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Journal of Zoology. Print ISSN 0952-8369
Morphology of the parotoid macroglands in Phyllomedusa
leaf frogs
M. M. Antoniazzi1, P. R. Neves1, P. L. Mailho-Fontana1, M. T. Rodrigues2 & C. Jared1
1 Laboratório de Biologia Celular, Instituto Butantan, São Paulo, Brazil
2 Depto. Zoologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil
Keywords
Amphibia; Anura; Phyllomedusa; parotoid;
poison glands; skin.
Correspondence
Carlos Jared, Laboratório de Biologia
Celular, Instituto Butantan, Av. Vital Brazil,
1500, CEP: 05503-900 São Paulo, SP, Brazil.
Tel/Fax: 55-11-26279772
Email: [email protected]
Editor: Andrew Kitchener
Abstract
The parotoid macroglands of toads (bufonids) and leaf frogs (hylids) are used in
passive defence against predators. The parotoids release poison when the amphibian is bitten by a predator. Despite the apparent similarity, the anatomical and
histological structure of these macroglands in hylids is poorly studied when compared with those of bufonids. In this paper, we focused on the morphology of the
macroglands of P. distincta, a leaf frog endemic to the Brazilian Atlantic rainforest, comparing their structure with those of bufonids. In addition, we compared
the macrogland morphology of P. distincta with those from major clades of Phyllomedusa. All results revealed a macrogland morphology in leaf frogs distinct from
that of toads, suggesting that the term parotoid should be used only for those of
bufonids.
Received 19 July 2012; revised 25 March
2013; accepted 4 April 2013
doi:10.1111/jzo.12044
Introduction
Amphibian skin is characterized by the presence of mucous
glands, mainly associated with respiration and protection
against desiccation, and granular (or poison) glands, which
provide an arsenal of chemical compounds used in defence
against microorganisms and predators (Duellman & Trueb,
1986; Fox, 1986; Toledo & Jared, 1993, 1995; Zug, 1993;
Stebbins & Cohen, 1995; Clarke, 1997). In addition to these
single microscopic glands spread over the whole integument,
poison glands of certain areas of the skin are greatly
enlarged and form accumulations that were named macroglands by Toledo & Jared (1995) in order to differentiate
them from regular, isolated skin granular glands. The parotoids, located in the postorbital and supratympanic region,
are the best-known macroglands (Brazil & Vellard, 1925,
1926; Wilber & Carroll, 1940; Tronchet, 1952; Lutz, 1971;
Hostetler & Cannon, 1974; Duellman & Trueb, 1986; Toledo
& Villa, 1987; Toledo, Jared & Brunner, 1992; Hutchinson &
Savitzky, 2004; Almeida et al., 2007; Jared et al., 2009,
2011). Cannon & Palkuti (1976) and Tyler, Burton & Bauer
(2001), based on the topographical anatomy of these glands,
gave etymological reasons for using the term parotoid
instead of paratoid or parotid and we follow them. In addition, following Toledo & Jared (1995), we prefer to use
‘parotoid macrogland’ instead of ‘parotoid gland’ to avoid
ambiguity in the identification of the cutaneous glands,
because parotoids are clearly multiglandular structures;
42
accordingly we restrict the use of the term ‘gland’ for skin
mucous, granular and lipid glands, which consist of single
glandular types.
Parotoid macroglands are commonly found in anurans,
particularly bufonids (Lutz, 1971; Toledo & Jared, 1995;
Almeida et al., 2007; Jared et al., 2009, 2011) and hylids (Phyllomedusinae) (Lutz, 1966), but they are also present in other
amphibians, such as salamanders (Brodie Jr & Gibson, 1969;
Luther, 1971; Brodie Jr, 1983). They are, in general, poorly
studied, possibly because they are believed to be similar to the
parotoids of bufonids, for which morphological descriptions
are more detailed (Toledo et al., 1992; Almeida et al., 2007;
Jared et al., 2009, 2011).
