas serous glands have syncytial secretory compartments (Fig. 1(B) and (D)). Furthermore, mucous glands always possess obvious lumina—which, however, may vary considerably in width (Fig. 1(A)), depending on the height (and related functional phase) of the mucus producing cells (mucocytes, Fig. 1(B) and (C)). Active mucocytes exhibit expanded apical portions, containing densely stained, secretory product (Fig. 1(C)). The serous secretory units, on the other hand, always lack lumina, and hold their specific products in syncytial cytoplasm bulks (Fig. 1(B)). Typical serous gland regions can be recognised by their morphology, structure and position (Fig. 1(B)). The contractile sheath (myoepithelium) consists of spindle-like smooth myocytes (myoepithelial cells), provided with elongate nuclei. These muscle cells have a remarkably dense cytoplasm, due to the myofilaments of their contractile apparatus, and can therefore be recognised from the paler background of the secretory syncytium (Fig. 1(D)). Furthermore, enlarged interstices emphasise the boundary between secretory and contractile compartments (Fig. 1(D)). The syncytial cytoplasm bulk of the secretory unit
Ultrastructural observations at the boundary between the intercalated tract and syncytium reveal a sharp contrast between the cytoplasm backgrounds of the neck adenoblasts and the secretory syncytium, which are opaque and transparent, respectively (Fig. 2(A)). The electron density of the stem cells is due to the remarkable content of free ribosomes, whilst the syncytial cytoplasm contains complements of the endoplasmic reticulum, both smooth and rough in nature (Fig. 2(A) –(C)), as well as rod-shaped mitochondria with a dense matrix (Fig. 2(B) – (D)). Therefore, it appears that adenoblasts are capable of assembling free intracytoplasmic proteins, whereas the secretory unit synthesises molecules in compartments bounded by membranes. Smooth endoplasmic reticulum consists of randomly oriented tubules, consistent in shape and diameter (approximately 70 – 80 nm). Cisterns of the rough endoplasmic reticulum are usually flat (Fig. 2(C)) and parallel in arrangement (Fig. 3(B)), although in later phases of gland activity rer profiles may become wider and irregular in shape (Fig. 2(D)). According to the centripetal functional polarisation, stacks of the Golgi apparatus occur toward the centre (i.e. just distally from the endoplasmic reticulum) and exhibit patterns of intense secretory activity (Fig. 2(E)). Usual features appear on the proximal side, where transfer vesicles, devoid of ribosomes, merge together to form cis saccules. Peculiar patterns are, indeed, obvious on the trans face of the organelles, where secretory vesicles can be detected holding the early poison with a characteristically dimorphic aspect. Early poison consists of a finely grained material, apart from single electrondense bodies (speheroidal in shape and 300– 500 nm in diameter, Fig. 2(E)). This secretory dimorphism represents a real marker which serves to identify deposits of early poison (Fig. 2(F)) and follow them throughout the maturation processes (Fig. 3(A) – (C)). Preliminary steps in maturational change involve condensation of the finely grained product (Fig. 3(A)), a gradual process that leads to thickening of this material,
32
R. Angel et al. / Toxicon 41 (2003) 29–39
Fig. 1. Leg skin, 42– 43. Structural aspects of larval cutaneous glands; dd ¼ dense dermis; fb ¼ fibroblast; im ¼ interstice between mucocytes; isc ¼ interstice between myoepithelium and serous syncytium; it ¼ intercalary tract (gland neck); ld ¼ loose dermis; m ¼ mucous gland with low mucocyte layer; mI ¼ mucous gland with high mucocyte layer; mc ¼ mucocyte; mec ¼ myoepithelial cell; mph ¼ sub-epidermal melanophore; s ¼ serous gland; sy ¼ serous secretory syncytium; v ¼ blood vessel. (A) Tangential section showing cutaneous gland apparatus and its relationships with skin structures. Cutaneous glands are inserted in the loose (spongy) dermis layer, and can be identified as serous or mucous. Arrows point to intraepidermal ducts sectioned on the transverse plane; rectangular areas enlarged in (B), (C) and (D). (B) Details of (A) which allows close comparison between mucous and serous glands. The mucous gland possesses an obvious lumen, although reduced in width by tall, pyramidal mucocytes with large interstices between; the myoepithelial sheath is usually thin. The serous gland has a syncytial, secretory unit lacking any lumen, encircled by obvious myoepithelial cells and separated from the overlying epidermis by stratified cells of the intercalated tract. Notice duct (arrow) within epidermis which seems to be multi-layered as an effect of the tangential section. Also notice interstices between mucocytes. (C) Details of (A) showing mucous gland with high, secreting mucocytes; these cells are characterised by expanded apical cytoplasms, containing densely stained product (arrowheads). Notice interstices between secretory cells. (D) Further details of (A), showing serous secretory unit at higher magnification. The syncytial cytoplasm contains central serous product and peripheral nuclei, according to the centripetal polarisation of these glands. Notice that serous aggregates are weakly stained in comparison with the mucous product in (C). Also notice enlarged interstices between contractile and secretory compartments.
