Contributions to Zoology, 76 (3) – 2007
Morphological re-evaluation of the parotoid glands of Bufo ictericus (Amphibia, Anura, Bufonidae)
Pablo G. de Almeida , Flavia A. Felsemburg , Rodrigo A. Azevedo , Lycia de Brito-Gitirana
Keywords: morphology, amphibian integument, exocrine glands, bufonid.
Multicellular glands in the amphibian integument represent a significant evolutionary advance over those of fishes. Bufonids have parotoid glands, symmetrically disposed in a post-orbital position. Their secretion may contribute to protection against predators and parasites. This study provides a re-evaluation of the morphology of the Bufo ictericus parotoid glands. The parotoid gland integument of the medial surface shows rounded depressions with small pores that connect with the duct openings of the larger granular glands. Under light microscopic evaluation the integument is constituted by typical epidermis, supported by dermis subdivided into a spongious dermis, a reticular dermis, and a compact dermis. The Eberth-Katschenko layer is identified as a basophilic material scattered throughout the superficial spongious dermis. The parotoid gland is an integument region, in which three exocrine glandular types occur: mixed glands, smaller granular glands and larger granular glands. The mixed gland is formed by mucous and serous cells while the small granular glands contain a homogene acidophilic intake. The larger granular glands produce a basophilic and alcianophilic material, and are responsible for the macroscopic protuberances designed as parotoid glands. Thus, the end product released by the parotoid glands is a mix of secretions produced by the three glands.
One of the main evolutionary advances of the amphibian integument over that of fishes is the presence of a large number of multicellular alveolar and, in some cases, tubular glands (Porter, 1972). The established histologic classification of exocrine glands is based on different criteria. According to the secretion mechanism, the exocrine gland which releases its secretory product by exocytosis, is classified as a merocrine gland, such as in the case of pancreatic secretion of zymogen granules. When the secretory mechanism involves partial loss of the apical portion of the cell, the gland is named apocrine. The lipid secretion by epithelial cells of the mammary gland is an example of this glandular type. In addition, if the end secretion is constituted by the entire cell and its secretory product, the exocrine gland is designated as an holocrine gland such as the sebaceous glands of the mammalian skin or the avian uropygial gland. Considering the secretory cell type, the merocrine gland is a mucous gland when its product is rich in glycoprotein, which is responsible for the cytoplasm basophilic staining. The merocrine serous gland shows acidophilic cytoplasm and its secretion is enriched with proteins. Thus, the mixed gland contains both mucous and serous cells (Kierszen-baum, 2002; de Brito-Gitirana, 2004).
Cutaneous glands of all living amphibians usually belong to four main types located in the spongious dermis: mucous, serous (granular or poison), lipid (or wax), and mixed (seromucous) glands (Duellmann and Trueb, 1994; Brizzi et al., 2002).
Mucous glands secrete a clear, watery to viscous substance. The mucous secretions keep the integument moist in terrestrial conditions and lubricate it when in water (Porter, 1972). Mucous secretions of amphibian integument glands can also have a bacteriostatic effect (Pough et al., 2003), and they possess the ability to mechanically retain microbial and fungal pathogens (Clarke, 1997; Fontana et al., 2006).
Granular glands produce a milky secretion that is much more toxic than that of the mucous, thereby providing protection against bacterial and fungal infection as well as defense against predators. In addition, there are other subtypes of serous glands, indicating high morphological variability (Warburg et al., 2000). Cutaneous lipids are present in some glands of phylomedusine anurans (Blaylock et al., 1976), hylids (Warburg et al., 2000) and in one rhacophorid frog (Lillywhite et al., 1997). Cutaneous lipid secretion appears to be the main physiological adaptation of xeric-inhabiting arboreal frogs, enabling them to remain exposed throughout the year, even in the dry season (War-burg et al., 2000). For Barbeau and Lillywhite (2005), the lipid secretory glands are related to specialized adaptations of the skin in order to provide resistance to evaporative water loss.