The parotoids of the leaf frogs (Hylidae, Phyllomedusinae)
have an apparently similar structure to those of toads, being
longer and less conspicuous, but they have never been studied
morphologically. In both toads and leaf frogs, their position
suggests association with passive defence, an adaptation to
avoid predation in frontal attacks by predators (Toledo &
Jared, 1995; Jared et al., 2009). In both cases the triggering
mechanism for poison release seems to be activated when the
predator bites the amphibian (Jared et al., 2009, 2011). This
paper focuses on the morphology of the parotoid macroglands
of Phyllomedusa distincta, a leaf frog endemic to the Brazilian
Atlantic rainforest (Frost, 2013). The structural similarity
between the macroglands of P. distincta and those of representatives of major clades of Phyllomedusa reveals a distinctive and different organization from those of toads, and leads
Journal of Zoology 291 (2013) 42–50 © 2013 The Zoological Society of London
M. M. Antoniazzi et al.
us to suggest that the use of the term ‘parotoid macrogland’
should be restricted to the bufonids.
Materials and methods
Five specimens of P. distincta Lutz (1950; Fig. 1), collected at
Estação Biológica de Boracéia, São Paulo State, Brazil, had
their left parotoid macroglands manually compressed for
poison release. Subsequently, the animals were sacrificed with
an overdose of thionembutal, and fragments of the dorsal
and ventral skin and both entire macroglands were removed,
fixed and prepared for light microscopy. Specimens of Phyllomedusa, P. distincta (MZUSP 35048), P. vaillant (MZUSP
86303, MZUSP 86309), P. hypochondrialis (MZUSP 38452),
P. burmeisteri (MZUSP 81254), P. tomopterna (MZUSP
80910), P. bicolor (MZUSP 66178, MZUSP 66180), P. megacephala (MTR 21354) were used for a comparison of macrogland microanatomy.
Four pairs of parotoids were cut transversely to the longitudinal axis in four pieces and fixed together with the skin frag-
Parotoid morphology in leaf frogs
ments in 4% formaldehyde (made from paraformaldehyde)
buffered in 0.1 M phosphate buffer, pH 7.2 (Junqueira, 1995)
for 48 h. The skin samples and the macroglands were embedded in glycol metachrylate (Leica historesin, Leica TM,
Wetzlar, Germany), sectioned 2–4-mm thick and stained with
toluidine blue-fuchsin. A pair of parotoids was also embedded
in paraffin both in transverse and longitudinal orientations,
and the sections were stained with picrosirius and examined by
polarized microscopy for collagen fibres (Junqueira, Bignolas
& Brentani, 1979). Glycol metachrylate sections were also
submitted to bromophenol blue, periodic acid-Schiff (PAS)
combined with alcian blue pH 2.5 and the von Kossa method
(Bancroft & Steven, 1990), for detection of proteins, neutral
and acid mucosubstances, and calcium, respectively. Some
fresh fragments of the dorsal skin were fixed in 10% formaldehyde with calcium chloride 1.3% and sucrose 7.5%, sectioned in
a cryostat after embedding in Jung-Tissue Tec (Leica), and
stained with Sudan black B, for identification of lipids.
Light micrographs were obtained in a Leica DMLB
microscope, equipped with a MPS 60 photographic system.
(a)
(b)
(c)
Figure 1 Phyllomedusa distincta (a) Lateral
view of an adult specimen showing the
whole length of one of the parotoids along
the body; (b) Cephalic portion of a parotoid
(arrow) forming a postorbital and supratympanic protuberance; (c) Parotoid opened in
horizontal plane showing the honeycomb-like
arrangement of the alveoli (*).
Journal of Zoology 291 (2013) 42–50 © 2013 The Zoological Society of London
43
Parotoid morphology in leaf frogs
M. M. Antoniazzi et al.
Polarized microscopy was done in an Olympus BX60 light
microscope equipped with PM-C 35 DX photographic system.
The anatomy of the parotoids in Phyllomedusa hypochondrialis, P. tomopterna, P. bicolor, P. burmeisteri, P. vaillanti
and P. megacephala was investigated under a stereomicroscope after making an incision in the skin.
For comparison, the parotoids of the toad Rhinella marina
were studied as a representative species of genus Rhinella.
Following similar methods used for P. distincta, two pairs of
parotoids of two Rhinella marina collected in Belterra, Pará
state, Brazil, were dissected, fixed in 4% buffered formaldehyde and embedded in paraffin. After microtomy, the sections
were stained with haematoxylin-eosin and photographed in an
Olympus BX51 light microscope equipped with a digital
camera and with the software Image-Pro Express (Media
Cybernetics, Rockville, MD, USA).