although the electron-opacity of the single, spheroidal bodies still prevails (Fig. 3(B) and (C)). Actually, condensation of the grained material is not homogeneous and results in a peculiar substructural pattern with a
somewhat repeating arrangement (Fig. 3(C)). Poison aggregates tend to acquire a roundish shape with several deep indentations on their profiles (Fig. 3(B) and (C)). Higher power magnification reveals the peculiar substructure
R. Angel et al. / Toxicon 41 (2003) 29–39
33
Fig. 2. Ultrastructural aspects of early poison biosynthesis i ¼ interstice between intercalary cell and secretory syncytium; rer ¼ rough endoplasmic reticulum; ser ¼ smooth endoplasmic reticulum. (A) Leg skin, 38–40. Notice sharp contrast between intercalary tract adenoblast (left), rich in ribosomes, and secretory syncytium, where endoplasmic reticulum complements can be seen against the pale hyaloplasmic background; compare with Fig. 4(A). (B) Leg skin, 38 –40. Smooth endoplasmic reticulum consists of randomly oriented tubules. (C) Leg skin, 38–40 and (D) trunk skin, 44–45. Early (C) and advanced (D) features of the rough endoplamic reticulum cisterns. (E) Leg skin, 38–40. Active Golgi stack: notice transfer vesicles on the cis face (arrowhead) and early product in secretory ‘vacuoles’ on the trans face (arrow). Just distally from this area, Golgian activity leads to the formation of peculiar secretory vesicles, with a single, dense body in a finely grained background. Large arrow points to a mature secretory deposit. (F) Trunk skin, 44–45. These dimorphic secretory aggregates are still produced in glands of juvenile specimens which already contain remarkable amounts of product with advanced features (compare with Fig. 3(A)–(C)).
of these poison deposits. They resemble intricate glomerules, although they are actually knots of short, discrete rods (serous subunits), provided with a dense axial rod (Fig. 3(D)). Although serous subunits are identical both in feature
and transverse diameter, they are arranged at random, and there is no close, periodic pattern. These glomerular-like poison aggregates, appear as real secretory granules on account of their limiting membrane and relatively compact
34
R. Angel et al. / Toxicon 41 (2003) 29–39
Fig. 3. Suggested sequence of poison maturation. (A) Leg skin, 38–40. Notice thickening of grained material in early secretory aggregate. (B) Trunk skin, 38–40. Further stage of condensation in an intermediate secretory granule at the periphery of the syncytium; notice flattened, parallel rer cisterns (left) and secretory aggregates with transparent compartment (right). The condensing granule shows a fine substructure. (C) Leg skin, 38–40. This substructure consists of a recurrent array, with pale subunits on an opaque background; notice mature secretory deposit containing discret particles (arrows). (D) Trunk skin, 38–40. Higher power magnification discloses the peculiar substructure in a intermediate, secretory granule, resembling a glomerular arrangement of short, rather transparent rods, ‘tubules’, which possess an electron-dense axial rod (arrows). This is possibly a peripheral section of the granule, not involving the dense body. (E) Leg skin, 42–43. In a later maturational step, the thick aggregation of subunits loosens, whilst the dense bodies split into fragments (arrows). (F) Trunk skin, 44–45. As a result of this change, serous aggregates tend to transform into secretory vesicles holding minute, discrete particles in a transparent background. (G) Trunk skin, 44– 45. Peculiar aspect of mature serous product, which resembles a loose network.