A remarkable glandular type, different from the mucous, serous, and seromucous glands in anuran integument, was described in Odontophrynus americanus (Felsemburgh et al., 2006). These glands have an acidophilic secretion constituted by a granular intake scattered by a homogeneous secretion. The homogeneous secretion showed continuity to glandular cell cytoplasm, and this continuity characterizes a glandular type with an apocrine mechanism of secretion, which was not previously described for the amphibian cutaneous glands.
Bufonids have peculiar glandular structures symmetrically disposed in a post-orbital position, known as the parotoid glands (Young, 1985; Pough et al., 2003). Although toads do not have a venom inoculation system, they are considered poisonous animals due to their cutaneous glandular secretions that contain a variety of compounds such as proteins, peptides, steroids, alkaloids and biogenic amines (Sakate and Lucas de Oliveira, 2000; Maciel et al., 2003). These glands are composed of large aggregations of granular glands responsible for the production and storage of a thick and creamy secretion which may contribute to the anuran protection against predators and parasites (Porter, 1972; Croce et al., 1973; Duellman and Trueb, 1994; Clarke, 1997; Sakate and Lucas de Oliveira, 2000).
On account of the Bufo ictericus parotoid gland relevant role, the aim of this work was to characterize the gross anatomy and the microscopic structure using stereoscopic microscopy, light microscopy, and histochemical methods.
Fig. 4. Light micrograph showing the mixed gland (’) associated to the duct (→) of the larger granular gland (). SD = spongious dermis; RD = reticular dermis. Scale = 100 µm. Gomori´s trichrome stain.
Fig. 5. Myoepithelial cells () around the secretory portion of mixed gland (’) and smaller granular gland (). Note the Eberth-Kathschenko layer (). Scale = 100 µm. Immunolabeled for smooth muscle α actin.
Fig. 9. Note the clear tinctorial difference between the intake of the smaller granular gland () and the larger granular intake (‚). Note collagenous fiber of a septum (’) separating the secretory portions. Scale = 50 µm. Gomori´s trichrome stain.
Fig. 10. Note alcianophilic reaction of the mucous cells () of the mixed gland (’) and serous cells (➔) which show no reaction. The smaller granular gland () demonstrate no reaction to AB-method. Scale = 50 µm. AB- stain.
Material and methods
Five adult males of Bufo ictericus Spix, 1824 were collected in Teresópolis, Rio de Janeiro State, according to the Brazilian laws (no. 191/2005 - IBAMA). The average weight of the toads was 120 grams and the average length (snout-vent) was 11 centimeter. For stereoscopic microscopy (SM) the entire parotoid gland region was excised (in toto), cut into 5 millimeter sections with a sharp blade, and fixed in 10% buffered neutral formaldehyde solution. The sections were analyzed under a Stemi SV11 Zeiss. The images were captured by the AxioVision 2.05 system. This procedure was used to characterize the gross anatomy of the internal arrangement of the parotoid gland region. For light microscopic (LM) analysis, parotoid gland fragments were fixed with 10% buffered formaldehyde and/or Bouin’s liquid, and processed according to standard histological techniques for paraffin embedding. Five-micrometer thick serial slices were stained with haematoxylin-eosin (HE) and Gomori’s trichrome (Lillie and Fulmer, 1976). Staining with 0.1% Alcian Blue (AB) 8GX at pH 2.5 (Kiernan, 1990) was employed for demonstration of sulfated and carboxylated forms of acid mucosubstances. The periodic acid Schiff (PAS) staining was employed to detect neutral glycoproteins (Mowry, 1963). The von Kossa method (Prophet et al., 1994) was used to detected calcium. The localization of smooth muscle α-actin positive cells was performed using the standard avidin biotin conjugate (ABC) immuno-assaying procedure. Paraffinized sections were de-waxed, dehydrated and then washed in phosphate buffered saline (PBS). Nonspecific binding sites were blocked with 3% hydrogen peroxide solution for 30 minutes. The sections were incubated in a humid chamber for 30 minutes with 1% bovine serum albumen (BSA) at room temperature. The sections were then incubated with monoclonal antibody anti-smooth muscle α-actin (Dako, HHF35 code no. M0635) diluted to 1:50 overnight. After washing with PBS, the sections were incubated for 1 hour at room temperature with the biotinylated secondary antibody (1:100) (Dako, Multilink swine anti-goat, mouse, rabbit immunoglobulins/biotin, code no. E0453). The sections were then incubated with ABC complex (extravidin 1:100) for 30 minutes. The sections were washed in PBS and revealed by treatment with a 3´3-diaminobenzidine tetrahydrochloride (DAB) solution containing 0.1% hydrogen peroxide. The sections were counterstained with haematoxylin, and permanent preparations are made as usual for standard technique. As control procedure, the treatment with the primary antibody was omitted. Slices were observed under an AxioVision 2 Zeiss microscopy, and the images were captured using the AxioVision 2.05 system.
