Ladies in armor: A micro-computed tomographic study of skin calcification in European toads (genus Bufo)

Micro-computed tomography is a powerful tool toward the detailed reconstruction of internal and external morphology, in particular for ossified and other dense tissues. Here, we document and compare the level of calcification in the skin of the head and the parotoids (the external skin glands) in males and females of common and spined toads, Bufo bufo and B. spinosus. In some anurans, including Bufo species, a specific acellular calcified tissue layer within the dermis has been documented (the Eberth-Katschenko, or EK-layer). By a combination of micro-computed tomography and classical histology, we detected additional calcium deposits located in the dermal layer stratum spongiosum, positioned above the EK-layer. We showed that the level of calcification and the presence of additional calcium deposits are size and sex related, increasing in the order B. bufo males, B. spinosus males, B. bufo females to B. spinosus females. The last of these groups is the least variable. Bufo spinosus females have dense calcium deposits in the parotoids and the dorsal and ventral skin. Three-dimensional volume renderings and cross-sectional slices obtained by micro-CT scanning indicate that this approach is a promising technique for further studies on bufonid skin anatomy and geographic variation in skin calcification.