R. Angel et al. / Toxicon 41 (2003) 29–39
arrangement of their product. However, they are ephemeral as the secretory product undergoes obvious morphological thinning, according to the centripetal gradient of maturation typical in serous glands. This process involves both rod-like subunits and dense bodies: the former give rise to a structureless array of moderately electron-opaque, dispersed material, the latter apparently split away in smaller fragments (Fig. 3(E)). As a result, the secretory deposits transform into large secretory vesicles, holding minute particles in a pale compartment (Fig. 3(F)). Since neosynthesis and maturation coexist in larval glands, early and advanced secretory aggregates can be found closely contiguous at the periphery of the secretory syncytium (Figs 2(E) and (F) and 3(C)). As a rule, the minute particles inside the mature serous deposits appear as discrete structures, but they may sometimes join together, resulting in a rather irregular, discontinuous network (Fig. 3(G)) in a pale compartment. Glands in advanced tadpoles and toadlets resemble miniature adult glands: the centripetal functional polarisation is masked by large amounts of mature secretory material, which tends to occupy most of the secretory syncytium, including the peripheral cytoplasm (Fig. 4(A)), formerly rich in biosynthesis organelles. On the skin surface, local areas can be observed with external epidermal cells (keratinocytes) that invaginate and form a longitudinal channel oriented towards the dermis (Fig. 4(B)). This is the duct primordium, a large interstice which will later acquire a keratinised lining, continuous with the external horny layer of epidermis. The intraepidermal channel reaches the level corresponding to the epidermal – dermal boundary and meets the gland neck, with cells arranged in a few, stratified layers. In the absence of obvious ultrastructural differences between component cells of the duct and neck primordia, the two regions can be identified by the orientation of their nuclei (Fig. 4(C)). The nuclei of the duct keratinocytes are oriented with their major axis orthogonal to the epidermis – dermis interface. The nuclei of the neck cells are either parallel or oblique to this boundary, following the external surface of the secretory unit upper pole. The funnel-shaped intercalary tract has a proper lumen, which continues into the duct lumen on the side of epidermis; on the opposite, dermal side it meets the cytoplasm bulk of the secretory syncytium (Fig. 4(C)). In resting glands, a thin screen, formed by cytoplasmic processes from the inner, lower adenoblasts, separates the syncytium from the intercalary lumen. However, these cell laminae, resembling cytoplasmic flaps, are easily forced upwards when the syncytium herniates into the lumen (Fig. 4(C)), following contraction of the myoepithelial layer around the secretory unit. Transverse sections confirm the above pattern of syncytial portions displaced in the neck lumen and show cytoplasm profiles in this cavity (Fig. 4(D)). This contractile response, possibly triggered by animal handling before sacrifice, pertains to the repertory of chemical skin defence, which in
35
adult specimens leads to discharge of the poison deposits held in the syncytium.
4. Discussion These findings demonstrate that an ontogenetic approach is suitable for investigating the ultrastructural aspects of cutaneous poison production in anurans, since it furnishes a dynamic series of secretory patterns, including biosynthetic as well maturational phases. In addition, recapitulation of ancestral steps of serous maturation during ontogenesis proposes appropriate lines for a phylogenetic comparison between taxa that exhibit similar ultrastructural patterns in larval glands. Developmental information is highly useful in tracking the evolutionary histories of more comprehensive taxonomic groups of anurans (Maglia et al., 2001). Serous biosynthesis processes in larval glands of D. auratus. follow a pathway consistent with protein and peptide production in membrane-bounded compartments of the rough endoplasmic reticulum. Furthermore, nonpolar (lipophilic) molecules appear to be produced in the smooth endoplasmic reticulum. Apart from single, dense bodies, most of the secretory product in the Golgi stacks is fine grained in consistency. Therefore, the prevailing activity in these organelles is to segregate the products manufactured in the endoplasmic reticulum from the hyaloplasm by membrane neosynthesis, rather than condense them. Although active polypeptides have usually been detected in frog poisons, carnosin (a dipeptide) is the representative molecule of this class of metabolites in Dendrobates serous glands, whereas