Macroscopically, the parotoid glands of Bufo ictericus are paired, elongated protuberances in post-orbital position (Fig. 1a). The parotoid glands are, on average, 3.5 ± 0.2cm in length, 1.5 ± 0.2cm in wide and 0.7 ± 0.2cm in thickness. On the external surface, numerous and slightly rounded concave depressions (0.7 ± 0.1mm in diameter) occur, containing elongated glandular openings (0.3-0.4mm) (Fig. 1b). The rounded depressions predominate on the parotoid medial surface, but they are absent on the lateral surface (Fig. 1a). In the middle of the rounded depressions, small pores are visible and these correspond to the duct openings of the larger granular glands.
Parotoid gland transverse sections reveal that the largest secretory units of exocrine glands are separated by connective tissue septa (Fig. 2). These glandular alveoli (4 ± 1mm in wide) occupy the middle portion of the parotoid gland and become smaller towards the periphery. Light microscopic analysis shows that the parotoid gland region is covered by an epidermis which is supported by the dermis. The epidermis shows similar features of the dorsal integument, i.e., the keratinocytes are organized in four to six cellular layers and they form sporadic conical projections that correspond to the cornified tubercles. In the epidermis, flask cells are also visualized (Fig. 3).
Just beneath the epidermis, there is a thin region in which the connective tissue is looser. This subepidermic region is separated from the spongious dermis by a pigment cell layer (Fig. 3). The spongious dermis of the parotoid gland region consists of loose connective tissue which contains small mixed glands (Figs. 3, 10).
Due to the collagenous fiber organization, an additional layer of irregular dense connective tissue is evidently present below the loose connective tissue. This region is characterized by coarse, thick, and intertwined bundles of collagenous fibers randomly organized (Fig. 4). This dermic region, reticular dermis, contains alveoli of smaller and larger granular glands. From the reticular dermis, septa of connective tissue containing blood vessels separate the larger glandular units (Fig. 8). Below the reticular dermis, there is a thin compact dermis. However, the collagenous fibers do not exhibit the criss-crossed arrangement.
In the integument of the parotoid gland region, the Eberth-Katschenko (EK) layer is evident through its typical basophilia (Fig. 5). Using the von Kossa method, calcium deposits are visualized in the EK-layer of the parotoid gland integument (Fig. 7).
The parotoid gland region contains three exocrine glandular types: mixed glands, small granular glands and large granular glands (Figs. 3, 4, 5, 8, 9). The glandular duct of the mixed and small granular glands is lined with bi-stratified cubic epithelium and the epidermal horny layer penetrates into the initial portion of the glandular duct. The duct of the larger granular glands is formed by stratified epithelium, because of the epithelial invagination during glandular development. Secretory portions of these three glandular types are surrounded by myoepithelial cells (Figs. 5, 6).
The mixed glands are located in the spongious dermis as single glands (Figs. 3, 4, 5). They can also be seen in small groups around the duct of the larger granular gland (Fig. 4). They are constituted by two cellular populations: mucous cells and serous cells. The mucous cells of the mixed glands are PAS- (Fig. 12) and AB-positive (Fig. 10). The serous cells are acidophilic and show no reaction to neither PAS nor AB methods (Figs. 10, 12).
Smaller granular glands are located in the outermost region of the reticular dermis. The secretory portion is made up of acidophilic cells and contains a homogeneous acidophilic granular intake (Figs. 5, 7, 9, 10). They are identified preferentially in the lateral region of the parotoid gland integument and are PAS- and AB-negative (Figs. 10, 11). Besides, the secretory cells constitute a syncytium.