Results
Parotoid macrogland anatomy of
P. distincta
The parotoids form a pair of well-defined and elongated dorsolateral skin structures, which stand out from the rest of the
(a)
Skin histology of P. distincta
The skin of P. distincta is characterized by the presence of
many glands penetrating the dermis; glands are more numerous dorsally (Fig. 2a). The dorsal skin also possesses a large
number of pigment cells, arranged in characteristic dermal
chromatophore units (Duellman & Trueb, 1986) (Fig. 2a).
The epidermis is thin and composed of four to five cell
layers, with a discrete stratum corneum. The dermis is composed of two layers, the stratum spongiosum, which is more
superficial, and the stratum compactum, below. The stratum
spongiosum is characterized by a layer of pigment cells (mainly
in the dorsum), underlying the epidermis, blood vessels,
and mucous, granular and lipid glands, which are distributed
both in the dorsal and ventral skin (Figs 2a–g and 3a–d). The
stratum compactum is poorly organized and is mainly
(b)
20 mm
(c)
20 mm
(d)
20 mm
(e)
20 mm
(f)
20 mm
44
skin. In the cephalic region, they occur in a postorbital and
supratympanic location (Fig. 1a,b), and are quite wide and
prominent. As they extend backwards on both sides of the
dorsum, they become thinner and eventually end in the posterior dorsal region (Fig. 1a). When longitudinally sectioned,
the macroglands show a honeycomb form composed of glandular alveoli (Fig. 1c).
(g)
20 mm
20 mm
Figure 2 Phyllomedusa distincta (a) Histological general view of the dorsal skin
showing a type 1 mucous gland (M1) and a
lipid gland (Li) inserted in the dermis (D).
Below the epidermis (E) the pigmentary cells
(P) show a characteristic arrangement. Blood
vessel (BV). Toluidine blue-fuchsin staining.
(b) Type 1 mucous glands (M1) with secretion
in the lumen (*). Toluidine blue-fuchsin staining. (c) Type 2 mucous glands (M2) composed of different cells, some of them with
metachromatic granules (arrows). Toluidine
blue-fuchsin staining. (d) Type 1 mucous
gland (M1) with all secretory cells positive to
protein. Bromophenol blue method. (e) Type
2 mucous gland (M2) with a few cells positive
to bromophenol blue (arrows). Bromophenol
blue method. (f) Type 2 mucous gland (M2)
with all the secretory cells positive to neutral
mucopolysaccharides, but only a few cells
positive to acid mucopolysaccharides
(arrows). PAS, + Alcian Blue pH 2.5 method.
(g) Type 1 mucous gland (M1) with all the
secretory cells exclusively positive to PAS.
PAS + Alcian blue, pH 2.5 method. PAS, periodic acid-Schiff.
Journal of Zoology 291 (2013) 42–50 © 2013 The Zoological Society of London
M. M. Antoniazzi et al.
Parotoid morphology in leaf frogs
(a)
(b)
100 mm
20 mm
(c)
20 mm
(d)
All glandular types are enveloped by a monolayer of
myoepithelial cells (Fig. 3c) and communicate with the skin
surface through epithelial ducts (Figs 2a,g and 3b,d).
The mucous glands are spherical and acinar, and are
formed by a single layer of secretory cells with basal nuclei and
an evident lumen; two distinct types are recognized according
to the lumen and secretory cells they possess. Type 1 glands
measure about 37.0 ⫾ 2.4 mm in diameter, have a wide lumen
and contain only one type of secretory cell (Fig. 2b), which is
positive to PAS (Fig. 2g) and bromophenol blue (Fig. 2d).
Type 2 glands measure about 34.5 ⫾ 3.5 mm in diameter, have
a narrower lumen and are formed by two different cell types
(Fig. 2c), both positive to bromophenol blue (Fig. 2e) and
PAS, but distinguishable by the secretory granules preferentially accumulated in an apical position, which are also positive to alcian blue, pH 2.5 (Fig. 2f).
The lipid glands are larger than the mucous glands, with an
acinar arrangement and a narrow lumen. The secretory epithelium is formed by a monolayer of columnar cells of only
one type, with basal nuclei and granules of irregular size, and
showing a pale content when stained with toluidine bluefuchsin (Fig. 2a). The histochemistry reveals that these granules are highly positive to Sudan black B (Fig. 3a), and slightly
positive to PAS and alcian blue pH 2.5 (Fig. 3b).