decahydriquinolines, histrionicotoxins, indolizidines and pumiliotoxins are consistent lipohilic compounds, assigned to the major classes of dendrobatid alkaloids (Daly et al., 1987). After metamorphosis, biosynthesis organelles in anuran serous glands tend to be segregated at the very periphery of the secretory syncytium and seem to be inactive: rough endoplasmic cisterns are scanty and short (Delfino et al., 1999), usually slender in shape (Delfino et al., 1993), whereas the smooth endoplasmic tubules appear in paracrystalline array (Neuwirth et al., 1979); the Golgi stacks consist of flattened sacculi, holding an electron-transparent compartment (Delfino et al., 1993). The shift of secretory organelles from active to inactive conditions appears to be related to the onset of the storage phase, when transport processes between membrane-bounded secretory aggregates and the syncytial cytoplasm prevail (Delfino et al., 2001b). During serous storage in Rana esculenta cutaneous glands, both morphology and chemical composition of secretory granules undergo obvious rearrangement, seemingly consistent with a seasonal cycle (Barni et al., 1987). The occurrence of remarkable amounts of serous product in the glands of tadpoles and froglets of D. auratus reared in captivity, and thus in a predator-free environment, suggests
36
R. Angel et al. / Toxicon 41 (2003) 29–39
Fig. 4. Ultrastructural aspects of serous glands in advancd tadpoles and juveniles; dc ¼ duct cells; ic ¼ intercalary tract cells. (A) Trunk skin, 44–45. Boundary region between intercalary tract and secretory unit: mature secretory deposits occupy the whole syncytium, reaching the peripheral cytoplasm; compare with Fig. 2(A). (B) Trunk skin, 44 –45. This wide interstice opening onto the body surface is the upper third of the gland duct primordium. In adult specimens, the inner cell lining of the duct (arrow) is continuous with the horny surface layer. (C) Trunk skin, 44 –45. Transitional region between duct and intercalated tract: compression by myoepithelial cells—resulting from a contractile response to a local noxa during animal handling—pushes the syncytial cytoplasm containing the secretory aggregates into the duct lumen (bowed arrows). Notice laminar cytoplasm processes adhering to the neck wall (arrowheads). (D) Leg skin, 42–43. Transversal section of the transition zone between duct and neck: notice portions of the syncytial cytoplasm in luminal cavity (arrows).
that their secretory activity is a constitutive, functional trait (i.e. not induced by external causes). However, Daly et al. (1987) report the apparent lack of active alkaloids in captive-raised anurans, whereas their wild-caught parents retain skin toxicity for several years. These results suggest that regulatory (inductive) environmental factors may play a role in the initiation of the biosynthesis of some poison fractions during ontogeny. In addition, many of the
dendrobatid alkaloids (or alkaloid precursors) might derive from dietary sources (Daly et al., 1994). The patterns of secretory release we have described in serous glands of larval D. auratus foreshadow the peculiar emission mechanism in adult specimens (Delfino et al., 2002; Nosi et al., 2002), referred to as holocrine discharge since it involves secretory product alongside masses of syncytial cytoplasm and requires regenerative processes
R. Angel et al. / Toxicon 41 (2003) 29–39
(Faraggiana 1938a, 1939; Delfino, 1980). On the basis of her pioneering studies on anuran serous glands, Faraggiana (1938b) developed the concepts of both syncytial structure and holocrine secretion. Considering that the formation of a secretory syncytium involves plasma-membrane break-down, this multinuclear organisation could be regarded as a preliminary step in complete cell degeneration and transformation into a secretory product, according to conventional holocrine processes (such as production and release of sebaceous material in mammalians). Actually, ultrastructural studies on several anuran species in larval development revealed that the syncytial structure does not result from degenerative change; it is, on the contrary, a specialised arrangement, capable of co-ordinating maturational processes (Delfino et al., 1988). On the other hand, there is no ultrastructural evidence for the transformation of syncytial cytoplam into secretory product. Serous deposits in anuran gland are membrane-bounded—namely segregated from the cytoplasm—and can be collected in saline as discrete structures after massive release induced by norepinephrine stimulation of peripheral myocytes (Dockray and Hopkins, 1975, Delfino et al., 2002, Nosi et al., 2002). Under these dramatic conditions, active compounds usually contained in the hyaloplasm can also be collected (Bols et al., 1986) whereas granules have to be sonicated (Delfino et al., 1982), or freeze – thawed and sonicated, to breakdown their limiting membranes and process their poison components (Barberio et al., 1987; Mastromei et al., 1991; Balboni et al., 1992; Sanna et al., 1993). It appears therefore that serous gland activity in anurans cannot be adequately described as belonging to the usual holocrine patterns. Therefore the term ‘bulk discharge’ has been introduced to define the massive release due to muscle sheath contraction (Delfino et al., 1996). The high metabolic costs incurred in a diffuse exocrine apparatus that adopts bulk discharge (Delfino, 1991) may be selectively balanced by the effectiveness of a wide-ranging chemical defence. Anuran cutaneous poisons perform cytolytic activity against prokaryotic (Bachmayer et al., 1967; Csorda´s and Michl, 1969; Barteczko and Kuziemski, 1970; Croce et al., 1973; Michl, 1978; Barberio et al., 1987; Zasloff, 1987, 1988; Mastromei et al., 1991) and eukaryotic cell lines (Kiss and Michl, 1962; Bachmayer et al., 1967; Kaiser and Kramar, 1967; Mar and Michl, 1976; C ¸ evikbas¸, 1978; Michl, 1978; Balboni et al., 1992; Sanna et al., 1993). Some alkaloids of the cutaneous poisons cause nerve and muscle cell failure in higher vertebrates (Myers and Daly, 1983), whereas other component molecules act as repellents (Brodie et al., 1978; Garton and Mushinsky, 1979; Formanowicz and Brodie, 1982; Szelistowski, 1985). Finally, serous skin products include regulative molecules that control Naþ and water exchange (Flier, 1980), and are relevant to adaptation to a terrrestrial habitat (Barni et al., 1987). Although granular secretory cells in the epidermis of bony fishes perform some roles common to serous
37
cutaneous glands of amphibians (Quay, 1972), these organs pertain to a set of novel adaptations which came about during the transition from freshwater to subaerial environments (Toledo and Jared, 1995). However, the real phylogenetic significance of this exocrine apparatus, be it an ancestral or derived trait, is still uncertain (Daly et al., 1987). Whereas skin poison biosynthesis in anurans follows the usual pathway found in the exocrine glands of higher eumetazoans, the post-Golgian patterns are peculiar and include production of serous deposits with unique features. The ephemeral granules with repeating substructures occurring in serous glands of D. auratus tadpoles closely resemble those observed in Hylidae and Pseudidae (Terreni et al., 2002), Leptodactylidae (Delfino et al. 1999, 2001a, Terreni et al., 2001), and some dendrobatid tree-frogs (Neuwirth et al., 1979). It appears that rearrangement of post-Golgian structureless products during maturation, to form granules with repeating substructures, is a functional trait shared by related taxa of Neobatrachia (see the dendrogram of Ford and Cannatella, 1993), possibly inherited from a common ancestor. In later stages of maturation, these recurrent patterns may disappear, as the secretory product undergoes fluidisation or extreme condensation, depending on the species and/or gland anatomical localisation (Delfino et al., 1999; Terreni et al., 2002). These differences in ultimate granule features were possibly acquired along the specific evolutionary paths which led to the current position of these related lines in distinct, but not natural groups (Delfino et al., 2001a).
References Bachmayer, H., Michl, H., Roos, B., 1967. Chemistry of cytotoxic substances in amphibian toxins. In: Russel, F.E., Saunders, P.R. (Eds.), Animal toxins, Pergamon Press, Oxford, pp. 395–399. Balboni, F., Bernabei, P.A., Barberio, C., Sanna, A., Rossi Ferrini, P., Delfino, G., 1992. Cutaneous venom of Bombina variegata pachypus (Amphibia: Anura): effects on the growth of the human HL 60 cell line. Cell Biol. Int. Rep. 16, 329 –338. Barberio, C., Delfino, G., Mastromei, G., 1987. A low molecular weight protein with antimicrobial activity in the cutaneous venom of the yellow-bellied toad (Bombina variegata pachypus ). Toxicon 25, 899–909. Barni, S., Bernocchi, G., Bottiroli, G., 1987. Histochemistry and morphology of the secretory granules of skin venom glands of Rana esculenta during the active and hibernating period. Arch. Biol. 98, 391 –406. Barteczko, I., Kuziemski, H., 1970. Anti-bacterial properties of cutaneous secretions of the frog Rana esculenta (L.) and some compounds of amphibian skin secretions. Zool. Pol. 20, 189 –198. Bols, N.C., Roberson, M.M., Haywood-Reid, P.L., Cerra, R.F., Barondes, S.H., 1986. Secretion of a cytoplasmic lectin from Xenopus laevis skin. J. Cell Biol. 102, 492– 499. Bovbjerg, A.M., 1963. Development of the glands of the dermal plicae in Rana pipiens. J. Morph. 231, 231 –243.
