JOURNAL OF MORPHOLOGY 263:247–258 (2005)
Ultrastructure of Poison Glands of South American frogs: A Comparison Between Physalaemus albonotatus and Leptodactylus chaquensis (Anura: Leptodactylidae) Blanca Beatriz Alvarez,1 Giovanni Delfino,2 Daniele Nosi,3* and Alessandro Terreni4 1
Departamento de Biologı´a, Universidad Nacional del Nordeste, 3400 Corrientes, Argentina Dipartimento di Biologia Animale e Genetica dell’Universita`, 50125 Firenze, Italy 3 Dipartimento di Anatomia, Istologia e Medicina legale dell’Universita`, 50134 Firenze, Italy 4 Laboratorio Centrale di Analisi Biochimico-Cliniche, Azienda Ospedaliera di Careggi, 50134 Firenze, Italy 2
ABSTRACT Serous (poison) cutaneous glands of the leptodactylid species Physalaemus albonotatus and Leptodactylus chaquensis were compared using light and transmission electron microscopy. Glands in the two species share structural traits common in anurans, including the peripheral contractile sheath (myoepithelium) and the syncytial secretory unit that produces, stores, and modifies the poison. At the ultrastructural level, early steps of poison production are also similar and fit the usual path of proteosynthesis, involving rough endoplasmic reticulum (RER) and Golgi stacks (dictyosomes) in the peripheral syncytial cytoplasm. However, several differences are obvious during the maturational processes that lead postGolgian products to their ultimate ultrastructural traits. In P. albonotatus, the dense product released from the dictyosomes acquires a thick repeating substructure, which, however, becomes looser in the inner portion of the syncytium. In L. chaquensis, serous maturation involves gradual condensation, and opaque, somewhat “vacuolized” granules are formed. These different maturational paths expressed during poison manufacturing in the two species agree with the polyphyletic origin of the family Leptodactylidae. On the other hand, data collected for P. albonotatus fit previous findings from P. biligonigerus and stress the view that poisons produced by congeneric species share similar (or identical) ultrastructural features. J. Morphol. 263:247–258, 2005. © 2004 Wiley-Liss, Inc. KEY WORDS: ultrastructure; serous glands; skin; Leptodactylidae
Poison cutaneous glands in anurans are referred to the main serous (or proteinaceous) cell line type occurring in the skin of anamniotic aquatic vertebrates (Quay, 1972). Poison glands are usually described as granular because of light microscopic features of their products, consisting of discrete structures, subspherical to elongate in shape, and characterized by various degrees of density and stain affinity. Based on transmission electron microscope criteria, these structures can, however, be ascribed to various morphological categories (Delfino et al.,1992), so that the unifying concept of secretory granules seems inadequate to define such a hetero© 2004 WILEY-LISS, INC.
geneous class of products. Actually, these “granules” represent the storage sites of various classes of active molecules (involved in both skin homeostasis and chemical defense, Daly et al., 1987) responsible for adaptation to terrestrial habitats (Toledo and Jared, 1995). They should be properly regarded as complex organelles that hold active molecules produced within the inner cell membrane system (namely, rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), and Golgi apparatus: the biosynthetic machinery) or in the secretory unit hyaloplasm, to be later transferred into these storage structures (Delfino, 1979, 1991). The occurrence of different morphological classes of serous gland products, among anurans, contrasts with the common biosynthetic machinery shared by serous glands throughout the order. The different features of the serous products mostly depend on the post-Golgian (maturational) phase, which proceeds along specific pathways in different anuran families. Since a comparison between various genera revealed that the production of skin poisons with similar features is a consistent trait in species of the same genus (Table 1), we desired to extend our investigations to different genera of the same family to verify any phylogenetic relevance of this morphological similarity. Accordingly, we studied the Argentine leptodactylid frogs Leptodactylus chaquensis and Physalaemus albonotatus (family Leptodactilylidae). Leptodactylidae is regarded as a paraphyletic taxon that includes hyloid frogs that do not share the derived traits usually indicative of the family status in anurans (Ford and Cannatella, 1993). The genera investigated are large groups, with broad spectra of diversity, representative of their peculiar
*Correspondence to: Daniele Nosi, Dipartimento di Anatomia, Istologia e Medicina legale, Universita` degli Studi di Firenze, Viale Morgagni 85, 50134 Firenze, Italy. E-mail:
[email protected] Published online 21 December 2004 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jmor.10301
248
B.B. ALVAREZ ET AL. TABLE 1. Anuran genera and cutaneous poison features: species in each genus possess glands producing poisons with similar features.
