Contributions to Zoology, 76 (3) – 2007Pablo G. de Almeida; Flavia A. Felsemburg; Rodrigo A. Azevedo; Lycia de Brito-Gitirana: Morphological re-evaluation of the parotoid glands of Bufo ictericus (Amphibia, Anura, Bufonidae)

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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.