The alveoli of the larger granular glands are macroscopically visualized (Fig. 2) and contain both basophilic and alcianophilic granular content (Fig. 11). Their secretory cells are represented by an acidophilic cellular syncytium with rounded nuclei at the basal region (Fig. 11). Large blood vessels are observed below the larger granular glands in a connective tissue layer which corresponds to the hypodermis (Fig. 2).
The parotoid gland of bufonids has received different designations. Firstly, the parotoid gland was designated as parotid gland; later it was named parotoid gland by Boulanger in order to differentiate it from the mammal salivary gland (Vital-Brazil and Vellard, 1925). However, diverse denotations are still observed, such as paratoid gland (de Assis et al., 1985), parotid gland (Santa Coloma et al., 1984; Fox, 1986), parotoid gland (Mahan and Biggers., 1977; Cannon and Hostetler, 1976; Pasquarelli et al., 1987; Duellman and Trueb, 1994; Hutchinson and Savitzky, 2004), and parotoid organ in Bufo (Delfino et al., 1999). In spite of the different designations, researchers agree that these glands represent an aggregate of granular glands in the post-orbital region (Cannon and Hostetler, 1976; Toledo et al., 1992; Duellman and Trueb, 1994; Hutchinson and Savitzky, 2004). In the present work, the term parotoid gland is used to designate large elevations on both sides of the toad body in post-orbital position.
In Bufo ictericus, the parotoid gland epidermis is similar to that of the dorsal integument as already described by de Brito-Gitirana and Azevedo (2005). Toledo et al. (1992) described rounded depressions on the parotoid gland surface as pores. In this work, we showed that the pores are located at the bottom of the rounded depressions. De Brito-Gitirana and Azevedo (2005) described rounded orifices on the dorsal and ventral integument, which corresponded to the openings of the glandular ducts. Thus, the pores in the bottom of the depressions are the excretory duct openings through which the milky venom is extruded.
In the spongious dermis of the anuran integument, two exocrine glands occur: mucous gland and granular (venom) gland (Porter, 1972; Delfino et al., 1987; Duell-man and Trueb, 1994; de Brito-Gitirana and Azevedo, 2005; Felsemburgh et al., 2006).
In B. ictericus, the present work shows that the parotoid gland is a specialized region in the integument in which an additional glandular type also occurs. The glandular units are well-developed and occupy the entire dermis. Their secretory portion can be macroscopically visualized, and is microscopically characterized by larger granular glands. Besides the larger granular glands in the parotoid gland of B. ictericus, mixed and smaller granular glands also occur, and they are visualized only through light microscopy.
Mixed glands are constituted by mucous and serous cells. According to the established histology which uses histochemical and structural parameters (Weiss and Greep, 1977; Kierszenbaum, 2002), mixed secretory units are made up of mucous cells and serous cells; they produce a seromucous secretion delivered into the same lumen. The cytoplasm of mucous cells is basophilic provided that they contain neutral glycoproteins and acid mucosubstances. The nucleus is flattened, and appears at the basal portion of the cell (Kiers-zenbaum, 2002). The serous cells have a round nucleus centrally placed and its cytoplasm is acidophilic.
In the B. ictericus parotoid gland, the small mixed glands have the same morphology as those distributed through the B. ictericus integument (de Brito-Gitirana and Azevedo, 2005). Although they are formed by two cell types, they are usually named mucous glands. Here, we prefer the designation mixed gland to emphasize this glandular type. In addition, the histochemical methods revealed that the end product of the mixed gland is enriched with proteins produced by serous cells and neutral glycoproteins as well as sulfated and carboxylated acid mucosubstances produced by mucous cells.
Toledo et al. (1992) pointed out that the secretion of granular glands is PAS-negative and AB-positive. However, they did not relate structural differences between the smaller granular gland and the larger granular gland. For Toledo et al. (1992), the granular glands produce an alcianophilic secretory product. Their results are in contrast to our histochemical data once the tinctorial affinities of the secretory intake of both glandular types are different, i.e., the smaller granular glands produce an acidophilic secretion but the larger granular glands have a predominant granular basophilic intake. Thus, the histochemical methods provide evidence that both granular glands differ in the chemical nature of their secretion.