The granular glands are larger than the mucous and lipid
glands and are flattened, with the larger axis measuring about
166.6 ⫾ 26.3 mm and 54.3 ⫾ 13.2 mm in height. They are
syncytial, with peripheral nuclei, and are completely filled with
a large number of spherical granules. These granules show low
affinity to toluidine blue (Fig. 3c) and are highly positive to
bromophenol blue (Fig. 3d).
Parotoid macrogland histology of
P. distincta
20 mm
Figure 3 Phyllomedusa distincta (a) Lipid glands (Li) are located just
below the epidermis (E) and are positive to Sudan Black B method; (b)
Lipid gland (Li) shows a few cells near to the glandular duct (*) that are
positive to acid mucopolisaccharides (arrows). PAS + Alcian Blue pH 2.5
method; (c) Granular gland (G) with nuclei of the secretory syncytium
(large arrows) and myoepithelial cell layer (thin arrow). Epidermis (E);
dermis (D). Toluidine blue-fuchsin staining; (d) Granular gland (G) with
the secretory granules positive to bromophenol blue indicating protein
content. Dermal stratum spongiosum (Ss) and stratum compactum
(Sc); secretory duct (*); myoepithelial layer (arrow). Bromophenol blue
method. PAS, periodic acid-Schiff.
comprised of collagen fibres. Finally, the innermost skin layer,
the subcutaneous tissue, is rich in blood vessels and nerves.
There is no evidence of the presence of a calcified dermal layer
between the stratum spongiosum and the stratum compactum,
even when the von Kossa method is applied.
Journal of Zoology 291 (2013) 42–50 © 2013 The Zoological Society of London
In transverse histological sections, the macroglands are characterized by the accumulation of large, elongated, syncytial
granular glands, measuring about 371.4 ⫾ 25.0 mm in diameter and 1277.8 ⫾ 327.8 mm in height. These are arranged side
by side deep in the dermis, below the layer formed by the much
smaller regular skin glands (Fig. 4a). In longitudinal sections,
the macroglands show the circular profiles of the elongated
granular glands immersed in a homogeneous matrix of connective tissue rich in blood vessels (Fig. 4b). Polarized microscopy of transverse and longitudinal sections, stained with
picrosirius, show that the matrix of connective tissue forms a
framework of collagen fibres mainly of type I, which are recognized by their reddish colour (Fig. 4c). The characteristic
granular glands that comprise the macroglands, similarly to
the regular skin glands, are enveloped by a myoepithelial
layer, which seems to be more developed when compared with
the other cutaneous glands (Fig. 4d) and with elements in the
cytoplasm that are quite positive to PAS (Fig. 4e). The large
syncytia are composed of a peripheral region, where the nuclei
are arranged, and of an internal region, full of spherical granules morphologically distinct from those observed in the
granular glands present in the rest of the skin; they are loosely
arranged, more spherical and heterogeneous in size, and with
45
Parotoid morphology in leaf frogs
M. M. Antoniazzi et al.
(a)
150 mm
(b)
(c)
200 mm
100 mm
(d)
(f)
(e)
10 µm
10 mm
a high affinity to toluidine blue-fuchsin (Fig. 4d). Although
positive to bromophenol blue, they are not so intensely stained
as the granules of the granular glands in the rest of the skin
(Fig. 4f).
Each secretory unit of the macroglands is connected to the
exterior through an epithelial duct, forming a canal through
which the secretion is liberated (Fig. 5a,b). Transverse and
longitudinal serial sections of the ducts reveal the flattened
shape of the ductal canal and of the ductal wall that is composed of three layers of epithelial cells (Fig. 5b). The whole
length of the ductal canal is open to the passage of the gland’s
secretion (Fig. 5a); in deeper sections passing through the
pigment layer some secretory granules were observed inside
the ducts (Fig. 5b).
When a phyllomedusine macrogland is manually compressed, its poisonous secretion is liberated through pores in
the form of drops on the skin surface. The histological sections
46
10 mm
Figure 4 Phyllomedusa distincta (a) Transverse histological section of the macrogland
showing the accumulation of large, elongated
granular glands (G), making this region much
thicker than the rest of the skin. Epidermis
(E); dermis (D); pigmentary cell layer (P);
mucous gland (M); lipid glands (Li); subcutaneous tissue with blood vessels (arrow).