38
R. Angel et al. / Toxicon 41 (2003) 29–39
Brodie, E.D. Jr., Formanowicz, D.R. Jr., Brodie, E.D., 1978. The development of noxiousness of Bufo ameriacanus tadpoles to aquatic insect predators. Herpetologica 34, 302–306. C ¸ evikbas¸, A., 1978. Antibacterial activity in the skin secretion of the frog Rana redibunda. Toxicon 16, 195– 197. Croce, G., Giglioli, N., Bolognani, L., 1973. Antimicrobial activity in the skin secretion of Bombina variegata pachypus. Toxicon 11, 99 –100. Csorda´s, A., Michl, H., 1969. Primary structure of two oligopeptides of the toxin of Bombina variegata L. Toxicon 7, 103–108. Daly, J.W., Myers, C.W., Whittaker, N., 1987. Further classification of skin alkaloids from Neotropical poison frogs (Dendrobatidae), with a general survey of toxic/noxious substances in the Amphibia. Toxicon 25, 1023–1095. Daly, J.W., Secunda, S.I., Garaffo, H.M., Spande, T.F., Wisnieski, A., Cover, J.F. Jr., 1994. An uptake system for dietary alkaloids in poison frogs (Dendrobatidae). Toxicon 32, 657 –663. Delfino, G., 1977. II differenziamento delle ghiandole granulose cutanee in larve di Bombina variegata pachypus (Bonaparte) (Anfibio: Anuro: Discoglosside). Ricerca al microscopio ottico e al microscopio elettronico. Arch. Ital. Anat. Embriol. 82, 337–363. Delfino, G., 1980. L’ attivita` rigeneratrice del tratto intercalare nelle ghiandole granulose cutanee dell’ululone Bombina variegata pachypus (Bonaparte) (Anfibio, Anuro, Discoglosside); studio sperimentale al microscopio elettronico. Arch. Ital. Anat. Embriol. 85, 283–310. Delfino, G., 1991. Ultrastructural aspects of venom secretion in anuran cutaneous glands. In: Tu, A.T. (Ed.), Handbook of Natural Toxins, Reptile Venoms and Toxins, vol. 5. Marcel Dekker, New York, pp. 777– 802. Delfino, G., Melis, G., 1998. Serous cutaneous glands in the natterjack Bufo calamita (Anura, Bufonidae): the fundamental role of aggregation processes during poison maturation. Zoology (Anal. Complex Syst.) 101, 53–66. Delfino, G., Amerini, S., Mugelli, A., 1982. In vitro studies on the venom emission from the skin of Bombina variegata pachypus (Bonaparte) (Amphibia Anura Discoglossidae). Cell Biol. Int. Rep. 6, 843–850. Delfino, G., Brizzi, R., Borrelli, G., 1988. Cutaneous glands in anurans: differentiation of the secretory syncytium in serous Anlagen. Zool. Jb. (Anatomie) 117, 255 –275. Delfino, G., Brizzi, R., Jantra, S., Streitberger, M., 1993. Cutaneous venom glands in the Tyrrhenian painted frog Discoglossus sardus (Tschudi): ontogenetic evolution of the biosynthesis apparatus. Acta Biol. Benrodis 5, 129–139. Delfino, G., Brizzi, R., Calloni, C., 1994. Serous cutaneous glands in the tree-frog Hyla arborea arborea (L.): origin, ontogenetic evolution and possible functional implications of the secretory granule substructure. Acta Zool. (Stockholm) 75, 27 –36. Delfino, G., Brizzi, R., Jantra, S., Feri, L., 1995. Post-Golgian maturative process during the biosynthesis of poison secretion in cutaneous glands of the european common toad Bufo bufo. J. Nat. Toxins 4, 97–113. Delfino, G., Brizzi, R., Melis, G., 1996. Merocrine secretion from serous cutaneous glands in Rana esculenta complex and Rana iberica. Alytes 13, 179–192. Delfino, G., Brizzi, R., Alvarez, B.B., Kracke-Berndorf, R., 1998. Serous cutaneous glands in Phyllomedusa hypochondrialis (Anura, Hylidae): secretory patterns during ontogenesis. Tissue Cell 30, 30–40.