Our results show that the mixed glands are also associated to the duct of the larger granular glands. So, the final secretory product is a mixture of secretions of both glandular types. Besides, we can not discard the fact that the secretory product of the mixed gland is important for the effectiveness of the venom released by the larger granular gland.
In B. ictericus, myoepithelial cells occur around the secretory portions of all gland types, and probably play a role in extrusion of secretory product. Mills and Prum (1984) and Delfino et al. (1987) related that myoepithelial cells are important in the structural organization of the glandular secretory portion.
In this work, the EK layer in the parotoid gland integument region was also visualized. Toledo et al. (1992) did not mention the EK layer in the parotoid gland integument of B. ictericus, but they commented that the EK layer occurs in Bufo cruficer, and it is absent in Bufo typhonicus. The water retention capacity of the EK layer is pointed out by several authors (Kobelt and Linsenmair, 1986; Toledo and Jared, 1993). Azevedo et al. (2006a) indicated that the entire spongious dermis is the cutaneous region responsible for the hydric reservoir in the B. ictericus integument. Their study was based on the localization of hyaluronic acid, a non-sulfated glycosaminoglycan with a water-binding quality.
In the dermis of the parotoid region, the collagenous fibers are visualized as irregularly oriented bundles. Since the collagenous fiber organization is different from that of the compact dermis of the B. ictericus bodyintegument (Azevedo et al., 2006b), we introduced the designation reticular dermis to characterize this organization of the connective tissue in this region.
The cutaneous secretory products of bufonids have been characterized, and different compounds are described and divided into four main categories: biogenic amines, bufodienolides (bufogenines and bufotoxins), alkaloids, peptides and proteins (Daly et al., 1987; Toledo and Jared, 1995; Erspamer, 1994). Due to the structural glandular arrangement of the parotoid gland, its secretory product represents the cutaneous secretions elaborated by the smaller and the larger glandular glands as well as by the mixed glands. The mucous secretion participates in the animal defense due to its noxious or toxic compounds (Phisalix, 1923; Sawaya, 1940; Noble and Noble, 1944). Clarke (1997) and Fontana et al. (2006) suggested that mucus possesses a bacteriostatic effect, and it represents a potential mechanical trap for microbial and fungal pathogens. Eggert-Kruse et al. (2000) demonstrated that the human cervical mucus has a considerable antimicrobial activity. Many antimicrobial compounds have been isolated from mucous secretions of different tissues/organs (Brogden, 2005). Hutchinson and Savitzky (2004) commented that the large vessels provide the precursor molecules necessary to formation of toxins contained in the secretion of the parotoid gland. In B. ictericus, large vessels occur in the hypodermis of the parotoid gland region.
This work showed that the parotoid gland is a specialized region of the integument, in which three gland types occur: the mixed, the smaller granular, and the larger granular glands. The larger granular glands are the main glandular component, and are responsible for the macroscopic protuberances known as parotoid gland. Thus, the end product released by the parotoid gland, know as venom, represents a mix of secretions which are elaborated by these three glandular types.
Received: 1 September 2006.
Accepted: 11 June 2007.
Azevedo RA, Carvalho HF, de Brito-Gitirana L. 2006a. Hyaluronan in the epidermal and the dermal extracellular matrix: its role in cutaneous hydric balance and integrity of anuran integument. Micron doi: dx.doi.org/10.1016/j.micron.2006.09.008.
Azevedo RA, Santana ASJ, de Brito-Gitirana L. 2006b. Dermal collagen organization in Bufo ictericus and in Rana catesbeiana integument (Anuran, Amphibian) under the evaluation of laser confocal microscopy. Micron 37 (3): 223-228.
Barbeau TR, Lillywhite HB. 2005. Body wiping behaviors associated with cutaneous lipids in hylid tree frogs of Florida. The Journal of Experimental Biology 208: 2147-2156.