Toluidine-blue-fuchsin staining; (b) transverse
section of the macrogland with some large
granular glands (G) immersed in the connective tissue (Ct) of the dermis. Toluidine-bluefuchsin staining; (c) polarized microscopy of
the macrogland showing the dermal connective tissue mainly composed of collagen type
I (Col). Large granular glands (G). Picrosirius
staining; (d) detail of a large granular gland of
the macrogland showing the syncytium full of
granules (g) and with peripheral nuclei (Sy).
Myoepithelial layer (My). Toluidine bluefuchsin staining; (e) detail of the myoepithelial layer of the large granular gland of the
macrogland. The myoepithelial cells are full of
granules highly positive to neutral mucopolysaccharides. PAS method. (f) Same region of
Fig. 4e, showing the secretory granules (g)
highly positive to protein. Periphery of the
syncytium (Sy). Myoepithelial layer (arrow).
Bromophenol blue method. PAS, periodic
acid-Schiff.
show that, after compression, some granular syncytia remain
untouched, while others are observed in different stages of
filling (Fig. 5c). Poison release leads to the collapse of glandular myoepithelia and the nucleated peripheral region of the
syncytia. Sections of the compressed parotoids observed
under polarized microscopy after Picrosirius staining indicate
that no significant changes are seen in the homogeneous
arrangement of the collagen fibres that form the glandular
framework (Fig. 5d).
Parotoid macroglands of other
Phyllomedusa
All of the analysed species of Phyllomedusa have honeycomblike alveoli in the dorsolateral area. In Phyllomedusa hypochondrialis and P. tomopterna the macroglands are not
prominent and were only detected after an incision of the skin
Journal of Zoology 291 (2013) 42–50 © 2013 The Zoological Society of London
M. M. Antoniazzi et al.
Parotoid morphology in leaf frogs
(a)
(b)
20 mm
20 mm
(c)
(d)
100 mm
adjacent to cephalic region. In the other examined species
(Phyllomedusa bicolor, P. burmeisteri, P. vaillanti and
P. megacephala), macrogland structure is very similar to that
observed in P. distincta.
Parotoid macroglands of Rhinella marina
The parotoids of R. marina are large and irregular, with a
polygonal profile, and restricted to a more anterior position,
just behind the tympani (Fig. 6a). The external pores are very
prominent. When longitudinally sectioned, they show a
honeycomb-like internal organization very similar to that of
P. distincta (Fig. 6b).
As in P. distincta, transverse histological sections through
the parotoids of R. marina show large, elongated, syncytial
granular glands arranged side by side in the dermis, below a
layer of smaller regular skin glands juxtaposed to the epidermis (Fig. 6c). They are also enveloped by a myoepithelial layer
and their secretion granules, in contrast to those from the rest
of the skin, are highly positive to alcian blue pH 2.5, weakly
positive to PAS and negative to bromophenol blue.
Each secretory unit of R. marina parotoids is connected to
the exterior through an epithelial duct, which is lined by a very
thick epithelium, which obstructs the ductal canal and forms
an epithelial plug (Fig. 6d). The plug is better observed in
transverse sections through the duct (Fig. 6f). Superficial
Journal of Zoology 291 (2013) 42–50 © 2013 The Zoological Society of London
150 mm
Figure 5 Phyllomedusa distincta (a) Longitudinal section of the ductal region (*) of a large
granular gland (G) of the macrogland. Note
the open lumen along the whole length of the
duct. Epidermis (E); dermis (D); pigmentary
layer (P).Toluidine blue-fuchsin staining. (b)
Same region of Fig. 5a in a transverse section
through the duct at the level of the pigmentary cells (P). Note the secretory granules (g)
inside the lumen. Toluidine blue-fuchsin staining. (c) Manually compressed macrogland.
Many large glands are collapsed (G*) while
others are full of secretion (G). Dermis (D).
Toluidine blue-fuchsin staining. (d) Polarized
microscopy of the macrogland after manual
compression. The arrangement of the collagen fibers (Col) in the dermis is similar of that
observed in the intact macrogland. Large
granular glands of the macrogland (G). Picrosirius staining.
differentiated glands are arranged around the duct (Fig. 6d,e)
similar to those observed in other bufonids (see Jared et al.,
2009, 2011).
In contrast to P. distincta, when the parotoids of R. marina
and other Rhinella are submitted to manual compression,
their poisonous secretion is liberated in the form of jets. Histological sections show that, after compression, poison release
empties many of the large granular glands, causing the total
collapse of several syncytial alveoli (for details, see Jared et al.,
2009 and Mailho-Fontana, 2012).