Genera
Species
Gland types
Maturational processes
Ultimate poison features
References
Alytes
A. obstetricans A. cisternasii
Ordinary serous glands Ordinary serous glands
Not described Gradual condensation
Roundish, electron-dense granules
Delfino et al., 2001b Terreni et al., 2003
Bombina (*)
B. paclypus
Type I serous glands Type II serous glands Type I serous glands Type II serous glands
Small and dense granules Large and moderately opaque granules Small and dense granules Large and moderately opaque granules
Delfino, 1977
B. orientalis
Fast condensation Merging and weak condensation Fast condensation Merging and weak condensation
Bufo
B. B. B. B.
Ordinary serous glands Ordinary serous glands Ordinary serous glands Type a serous glands
Merging of vesicles containing minute subunits Gradual condensation and aggregation of subunits
Peculiar aggregates of lamellar and/or morular structures Complex structures with grained medulla and lamellar cortex
Delfino et al., 1995a, b Delfino and Melis, 1998 Delfino, pers. obs. Delfino et al., 1999a
Colostethus
C. inguinalis C. trinitatis
Ordinary serous glands Ordinary serous glands
Not described Not described
Granules with repeating aggregations of globular and elongated subunits respectively
Neuwirth et al., 1979 Neuwirth et al., 1979
Dendrobates
D. auratus
Ordinary serous glands Ordinary serous glands
Transparent vesicles holding minute particles with irregular shapes
Angel et al., 2003
D. pumilio
Condensation of subunits followed by rarefaction Not described
Discoglossus
D. pictus D. sardus D. galganoi
Ordinary Ordinary Ordinary Ordinary
serous serous serous serous
glands glands glands glands
Gradual condensation leading to transitory spongeous forms
Dense granules, irregular in shape
Delfino, 1979; Delfino et al., 2001b Delfino et al., 1988; 1993; 2001b Delfino et al., 2001b
Hyla
H. H. H. H.
Ordinary Ordinary Ordinary Ordinary
serous serous serous serous
glands glands glands glands
Not described Aggregation of subunits Aggregation of subunits Multi-point condensation
Phyllomedusa (*)
bufo. calamita danatensis granulosus (*)
arborea intermedia regilla nana
P. hypochondrialis
Type II (lipid) glands
P. iherengi P. pailona P. sauvagei
Type II (lipid) glands Type II (lipid) glands Type II (lipid) glands
Scinax
S. acuminata S. fuscovaria S. nasica
Rana
Granules with repeating aggregations of subunits
Delfino et al., 1990
Neuwirth et al., 1979
Grosse and Linnenbach, 1989 Delfino et al., 1994 Brizzi et al., 2004 Terreni et al., 2002
Accumulation of transparent, lipid subunits Not described Not described Accumulation of transparent, lipid subunits
Aggregations of rodlets
Delfino et al., 1998a, b; Blaylock et al., 1976
Not described Not described Aggregations of rodlets
Blaylock et al., 1976 Blaylock et al., 1976 Delfino et al., 1998b; Blaylock et al., 1976
Ordinary serous glands Ordinary serous glands Ordinary serous glands
Multi-point condensation of structureless product
Granules with repeating substructures
Terreni et al., 2002 Terreni et al., 2002 Terreni et al., 2002
R esculenta (complex
Ordinary serous glands
R. iberica
Ordinary serous glands
R. camerani R. dalmatina
Ordinary serous glands Ordinary serous glands
Non-homogeneous condensation Non-homogeneous condensation Not described Not described
Delfino et al., 1996 Dense, somewhat spongeous granules
Delfino et al., 1996 Delfino, pers. obs. Delfino, pers. obs.
*Serous polymorphism reported.
phyletic histories. These wide ranges of diversity reflect a single evolutionary line of adaptation in Leptodactylus but different evolutionary lines in Physalaemus (Cei, 1980). MATERIALS AND METHODS Adult specimens of Physalaemus albonotatus and Leptodactylus chaquensis were collected near Resistencia (Chaco, Republica Argentina) and kept in the Laboratory of Comparative Anatomy
(Departamento de Bio´logia, UNNE, Corrientes). Skin strips (4 mm2 in surface area) were removed from animals sacrificed with 0.2% chlorobutanol and treated (4 h, 4°C) with an aldehyde mixture according to Karnovsky (1965). The tissue fragments were then washed with 0.1 M, pH 7 cacodylate buffer (the same as the prefixative solution), and sent in 2– 4 ml of this buffer (with the addition of a drop of glutaraldehyde) to the Dipartimento di Biologia Animale e Genetica (Universita` di Firenze, Italy). Here, the skin specimens were rinsed once more, reduced in size, and postfixed in 1% OsO4 (90 min), again using the cacodylate buffer. After rinsing in this solution the samples were dehydrated in
POISON GLAND ULTRASTRUCTURE IN LEPTODACTYLIDAE graded ethanol, soaked in propyleneoxide, and infiltrated in Epon 812 to obtain flat blocks by polymerization. Epon blocks were cut with a NOVA LKB ultramicrotome into semithin (1–2 m) and ultrathin (yellow-white, interference color) sections. Semithin sections were stained with buffered Toluidine blue for preliminary light microscope (LM) observations. Ultrathin sections were collected on 300-mesh uncoated copper grids, then electron-dense stained in sequence with hydroalcoholic uranyl acetate and alkaline lead citrate solutions (saturated and 2 mg/ml, respectively). Samples were observed (80 kV) under a Siemens 101 transmission electron microscope (TEM).