Blaylock LA, Ruibal R, Platt-Aloia K. 1976. Skin structure and wiping behavior of Phyllomedusine frogs. Copeia 2: 283-295.
Brizzi R, Delfino G, Pellegrini R. 2002. Specialized mucous glands and their possible adaptive role in the males of some species of Rana (Amphibia, Anura). Journal of Morphology 254: 328-341.
Brogden KA. 2005. Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? Nature 3: 238-250.
Cannon MS, Hostetler JR. 1976. The anatomy of the parotoid gland in Bufonidae with some histochemical findings. Journal of Morphology 148: 137-157.
Clarke BT. 1997. The natural history of amphibian skin secretions, their normal functioning and potential medical applications. Biologial Review 72: 365-379.
Croce G, Gigliolo N, Bolognani L. 1973. Antimicrobial activity in the skin secretions of Bombina variedata pachypus. Toxicon 11: 99-100.
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 amphibian. Toxicon 25: 1023-1095.
de Assis AI, Barbosa JE, de Carvalho IF. 1985. Anticomplementary fraction from the poisonous secretion of the paratoid gland of the toad (Bufo marinus paracnemis Lutz). Experientia 41(7): 940-942.
de Brito-Gitirana L. 2004. Histologia: Conceitos básicos dos tecidos. São Paulo: Editora Atheneu: 21-44.
de Brito-Gitirana L, Azevedo RA. 2005. Morphology of Bufo ictericus integument (Amphibia, Bufonidae). Micron 36 (6): 532-538.
Delfino G, Brizzi R, Calloni C. 1987. Differentiation of myoepithelial cells during the development of cutaneous serous glands in Anura. Zoologische Anzeiger 218: 219-236.
Delfino G, Brizzi Alvarez BB, 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.
Duellman WR, Trueb L. 1994. Biology of Amphibians. Baltimore. The Johns Hopkins University Press Ltd. 610.
Eggert-Kruse W, Botz I, Pohl S, Rohr G, Strowitzki T. 2000. Antimicrobial activity of human cervical mucus. Human Reproduction 15: 778-784.
Erspamer V. 1994. Bioactive secretions of the amphibian integument. In: Heatwole G, Barthalmus T and Heatwole AY. Amphibian Biology. Surrey Beatty & Sons, Chipping Norton, 178-350.
Felsemburgh FA, Carvalho-e-Silva SP, de Brito-Gitirana L. 2006. Morphological characterization of the anuran integument of the Proceratophrys and Odontophrynus genera (Amphibia, Anura, Leptodactylidae) Micron, doi: dx.doi.org/10.1016/j.micron.2006.06.015.
Fontana MF, Ask KA, Macdonald RJ, Carnes AM, Staub NL. 2006. Loss of traditional mucous glands and presence of a novel mucus-producing granular gland in the plethodontid salamander Ensatina eschscholtzii. Biological Journal of the Linnean Society 87: 469-477.
Fox H. 1986. Dermis In: Bereiter-Hahn J, Matolsy AG, Richards S, eds., Biology of the integument. 2. Vertebrates. Berlin, New York: Springer-Verlag: 111-149.
Hutchinson DA, Savitzky AH. 2004. Vasculature of the parotoid glands of four species of toads (bufonidae: bufo). Journal of Morphology 260: 247-254.
Kiernan JA. 1990. Histological and Histochemical Methods - Theory and Practice. 2nd ed., Frankfurt, Pergamon Press: 172-177.
Kierszenbaum AL. 2002. Histology and cell biology. An introduction to pathology. Mosby, St Louis, Missouri: 55.
Kobelt F, Linsenmair KE. 1986. Adaptations of the reed frog Hyperolius viridiflavus (Amphibia, Anura, Hyperoliidae) to its arid environment. The skin of Hyperolius viridiflavus nitidulus in wet and dry season conditions. Oecologia (Berlin) 68: 533-541.
Lillie RD, Fullmer HM. 1976. Histopathologic Technique and Practical Histochemistry. 4th ed., New York: Mac Graw-Hill Book Co.: 208-701.