Discussion
Knowledge of the morphology of anuran parotoids is based
mainly on histological and ultrastructural studies in bufonids,
which describe the secretory syncytium, the secretory granules
and the myoepithelial layer (Hostetler & Cannon, 1974;
Cannon & Hostetler, 1976; Toledo et al., 1992; Almeida et al.,
2007; Jared et al., 2009, 2011). Despite the great differences in
size and in morphology of the secretion granules of skin
granular glands and the parotoid glandular units (alveoli), it is
believed that both structures have the same epithelial origin
(Toledo et al., 1992; Toledo & Jared, 1995). In P. distincta, the
different grades of positive reaction to bromophenol blue indicate qualitative and/or quantitative differences in the proteins
present in both types of glands. In the Rhinella species studied
47
Parotoid morphology in leaf frogs
(b)
(c)
M. M. Antoniazzi et al.
(a)
(e)
(d)
300 mm
(f)
100 mm
so far, secretions from the skin and from the parotoids are
totally distinct. While in the skin there is a predominance of
proteins, in the parotoids the secretion is composed of substances that are reactive to PAS and alcian blue pH 2.5, but
not to bromophenol blue, indicating a non-protein secretion.
The conclusion is that even having a common origin with
the glands in the rest of the skin, the parotoids, especially
in bufonids, are well-differentiated structures strategically
located for the rapid release of large quantities of poison in
case of a predator’s attack, and must not be considered as
simple accumulations of granular glands.
The connective tissue framework observed in the parotoids
of bufonids is responsible for a macrogland honeycomb architecture (Jared et al., 2009). The homogeneous collagen of the
bufonid parotoid is very elastic and resistant, because, even
after compression, the shape of the macrogland usually
remains unchanged, a phenomenon that was observed in the
histological sections examined under polarized microscopy
(Jared et al., 2009). In P. distincta, we detected a similar alveolar structure, but comparison of alveoli before and after compression shows that the secretory syncytia contain different
volumes of secretion, probably depending on the amount of
external pressure received. The differential content of postcompression phyllomedusine gland differs from that of bufonids (Jared et al., 2009), in which syncytia are totally emptied
48
100 mm
Figure 6 Rhinella marina (a) Specimen
showing one of the parotoids, just behind the
tympanus. Note the polygonal shape of the
macrogland. Parotoid pores (arrows). (b)
Parotoid sectioned in horizontal plane
showing the honeycomb-like arrangement of
the alveoli(*). (c) Low magnification of a
transverse histological section of the macrogland showing the arrangement of the large,
elongated granular glands (G). Haematoxylineosin staining (d) longitudinal histological
section of the ductal region of a large granular
gland (G) of the parotoid. Note the ductal
canal obstructed by an epithelial plug (pl). Calcified dermal layer (cc); pore (po); common
mucous glands (arrows); differentiated
mucous glands (*). Haematoxylin-eosin staining (e) transverse histological section of the
duct near the skin surface showing the
arrangement of the differentiated glands (*)
around it. Calcified dermal layer (cc). pore
(po). Haematoxylin-eosin staining (f) transverse histological section of the duct in a
deeper plane, where it is completely
obstructed by the epithelial plug (pl).
Haematoxylin-eosin staining.
after compression. In these toads, the pressurized poison
breaks a plug and is squirted in toto in the form of jets (Jared
et al., 2009). Although a plug is absent in P. distincta and the
successive phases of the macrogland recovery after compression have never been followed, it is expected that they regenerate gradually and recover the poison inside the secretory
syncytia, similar to what has already been observed in bufonids (Jared et al., unpubl. data).
Additionally, bufonid parotoids have a type of differentiated mucous gland that is arranged around the syncytial
ducts, forming a complex structure resembling a rosette (Jared
et al., 2009). These differentiated glands may play a role in the
physiology of the alveoli and/or in the final composition of the
toad poison. Such structures were not observed in the macroglands of P. distincta, reinforcing the idea that they are functionally distinct.
In contrast to the parotoids in bufonids (Toledo & Jared,
1995; Jared et al., 2009), in phyllomedusines the macroglands
are not associated with any evident defensive behavioural displays such as lung inflation, head butting or crouching.
