Histological Study of Glandular Variability in the Skin of the Natterjack Toad- Epidalea calamita (Laurenti, 1768)-Used in Spanish Historical Ethnoveterinary Medicine and Ethnomedicine

Abstract

Common toads have been used since ancient times for remedies and thus constitute excellent biological material for pharmacological and natural product research. According to the results of a previous analysis of the therapeutic use of amphibians in Spain, we decided to carry out a histological study that provides a complementary view of their ethnopharmacology, through the natterjack toad (Epidalea calamita). This species possesses a characteristic integument, where the parotoid glands stand out, and it has been used in different ethnoveterinary and ethnomedical practices. This histological study of their glandular variability allow us to understand the stages through which the animal synthesises and stores a heterogeneous glandular content according to the areas of the body and the functional moment of the glands. To study tegumentary cytology, a high-resolution, plastic embedding, semi-thin (1 micron) section method was applied. Up to 20 skin patches sampled from the dorsal and ventral sides were processed from the two adult specimens collected, which were roadkill. Serous/venom glands display a genetic and biochemical complexity, leading to a cocktail that remains stored (and perhaps changes over time) until extrusion, but mucous glands, working continuously to produce a surface protection layer, also produce a set of active protein (and other) substances that dissolve into mucous material, making a biologically active covering. This study provides a better understanding of the use of traditional remedies in ethnoveterinary medicine.

Keywords: Epidalea calamita; amphibian drug discovery; epistemological approach; ethnoveterinary medicine; histology; skin; zootherapy.

Figure 1

The images show the great variability in colour of the back of the natterjack toad that can also have a clear and light, narrow mid-dorsal line (photos by J.R. Vallejo).

Figure 2

Characteristic aspect of dorsal and ventral tegumentary surfaces and pigmentary pattern. Ventral skin (inset) with smaller cystic serous glands, less relief, and lighter pigment density than on the back, allowing a better binocular observation of the light-reflecting pattern and individual melanophores on a surface view (photos by J.M. López-Cepero).

Figure 3

Semi-thin sections of tegument epithelium and mucous glands. Toluidine-blue stained. Surface epithelium and pigmentary layers architecture (a); iridophores (b) of the previous section (arrowheads) under polarised light. Common (c) and large (d) mucous glands and morphology of the secretory granules (e). Near-adjacent sections (f,g) of the smaller glands show patchy clonal (homogeneous type) cell areas in the adenomere but they still contain every type of secretory granule (cell types) (g,h,i) (for legend references, see the text) (photos by J.M. López-Cepero).

Figure 4

Large adenomeres contain all cell types and differentiation stages (see text) (1 µm section, TolBlue) (a). Only a subset of mucous cells (b) are glucoprotein PAS+ secreting cells, whereas adjacent ones are PAS- (c) (2 µm sections). All PAS+ cells are caliciform-type cells (d) interspersed between other cell types (e), storing secretion in their apical pole, and immature basal cells with PAS+ secretory granules (inset). Low power (f) serous (venom glands), (2 µm section, iron haematoxylin) with a peripheral syncytium layer devoid of granules (g). Keratinised lumens of the secretory ducts (h) maintain a closed compartment under pressure. Granule staining showing similar aspects (i) and size distributions with iron haematoxylin, PAS, and toluidine blue methods, revealing their predominant protein nature (in sections) (photos by J.M. López-Cepero).