RESULTS Light Microscope Observations An LM comparison between serous glands in the species investigated disclosed identical structural traits along with obvious differences in secretory product features. We first describe common features followed by contrasting morphofunctional traits. The largest and most metabolically active component of the serous glands is the secretory unit (syncytial in structure and intradermal in position), which is provided with a muscular (myoepithelial) sheath (Fig. 1A,D,F). As usual in anurans, serous glands lack a proper lumen, which means that the poison is stored within the serous syncytium (Fig. 1A,D,F). The secretory units are roundish, although where glands crowd together they appear irregularly pear-shaped, with nuclei scattered in a single peripheral row (Fig. 1A). Starting from the periphery, secretory activity proceeds towards the upper pole of the secretory unit, at the epidermis dermis boundary. Here the secretory units joins the gland neck (or intercalated tract), where the tips of the myoepithelial cells converge (Fig. 1B,C,E). The intercalated tract consists of a few layers of cells with a high nuclear– cytoplasmic ratio (Fig. 1A,B,E), and represents the regenerative compartment involved in gland turnover. Although these stem cells may include both adenoblasts and myoblasts, turnover processes in adult specimens mostly involve secretory cytodifferentiation, as suggested by the occurrence of single adenoblasts containing secretory granules of various size and density (Fig. 1C). These single secretory cells will later merge into the syncytium. The gland neck possesses an inner compartment (the intercalated lumen, Fig. 1B,E), which includes a slightly enlarged cavity extending down into the secretory syncytium (Fig. 1D, insert). This extension—an extremely reduced gland lumen—is completely empty, because secretory granules collect around the limiting membrane that corresponds to the syncytium plasmalemma. The neck lumen exhibits a slender peripheral extension toward the epidermis, continuous with the gland duct (Fig. 1B,E). According to functional criteria, the duct represents the end portion of the gland and resembles a totally intraepidermal channel, lined by keratinized cells (Fig. 1F). Because neck and duct are not aligned along the main gland axis, their respective
249
lumina usually appear in sequential sections (Fig. 1A,B,E,F). Secretory granules in serous glands of Physalaemus albonotatus (Fig. 1A) are ellipsoidal to roundish in shape and are characterized by various degrees of density. A prominent maturational gradient is detectable among serous aggregates, with smaller and denser secretory granules at the periphery and larger and paler granules toward the interior. The most peripheral syncytial areas also contain small, moderately opaque granules. As later described in the TEM results, this thin secretory material derives from early (post-Golgi) activity. In contrast to the above findings, serous glands of Leptodactylus chaquensis contain secretory granules with homogenous features: these are dense, roundish particles, provided with an evident halo, that does not exhibit any obvious maturational gradient (Fig. 1D, insert, E,F). Smaller glands are scattered between the serous units in both species; these are mucous glands and possess an obvious lumen, its width depending on the functional stage of the encircling mucocytes (Fig. 1A,D,F). The mucous product consists of discrete, dense granules when intracytoplasmic (Fig, 1A,D,F), whereas it is more transparent and structureless when intraluminal (Fig. 1D,F). Electron Microscope Observations Physalaemus albonotatus. Because of the centripetal, functional gradient usual in serous glands, under TEM the most attention was paid to the peripheral areas of the secretory syncytium, where biosynthesis and early maturation are interlaced. The peripheral cytoplasm of the secretory unit is enveloped by a continuous layer of thin myoepithelial cells (Figs. 2A, 3B), which exhibit dense plaques among myofilaments as peculiar components of their cytoskeleton (Fig. 2B). Further typical features in these contractile cells are scattered profiles of sarcoplasmic reticulum (SR, Fig. 2C) and minute, spherical inpocketings (caveolae) from the plasma membrane. In tangential sections, these caveolae form a somewhat honeycomb pattern due to their thick arrangement (Fig. 2D). In some instances, single caveolae can be seen in close contiguity to complements of the sarcoplasmic reticulum (Fig. 2E). The narrow space between secretory and contractile compartments contains thin cell processes; that are cylindrical in shape and contain aligned microtubules: thus, they resemble axonal processes (Fig. 2B). A remarkable functional trait in adult glands is the sharp contrast between large amounts of mature granules and resting biosynthesis apparatus (Figs. 2A, 3B,F). Biosynthetic organelles consist of rough endoplasmic reticulum (RER, Fig. 2A,B,H) and Golgi stacks (Fig. 2F,G). RER cisterns are flat (Fig. 2A) and contain a slightly opaque product (Fig. 2B), whereas Golgi saccules are mostly dilated and irreg-
250
B.B. ALVAREZ ET AL.