Delfino, G., Brizzi, R., Alvarez, B.B., Gentili, M., 1999. Granular cutaneous glands in the frog Physalaemus biligonigerus (Anura: Leptodactylidae): comparison between ordinary serous and inguinal glands. Tissue Cell 31, 576– 586. Delfino, G., Nosi, D., Brizzi, R., Alvarez, B.B., 2001a. Serous cutaneous glands in the paludiculine frog Physalaemus biligonigerus (Anura, Leptodactylidae): patterns of cytodifferentiation and secretory activity in prematomorphic specimens. Acta Zool. (Stockholm) 82, 149 –158. Delfino, G., Nosi, D., Giachi, F., 2001b. Secretory granulecytoplasm relationships in serous glands of anurans: ultrastructural evidence and possible functional role. Toxicon 39, 1161–1171. Delfino, G., Brizzi, R., Nosi, D., Terreni, A., 2002. Serous cutaneous glands in New World hylid frogs: an ultrastructural study on skin poisons confirms phylogenetic relationships between Osteopilus septentrionalis and Phrynohyas venulosa. J. Morphol. 253, 176–186. Dockray, G.J., Hopkins, C.R., 1975. Caerulein secretion by dermal glands in Xenopus laevis. J. Cell Biol. 64, 724–733. Faraggiana, R., 1938a. Ricerche istologiche sulle ghiandole cutanee granulose degli Anfibi Anuri. I. Bufo vulgaris e Bufo viridis. Arch. Ital. Anat. Embriol. 39, 327–376. Faraggiana, R., 1938b. La struttura sinciziale e il meccanismo di secrezione delle ghiandole cutanee granulose di Anfibi Anuri. Monitore Zool. Ital. 49, 105 –108. Faraggiana, R., 1939. Ricerche istologiche sulle ghiandole cutanee granulose degli Anfibi Anuri. II. Rana esculenta, Rana agilis e Bombinator pachypus. Arch. Ital. Anat. Embriol. 41, 390–410. Flier, J., 1980. Widespread occurrence in frogs and toads of skin compounds interacting with the ouabain site of Naþ, KþATPase. Science 208, 503 –505. Flucher, B.E., Lenglachner-Bachinger, C., Pohlammer, K., Adam, H., Mollay, C., 1986. Skin peptides in Xenopus laevis: morphological requirements for precursor processing in developing and regenerating granular skin glands. J. Cell Biol. 103, 2299–2309. Ford, L.S., Cannatella, D.C., 1993. The major clades of frogs. Herpetol. Monogr. 7, 94 –117. Formanowicz, D.R. Jr., Brodie, E.D. Jr., 1982. Relative palabilities of members of a larval amphibian community. Copeia, 91–97. Garton, J.D., Mushinsky, H.R., 1979. Integumentary toxicity and umpalatability as an antipredator mechanism in the narrow mouthed toad, Gastrophryne carolinensis. Can. J. Zool. 57, 1965–1973. Gillois-Chevalier, M., 1960. Histogene`se des glandes cutane´es d’Alytes obstetricans Laur. Arch. Anat. Microsc. Morph. Exp. 49, 281–306. Gosner, K.L., 1960. A simplified table for staging anuran embryos and larvae with notes of identification. Herpetologica 16, 183 –190. Howes, N.H., 1947. The skin of the tadpole of the common toad Bufo bufo bufo (L.), during metamorphosis. Proc. Zool. Soc. Lond. 116, 602–610. Kaiser, E., Kramar, R., 1967. Biochemistry of the cytotoxic action of amphibian poisons. In: Russel, F.E., Saunders, P.R. (Eds.), Animal Toxins, Pergamon Press, Oxford, pp. 389–394. ¨ ber das Giftsekret der Gelbbauchunke Kiss, G., Michl, H., 1962. U Bombina variegata L. Toxicon 1, 33–39. Le Quang Trong, N.Y., 1967. Structure et de´veloppment de la peau