However, many species of Phyllomedusa, including P. distincta, show a dead feigning (tanatosis) behaviour (Sazima,
1974), in which the animal tightens the body and remains
static, acquiring a foetal position. This behaviour could make
the macroglands more prominent and could contribute to
Journal of Zoology 291 (2013) 42–50 © 2013 The Zoological Society of London
M. M. Antoniazzi et al.
poison release at the moment of a predator’s bite. The absence
in P. distincta of the characteristic epithelial plug that
obstructs the ducts in bufonids (Jared et al., 2009) might facilitate poison release. The transverse serial sections through the
whole length of the ducts in P. distincta showed that no plug is
present. In contrast, the epithelial plug is quite evident in
bufonid parotoids and must be responsible for the permanent
turgidity of the parotoid secretory syncytia (Jared et al., 2009).
In this case, pressure exerted by the bite of a potential predator works as a trigger for the explosive release of poison in the
form of powerful jets (Toledo et al., 1992; Toledo & Jared,
1995; Jared et al., 2009). Conversely, when the parotoids of
P. distincta are compressed, no jets are observed. The poison
is expelled from the pores in the form of droplets and accumulates on the macrogland skin surface.
In addition, phyllomedusines make use of an array of
defensive morphological, behavioural and toxinological strategies that are different from those of bufonids. The green
colour of the body, which serves as efficient camouflage
among leaves, is rapidly changed to aposematism when these
anurans move among vegetation, exposing the yellow, orange
and red colours of their flanks and limbs. Also, if a phyllomedusine is swallowed by a predator, many different bioactive compounds (including opioids and emetic substances) are
released, giving the swallowed anuran the chance to be regurgitated (Sazima, 1974; Erspamer et al., 1993).
Despite the tradition of designating all amphibian
postorbital/supratympanic macroglands as parotoids, we
have shown here that, at least between macroglands of bufonids and those of P. distincta, there are significant morphological differences that are supported by our preliminary
observations in other phyllomedusines (P. burmeisteri, P. bicolor, P. rohdei, P. tetraploidea, P. bahiana, P. megacephala).
In these species, and also in P. vaillanti, P. burmeisteri,
P. tomopterna and P. hypochondrialis, our preliminary results
indicate the presence of a honeycomb-like macrogland,
structurally similar to that of P. distincta. Therefore, the
consistent morphological similarity in the macroglands of
representatives of the major clades among Phyllomedusa
(Faivovich et al., 2010; Pyron & Wiens, 2011) suggests that,
despite variation in length and conspicuousness, this structure seems to be widely distributed in the genus. On the
other hand, there is still no consensus about the presence of
parotoid macroglands in all Phyllomedusinae (Lutz, 1950).
They have been reported to be present in Phyllomedusa, but
absent in Phasmahyla guttata by Cochran (1955), who
described only a glandular dorsolateral ridge in this species.
As commented by Faivovich et al. (2010), additional work
should be done to verify the homology between parotoids of
Phyllomedusa and these dorsolateral glands of Phasmahyla,
which is currently regarded as the sister group of the
monophyletic Phyllomedusa. Also, additional anatomical,
ultrastructural and histological data on these or similar
structures in Agalychnis, Cruziohyla and Phrynomedusa, the
successive external groups for Phyllomedusa and Phasmahyla, would be an important contribution to understanding further the phylogenetic history of phyllomedusines and
of their glands.
Journal of Zoology 291 (2013) 42–50 © 2013 The Zoological Society of London
Parotoid morphology in leaf frogs
Our results suggest that bufonid parotoids are much more
specialized structures for poison release than their equivalent
in phyllomedusines. Even considering the need for more
studies covering a taxonomically more representative sample,
we suggest restricting the term parotoid to bufonid postorbital
macroglands. Taking into consideration the characteristic
anatomy of the macroglands in most phyllomedusines, we
suggest for them the term ‘dorsolateral macroglands’. Considering the diversity and the span of morphological variation in
size, habitat and body form in bufonids and phyllomedusines,
it will be very interesting to know the variation of the structural differences observed in their macroglands.
Acknowledgements
We thank Beatriz Maurício and Simone Jared for their technical assistance. CNPq, INCTTOX-CNPq and Capes provided financial support. IBAMA provided permission to
collect animals to M.T. Rodrigues (#193/2001) and to M.M.
Antoniazzi (#15964-1).
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Journal of Zoology 291 (2013) 42–50 © 2013 The Zoological Society of London