Fig. 1. Features of serous cutaneous glands in Physalaemus albonotatus (A–C, serial section) and Leptodactylus chaquensis (D–F). LM. A: Main architecture of serous glands including duct, intercalary tract, secretory unit, and myoepithelium. Serous product is stored within the secretory unit syncytium and exhibits an obvious maturational gradient; arrow points to the duct lumen. B: Transitional region between intercalary tract (neck) and duct (compare with E); notice myoepithelial cells converging toward the gland neck; arrowhead and arrow point to the lumina of intercalary and duct, respectively. C: Peripheral region of the gland neck: notice adenoblast differentiating from the stem cell pool. D: Low- power magnification showing differences between serous and mucous glands: notice obvious lumina in mucous glands (closed and open, large asterisks) and the solid serous unit (left). Also notice the exiguous sub-intercalary lumen in serous gland (small, closed asterisk in the insert). E: Transition region between neck and duct (compare with B), as shown by continuous lumina: arrowhead points to the neck lumen and arrow to the duct lumen. F: Main organization of serous gland; the intercalary tract appears in tangential section since the functional axis of the gland is not aligned with the neck axis. Arrow points to the duct lumen. d, gland duct; it, intercalary tract; mec, myoepithelial cells; mg, mucous gland; SIII-VI, maturational stages of serous product; sc, differentiating secretory cell (adenoblast).
ular in profile (Fig. 2F). Slender saccules are, however, provided with enlarged ends, where a structureless material tends to aggregate in dense particles (Fig. 2G). Once released as vesicles from the Golgi apparatus, this product forms small granules exclusively found in the most peripheral cytoplasm (Fig. 2F). Both the amorphous product and dense granules are contained within post-Golgi secretory vesicles engaged in merging processes (Fig. 2H). Larger vesicles thus arise, where the secretory
material becomes rearranged in a labyrinthine pattern, resulting from alternating dense and pale products (Fig. 3A). Secretory vesicles gradually turn into secretory pregranules, while the labyrinthine pattern changes into a loose aggregation of subunits (Fig. 3A). As expected from LM microscope observations, under TEM the ultimate secretory granules exhibit an obvious range of ultrastructural features even in the peripheral cytoplasm, where they are irregular in shape and vary in density (Fig. 3B).
Fig. 2. Serous glands of Physalaemus albonotatus; early patterns of biosynthesis and ultrastructural features of myoepithelial cells. TEM. A: Peripheral gland region showing secretory granules, biosynthesis organelles, and myoepithelial cell. B: Detail of the boundary zone between contractile and secretory compartments; notice rough cistern in the secretory syncytium, a thin nerve ending in the interstice, and dense plaques among myofilaments. C: Profiles of the sarcoplasmic reticulum (arrows) in myoepithelial cell. D: Tangential section of a myoepithelial cell: this honeycomb-like pattern results from closely arranged caveolae (arrowheads). E: Notice contiguous sarcoplasmic reticulum tubule and caveola (arrow and arrowhead, respectively). F: These Golgi stacks release early secretory product in the form of minute, dense granules (arrows). G: This early product arises from marginal dilations of sacculi in the trans face of the Golgi stack. H: Merging processes (arrowheads) involve post-Golgi products, leading to large secretory product accumulations. dp, dense plaques; G, Golgi stack; mec, myoepithelial cell; nt, neurotubules in nerve ending; RER, rough endoplasmic reticulum; S-SI, SIV, maturational stages of serous product.
252
B.B. ALVAREZ ET AL.
Figure 3.