R. Angel et al. / Toxicon 41 (2003) 29–39 et des glandes cutane´es de Nectophrynoides occidentalis Angel. Arch. Zool. Exp. Ge´n. 108, 589 –610. Le Quang Trong, N.Y., 1973. Structure et de´veloppment de la peau et des glandes cutane´es de Bufo regularis Reuss. Bull. Soc. Zool. Fr. 98, 449–485. Maglia, A.M., Pugener, L.A., Trueb, L., 2001. Comparative development of anurans: using phylogeny to understand ontogeny. Am. Zool. 41, 538–551. Mar, A., Michl, H., 1976. A study of the high molecular weight hemolysin from the skin secretion of the amphibian Bombina variegata. Toxicon 14, 191 –195. Mastromei, G., Barberio, C., Pistolesi, S., Delfino, G., 1991. A bactericidal protein in Bombina variegata pachypus skin venom. Toxicon 29, 321–328. Michl, H., 1978. On hemolytic substances in the toxin of Bombina species. Period. Biol. 80 (Suppl. 1), 59–61. Myers, C.W., Daly, J.W., 1983. Dart-poison frogs. Sci. Am. 248, 96–105. Neuwirth, M., Daly, J.W., Myers, C.W., Tice, L.W., 1979. Morphology of the granular secretory glands in skin of poison-dart frogs (Dendrobatidae). Tissue Cell 11, 755 –771. Nosi, D., Terreni, A., Alvarez, B.B., Delfino, G., 2002. Serous gland polymorphism in the skin of Phyllomedusa hypochondrialis azurea Cope, (1862) (Anura, Hylidae): response by different gland types to nor-epinephryne stimulation. Zoomorphology 121, 139 –148. Quay, W.B., 1972. Integument and environment: glandular composition, function, and evolution. Am. Zool. 12, 95–108. Sabatini, D.D., Bensch, K., Barrnett, R.J., 1963. Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19–58. Sanna, A., Bernabei, P.A., Brunelli, T., Rossi Ferrini, P., Delfino, G., 1993. The cutaneous venom of Bombina orientalis:
39
cytotoxic effects on the human HL 60 cell line and a comparison with Bombina variegata. J. Nat. Toxins 2, 161–173. Seki, T., Kikuyama, S., Yanaihara, N., 1989. Development of Xenopus laevis skin glands producing 5-hydroxytryptamine and caerulein. Cell Tissue Res. 258, 483 –489. Spannhoff, L., 1954. Zur Genese, Morphologie und Physiologie der Hautdru¨sen bei Xenopus laevis Daudin. Wiss. Z. HumboldtUniv. Berl., matematische, naturwissenschaftliche Reihe, vol. 3, 295 –305. Szelistowski, W.A., 1985. Unpalatability of the poison arrow frog Dendrobates pumilio to the ctenid spider Cupiennius coccineus. Biotropica 17, 345 –346. Terreni, A., Alvarez, B.B., Brizzi, R., Nosi, D., Delfino G., 2001. Ghiandole sierose cutanee in Physalaemus albonotatus (Anura Leptodactylidae): maturazione secretoria durante lo storage dei granuli. In: Barbieri F. et al. (Eds.), Atti 38 Congr. Naz. S. H. I. (Pavia, 14–16 settembre 2000), Pianura, vol. 13, pp. 63–67. Terreni, A., Nosi, D., Brizzi, R., Delfino, G., 2002. Cutaneous serous glands in South American anurans: an ultrastructural comparison between hylid and pseudid species. Ital. J. Zool. 69, 115 –123. Toledo, R.C., Jared, C., 1995. Cutaneous granular glands and amphibian venoms. Comp. Biochem. Physiol. 111A, 11 –29. Toledo, R.C., Jared, C., Brunner, A. Jr., 1992. Morphology of the large granular alveoli of the parotoid glands in toad (Bufo ictericus ) before and after compression. Toxicon 7, 745 –753. Vanable, J.W. Jr., 1964. Granular gland development during Xenopus laevis metamorphosis. Dev. Biol. 10, 331 –357. Zasloff, M., 1987. Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms and partial cDNA sequence of a precursor. Proc. Natl. Acad. Sci. USA 84, 5449–5453. Zasloff, M., Martin, B., Chen, H.-C., 1988. Antimicrobial activity of synthetic magainin peptides and several analogues. Proc. Natl. Acad. Sci. USA (85), 910–913.