Lillywhite HB, Mittal AK, Garg TK, Agrawal N. 1997. Integumentary structure and its relationship to wiping behavior in the common Indian tree frog, Polypedates maculates. Journal of Zoology 243: 675-687.
Maciel NM, Schwartz CA, Pires Júnior OR, Sebben A, Castro MS, Sousa MV, Fontes W, Schwartz ENF. 2003. Composition of indolealkylamines of Bufo rubescens cutaneous secretions compared to six other Brazilian bufonids with phylogenetic implications. Comparative Biology and Physiology Part B 134: 641-649.
Mahan JT, Biggers CJ. 1977. Electrophoretic investigation of blood and parotoid venom proteins in Bufo americanus americanus and Bufo woodhousei fowleri. Comparative Biochemistry and Physiology C 57: 121-126.
Mills JW, Prum BE. 1984. Morphology of the exocrine glands of the frog skin. American Journal of Anatomy 171: 91-106.
Mowry RW. 1963. The special value of methods that color both acidic and vicinal hydroxyl groups in the histochemical study of mucins. With revised directions for the coloidal iron stain, the use of Alcian Blue G8X and their combinations with the periodic acid-Schiff reaction. Annals of New York Academy of Sciences 106: 402-423.
Noble GA, Noble ER. 1944. On the histology of frog skin glands. Transactions American Microscopic Society 63: 254-263.
Pasquarelli P, Mendes EG, Sawaya P. 1987. The action of parotoid venom on the heart of the toad (Bufo ictericus ictericus Spix 1824) and its effects on the inhibition caused by vagal stimulation. Comparative Biochemistry and Physiology C 87: 393-399.
Phisalix M. 1923. Le venin cutané muqueux du Bombinator pachypus Fitzs. var. brevipes Blasius. Bulletin du Muséum d’Histoire Naturelle 29: 40-44.
Porter KR. 1972. Herpetology. V.B. Saunders Company, United States of America: 18-25.
Pough FH, Andrews RM, Cadle JE, Crump ML, Savitzky AH, Wells KD. 2003. Herpetology. New Jersey, Prentice Hall: 158-159.
Prophet EB, Mills B, Arrington JB, Sobin LH.1994. Laboratory methods in histotechnology. Washington DC, Armed Forces Institute of Pathology: 159-197.
Sakate M, Lucas de Oliveira PC. 2000. Toad envenoming in dogs: effects and treatment. Journal of Venom Animal Toxins 6: 46-58.
Santa Coloma TA, Garrafo HM, Pignataro OP, Charreau EH, Gros EG. 1984. Biosynthesis of bufadienolides in toads. V. The origin of the cholesterol used by toad parotid glands for biosynthesis of bufadienolides. Steroids 44: 11-22.
Sawaya P. 1940. Sobre o veneno das glândulas cutâneas, a secreção e o coração de Siphonops annulatus. Boletim da Faculdade de Filosofia e Ciências da Universidade de São Paulo. Series Zoológica 4: 207-270.
Toledo RC, Jared C. 1993. A Contribution to the study of the cutaneous calcified layer in the Anurans. Revista Brasileira de Biologia 53: 619-624.
Toledo RC, Jared C. 1995. Cutaneous granular glands and amphibian venoms. Comparative Biochemistry and Physiology 111A: 1-29.
Toledo RC, Jared C, Brunner Jr A. 1992. Morphology of the large granular alveoli of the parotoid glands in toad (Bufo ictericus) before and after compression. Toxicon 30: 745-753.
Vital-Brazil O, Vellard J. 1925. Contribuíção ao estudo do veneno de batráchios do gênero Bufo. Brazil-Médico Rio de Janeiro 39: 175-180.
Warburg MR, Rosenberg M, Roberts JR, Heatwole H. 2000. Cutaneous glands in the Australian hylid Litoria caerulea (Amphibia, Hylidae). Anatomy and Embryology 201: 341-348.
Weiss L, Greep RO. 1977. Histology. United States of American: McGraw-Hill Book Company: 140-144.
Young JZ. 1985. La vida de los vertebrados. Ediciones Omega S.A. Barcelona.