POISON GLAND ULTRASTRUCTURE IN LEPTODACTYLIDAE
Higher magnification of these secretory deposits discloses a consistent repeating substructure, with minute subunits arranged in a thick pattern (Figs. 2A, 3A,C,H). Just distally from the myoepitheliumsecretory syncytium boundary, secretory granules are more homogenous in shape as well as density; they are densely packed, their flat surfaces facing each other (Fig. 3D), so that their circular profiles appear somewhat polygonal in section (Fig. 3C). As a result of crowding, extremely thin cytoplasm layers separate contiguous granules (Fig. 3D) and follow the complex membrane relationships that lead to merging processes (Fig. 3E). A characteristic trait of maturational processes is the relationship between central granules and the secretory syncytium. The slender halo that encircles the serous products extends locally toward the cytoplasm, isolating small areas that are circular to elliptical in section and exhibit complex patterns of parallel membranes (Fig. 3F). These membranes may belong to the cell inner membrane system (e.g., the endoplasmic reticulum) or originate from invagination of the granule limiting membrane. In some instances, these small cytoplasmic portions are regularly aligned around the granule periphery in a recurrent pattern (Fig. 3G). Proceeding toward the inner region of the syncytium, the thickly arranged subunits of the serous deposits gradually vanish (Fig. 3H), explaining the pale granules detected in the inner syncytium under LM. Leptodactylus chaquensis. Low-power TEM micrographs confirm the basic morphological traits in serous glands of this species, including secretory granule features. Secretory granules may vary in shape, but exhibit rather homogenous electron-opacities (Fig. 4A). Since they are provided with wide halos, the inner portion of the secretory syncytium contains an obvious cavitary system and sharply contrasts with the peripheral cytoplasm, which has a solid background and accommodates a single row of nuclei (Fig. 4A). Because the dilated perigranular halos merge together, the inner region of the secretory unit seems to contain a lumen with an irregular profile, in which serous product is stored (Fig. 4A). However, at higher magnification the peripheral syn-
253
cytium appears to be continuous with the cytoplasmic partitions inserted between the granules in the inner syncytium (Fig. 4B), demonstrating that the “lumen” is actually a complex of contiguous compartments. As in the former species, the space between secretory unit and contractile sheath is exiguous and holds small neurite endings with dense-cored synaptic vesicles (Fig. 4C). Contractile sheath and secretory unit can be easily distinguished by their different cytoplasmic contents: the myoepithelial cells hold dense plaques along the myofilament bundles (Fig. 4D), together with complements of the sarcoplasmic reticulum, both vesicular (Fig. 4C) and tubular in shape (Fig. 4E); the secretory syncytium contains slender cisterns of the RER (Fig. 4C). Furthermore, there are no plasma membranes intervening between nuclei in the peripheral syncytial cytoplasm (Fig. 4B), whereas the contractile compartment consists of discrete smooth muscle cells, linked by desmosomes that alternate with peripheralcaveolae (Fig. 4F). In addition to flattened RER profiles, containing moderately opaque material, the biosynthetic apparatus of the secretory syncytium includes stacked saccules of the Golgi apparatus (Figs. 4G, 5A), involved in condensation of serous product (Fig. 4H). In comparison with Leptodactylus chaquensis, poison biosynthesis and maturation in Physalaemus albonotatus proceeds according to a less obvious sequential development. Secretory granules derive from homogeneous thickening of the product contained in Golgi vesicles (Fig. 4H). Condensation may be either restricted to foci or diffuse, so that secretory granules show patterns of alternating paler and denser areas, or homogenous opacity (Fig. 5A). Advanced maturation leads to spongylike granules alternating with granules of compact structure (Fig. 4A). Small, opaque particles are seldom detected in large vesicles that also contain a finely dispersed material (Fig. 5B). The wide halo around secretory granules contains a network of minute structures (Fig. 5A); these are thin cytoplasmic outgrowths emanating from the syncytium (Fig. 5C) and arranged in a branched pattern (Fig. 5D).
Fig. 3. Maturational processes affecting the secretory product in serous cutaneous glands of Physalaemus albonotatus. TEM. A: The dense, discrete secretory aggregates contained in the post-Golgi vesicles tend to change into a labyrinthine-shaped material, due to random distribution of opaque and light products (SII). In later granules (SIII), discrete subunits appear, which acquire a thicker arrangement, with an obvious repeating substructure (SIV). B: As a result of this secondary condensation, the secretory deposits accumulating in the syncytium tend to resemble real granules, namely, opaque storage bodies. Secretory granules crowd in the peripheral cytoplasm and mask the maturational gradient; this low-magnification picture does not resolve any substructure in secretory granules. C: Higher magnification shows that the recurrent aggregation of subunits represents a common trait in these peripheral granules, which face each other with flat surfaces (opposite arrows). D,E: Thin cytoplasmic screens are interposed between contiguous serous deposits, molding their profiles and following granule gear-like relationships (arrows in E); notice the narrow halos encircling serous aggregates. F: These narrow compartments may evaginate into the syncytium, encircling small cytoplasmic portions, provided with inner membrane systems. G: In some instances, these small cytoplasmic portions exhibit a regular arrangement around the granules. H: As described under LM, the thick subunit arrangement tends to be progressively weaker in the inner syncytium (SIV-SVI). h, perigranular halo; mec, myoepithelial cell; RER, rough endoplasmic reticulum.
254
B.B. ALVAREZ ET AL.
Figure 4.
POISON GLAND ULTRASTRUCTURE IN LEPTODACTYLIDAE
255
Fig. 5. Maturational processes affecting the secretory product in serous cutaneous glands of Leptodactylus chaquensis. TEM. A: Higher-power magnification discloses differences between serous deposits, which are mostly related to their densities. B: Peculiar feature of serous product, consisting of an electron-opaque core inserted in a compartment holding finely dispersed material. C: The cytoplasm that borders the perigranular halo evaginates, forming thin, microvillous outgrowths. D: These minute cytoplasmic processes form a three-dimensional net; arrows point to branching points. G, Golgi stack; SII, SII/III, SIII, stages of serous maturation.
DISCUSSION The ultrastructural traits we have detected reveal that consistent differences exist between the secretory compartments of the two leptodactylid species under comparison. By contrast, myoepithelial cells in Leptodactylus chaquensis and Physalaemus albonotatus exhibit identical subcellular features (caveolar invaginations, sarcoplasmic reticulum, and
dense plaques). These traits fit the type II pattern described in cutaneous glands of several amphibians, referred to smooth contractile cells provided with phasic activity (Bani, 1976). Different traits in the syncytia mostly deal with serous products and post-Golgi (maturational) phases of the secretory cycle, because in both species secretory activity involves the same organelle ma-
Fig. 4. Main ultrastructural features in serous glands of Leptodactylus chaquensis: secretory unit (A–C,E,G,H) and myoepithelium (C–E). A: Low-power magnification of serous gland, showing homogeneous features of secretory granules and wide cavities (asterisks) in the cytoplasm. B: These cavities are dilated perigranular halos; notice contiguous nuclei in the secretory unit, with no intervening membrane. C: Secretory and contractile compartments are separated by slender interstices holding thin neurite endings; notice that dense-cored synaptic vesicles are distributed on both sides of the interstice, secretory and contractile as well. Arrows point to vesicular profiles of the sarcoplasmic reticulum. D: Myoepithelial cell cytoskeleton includes dense bodies interspersed among myofilaments. E: Notice elongate profiles of the sarcoplasmic reticulum, oriented along the main axis of this myoepithelial cell (arrows). F: Desmosome-like junctions occur between myoepithelial cells; the small vesicles close to the myoepithelial cell plasma membranes are permanent caveolar inpocketings continuous with the interstices (arrow). G: Slender RER complements and Golgi stack; the Golgi is releasing amorphous, secretory material. H: Early stages of post-Golgi condensation affecting the secretory product. c, caveola; dp, dense plaques in myoepithelial cell; G, Golgi stack; h, perigranular halo; i, interstice between secretory and contractile compartments; RER, rough endoplasmic reticulum; S-SII, stages of serous synthesis and maturation; ss, secretory syncytium.
256
B.B. ALVAREZ ET AL.
Fig. 6. Schematic, 3D representation (not to scale) of activity in serous glands of Physalaemus albonotatus (A–C), and Leptodactylus chaquensis (AI–CI). Based on present findings and data from Delfino et al. (2001). This comparison shows the main arrangement of the secretory units (A and AI), serous maturation (B and BI), and cytoplasm-secretory product relationships (C and CI). S-SV/VI, stages of serous maturation in both species.
chinery pursuing a common proteosynthetic pathway and their early products share similar features. In both species, the early secretory material contained in the rough cisterns shows a weak opacity, but later the Golgi apparatus performs an obvious condensing activity, because the serous product released from the sacculi of the trans side is more opaque. Starting from the Golgi step, serous maturation processes continue during the intracytoplasmic storage of the poison deposits (granules), and follow the differential pathways summarized in Figure 6, in accordance with the centripetal maturational gradient characteristic of serous anuran glands (Delfino, 1991). As a result of these matura-
tional processes, the skin poisons acquire the peculiar traits of the species observed. In Pysalaemus albonotatus the dense material released by the Golgi apparatus reaches a thick repeating substructure through sequential, intermediate features, consisting of alternations of dense and pale areas and labyrinthine patterns. This maturational sequence has also been described by Terreni et al. (2002) in hylid and pseudid serous glands, genera Hyla and Scinax, and Pseudis, respectively. Maturational changes detected in P. albonotatus are short-cuts of the post-Golgi processes occurring in tadpoles of the congeneric P. biligonigeros (Delfino et al., 2001a) and the hylid Scinax nasica (Terreni et
POISON GLAND ULTRASTRUCTURE IN LEPTODACTYLIDAE
al., 2003). Further maturational steps affecting granules of P. albonotatus lead to the repeating substructures gradually vanishing, as also observed in “ordinary” serous glands of P. biligonigeros (Delfino et al., 1999b). In the latter species, on the contrary, large serous units have been described (“inguinal glands”; Cei, 1980) where serous maturation involves extreme thickening of the repetitive subunit arrangement, until extremely opaque, structureless granules are formed. This process allows remarkable amounts of poison to be stored in a condensed (possibly inactive) state (Delfino et al., 1999b). The serous glands we have described in P. albonotatus belong to the ordinary type, because, in these species, inguinal glands are either extremely small or absent (Lynch, 1970). When compared with Pysalaemus albonotatus species, maturational processes in serous glands of Leptodactylus chaquensis display a less obvious sequential development. However, a fundamental trend is apparent, roughly along the centripetal gradient that leads to the formation of granules provided with a somewhat spongious substructure. Similar “vacuolized” granules have been described in serous glands of species pertaining to the families Discoglossidae and Ranidae (see Table 1): Discoglossus pictus (Delfino, 1979; Delfino et al., 2001b) and D. sardus (Delfino et al., 1993); Rana esculenta (Barni et al., 1987; Delfino et al., 1996), R. iberica (Delfino et al., 1996), R. camerani and R. dalmatina (Delfino, pers. obs.). This peculiar substructure seems to be related to the secretory product maturation. Among ultrastructural aspects of gland activity, which could better describe maturational changes, we detected enhanced serous productsyncytial cytoplasm relationships, linked to two general mechanisms consistent in several anuran species (Delfino et al., 2001b). These are: 1) evagination of the compartment (halo) encircling the serous material into the cytoplasm, and 2) the development of a network of branched and slender cytoplasmic processes (resembling minute microvilli) from the syncytium. Although both of these devices, which amplify the surface area of the granule-limiting membrane, may coexist in the same glands, it appears that evaginations of the halo prevail in granules with ordered substructures, whereas microvillous outgrowths predominate in glands producing spongy-like granules (Delfino et al., 2001b). An increase in the surface areas involved in exchange between cytoplasm and granules may be related to in/outflows of substances: some low molecular weight poison fractions are produced in the secretory syncytium cytosol to be later transferred into granules (Delfino, 1979, 1991). Furthermore, condensation of the serous product involves transport of watery fractions towards the cytoplasmic compartment. In dendrobatid frogs serous granules store products from various sites, both endo- and/or exogenous in origin, including dietary arthropods (Daly,
257
1998; Smith et al., 2001) and symbiotic microorganisms (Daly et al., 1997). Biochemical data on the composition of anuran skin poisons from a wide taxonomic distribution indicate several differences between Physalaemus and Leptodactylus, concerning biogenic amines and peptides (Daly et al., 1987). Nonetheless, major classes of active molecules produced by serous glands are widely distributed among anurans regardless of their taxonomic position. Furthermore, ordinary ultrastructural analysis can only provide information on the biosynthesis of large molecules, occurring in serous skin products, such as proteins (involving RER) or steroids (SER), whereas small active molecules cannot be detected because they are produced in the cytosol (Delfino, 1991). Despite the sites of origin of the active poison molecules, it should be remembered that the different maturational processes observed in P. albonotatus and L. chaquensis are related to changes in the physical state of the products, rather than to chemical composition. The remarkable differences between cutaneous poisons in the two genera under comparison agree with the possible non-monophyletic status of the family Leptodactylidae (Ford and Cannatella, 1993). At the generic level, it appears that poison biosynthesis in Physalaemus albonotatus closely resembles that of P. biligonigerus (Delfino et al. 1999b, 2001a; Terreni et al., 2003), which, in turn, support the data listed in Table 1. Results obtained from Physalaemus in the present study are even more relevant because we have described identical features in species pertaining to different groups of this genus: the smaller biligonigerus tribe and the larger cuvieri tribe including albonotatus (Cei, 1980).
LITERATURE CITED Angel R, Delfino G, Parra GJ. 2003. Ultrastructural patterns of secretory activity in poison cutaneous glands of larval and juvenile Dendrobates auratus (Amphibia, Anura). Toxicon 41: 29 –39. Bani G. 1976. Cellule mioepiteliali accluse alle ghiandole cutanee di alcuni Anfibi. Archo Ital Anat Embriol 81:133–184. 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. Blaylock LA, Ruibal R, Platt-Aloia K. 1976. Skin structure and wiping behaviour of phyllomedusine frogs. Copeia 1976:283– 295. Brizzi R, Corti C, Delfino G, Drewes R. 2004. Ultrastructural patterns of secretory activity in serous cutaneous glands of the Pacific tree-frog Hyla regilla (Anura, Hylidae). Ital J Zool 71 (suppl. 2). Cei JM. 1980. Amphibians of Argentina. Monit Zool Ital (Nuova Serie), Monografia 2. Daly JW. 1998. Thirty years of discovering arthropod alkaloids in amphibian skin. J Nat Toxins 61:162–172. Daly JW, Myers CW, 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.
258
B.B. ALVAREZ ET AL.
Daly JW, Padgett WL, Saunders RL, Cover JF. 1997. Absence of tetrodotoxins in a captive-raised riparian frog Atelopus varius. Toxicon 35:705–709. Delfino G. 1977. Il differenziamento delle ghiandole granulose cutanee in larve di Bombina variegata pachypus (Bonaparte) (Anfibio: Anuro: Discoglosside). Ricerca al microscopio ottico e al microscopio elettronico. Archo Ital Anat Embriol 82:337–363. Delfino G. 1979. Le ghiandole granulose cutanee di Alytes cisternasii Bosca` e Discoglossus pictus Otth (Anfibi, Anuri, Discoglossidi): Struttura, ultrastruttura e alcuni dati citochimici. Archo Ital Anat Embriol 84:81–106. Delfino G. 1991. Ultrastructural aspects of venom secretion in anuran cutaneous glands. In: Tu AT, editor. Handbook of natural toxins, vol. 5. Reptile venoms and toxins. New York: Marcel Dekker. p 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 101: 53– 66. Delfino G, Brizzi R, Borrelli G. 1988. Cutaneous glands in anurans: differentiation of the secretory syncytium in serous Anlagen. Zool Jahrbu¨ch (Anat) 117:255–275. Delfino G, Brizzi R, Calloni C. 1990. A morpho-functional characterization of the serous cutaneous glands in Bombina orientalis (Anura:Discoglossidae). Zool Anz 225:295–310. Delfino G, Brizzi R, De Santis R, Melosi M. 1992. Serous cutaneous glands of the western spade-foot Pelobates cultripes (Amphibia: Anura): an ultrastructural study on adults and juveniles. Archo Ital Anat Embriol 97:109 –120. 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 (L.): origin, ontogenetic evolution, and possible functional implications of the secretory granule substructure. Acta Zool (Stockh) 75:27–36. Delfino G, Brizzi R, Jantra S, Feri L. 1995a. Post-Golgian maturative processes 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, Feri L. 1995b. Chemical skin defence in Bufo bufo: an ultrastructural study during ontogenesis. Zool Anz 234:101–111. 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 BB, Kracke-Berndorff R. 1998a. Serous cutaneous glands in Phyllomedusa hypochondrialis (Anura, Hylidae): secretory patterns during ontogenesis. Tissue Cell 30:30 – 40.
Delfino G, Alvarez BB, Brizzi R, Cespedez JA. 1998b. Serous cutaneous glands of Argentine Phyllomedusa Wagler 1830 (Anura Hylidae): secretory polymorphism and adaptive plasticity. Trop Zool 11:333–351. Delfino G, Brizzi R, Alvarez BB, Taddei L. 1999a. Secretory polymorphism and serous cutaneous glands heterogeneity in Bufo granulosus (Amphibia, Anura). Toxicon 37:1281–1296. Delfino G, Brizzi R, Alvarez BB, Gentili M. 1999b. 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 BB. 2001a. Serous cutaneous glands in the paludiculine frog Physalaemus biligonigerus (Anura, Leptodactylidae): patterns of cytodifferentiation and secretory activity in prematomorphic specimens. Acta Zool (Stockh) 82:149 –158. Delfino G, Nosi D, Giachi F. 2001b. Secretory granule-cytoplasm relationships in serous glands of anurans: ultrastructural evidence and possible functional role. Toxicon 39:1161–1171. Ford LS, Cannatella DC. 1993. The major clades of frogs. Herpetol Monogr 7:94 –117. Grosse WR, Linnenbach M. 1989. Feinbau der Hautdru¨sen und Entwicklung des Melanophoren-Musters bei Laubfrosch, Hyla arborea L. (Amphibia, Anura). Zool Anz 223:211–219. Karnovsky MJ. 1965. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J Cell Biol 27:137A. Lynch JD. 1970. Systematic status of the American leptodactylid frog genera Engystomops, Eupemphix, and Physalaemus. Copeia 1970:488 – 496. Neuwirth M, Daly JW, Myers CW, Tice LW. 1979. Morphology of the granular secretory glands in skin of poison-dart frogs (Dendrobatidae). Tissue Cell 11:755–771. Quay WB. 1972. Integument and environment: glandular composition, function, evolution. Am Zool 12:95–108. Smith BP, Tyler MJ, Kanekko T, Garraffio M, Spande TF, Daly JW. 2001. Evidence for biosynthesis of pseudorhynamine alkaloids by an Australian myobathrachid frog (Pseudophryne) and for sequestration of dietary pumiliotoxins. J Nat Toxins 65: 439 – 447. Terreni A, Nosi D, Delfino G. 2002. Cutaneous serous glands in South-American anurans, an ultrastructural comparison between hylid and pseudid species. Ital J Zool 69:115–123. Terreni A, Nosi D, Greven H, Delfino G. 2003. Development of serous cutaneous glands in Scinax nasica (Anura, Hylidae): patterns of poison biosynthesis and maturation in comparison with larval glands in specimens of other families. Tissue Cell 35:274 – 87. Toledo RC, Jared C. 1995. Cutaneous granular glands and amphibian venoms. Comp Biochem Physiol 111A:11–29.
