Skin Gland Morphology and Secretory Peptides in Naturalized Litoria Species in New Zealand
The defensive secretions of anurans contain a large array of chemical compounds that are synthesized in the granular glands and released onto the skin. We used histological and peptidomic analyses to investigate the skin glands and their products in Litoria aurea, Litoria ewingii, and Litoria raniformis, which were introduced to New Zealand from Australia approximately 150 yr ago. The skin glands were induced to release their product by either norepinephrine or electric stimulation. Granular glands in all three species are distributed evenly in both dorsal and ventral skin and share morphological features common in other anurans, such as a contractile myoepithelium that surrounds the syncytial secretory unit. However, differences are observed in the granular ultrastructure between L. ewingii and the more closely related L. aurea and L. raniformis. The latter have larger glands with granules that are opaque and contain homogeneously spaced, diaphanous vesicles, whereas the substructure of the granules in L. ewingii is homogeneous and consists of miniscule vesicles that are either electron opaque or diaphanous. Comparatively large mucous glands in the small-bodied L. ewingii may be attributed to increased mucous requirements due to differences in microhabitat use. Nanospray mass spectrometric analyses confirmed the presence of several unidentified peptides, as well as 11 peptides described previously. Both exposure to norepinephrine and mild electric stimulation of the skin triggered the bulk discharge of gland contents. We discuss potential functional specializations of gland structure and peptide content as mechanisms for predator or pathogen defense.Abstract
(A) Cross-sections of Litoria ewingii dorsal skin showing structural aspects: e, epidermis; G, granular gland; M, full mucous gland; M'; empty mucous gland; mec, myoepithelial cells; Black arrow pointing toward mucocytes surrounding the mucous glands. (B) Granular gland of L. ewingii releasing secretory granules through the duct–intercalary tract unit (arrowhead) onto the surface of the skin. (C) Granular glands in L. aurea showing various stages of regeneration after treatment with norepinephrine, ranging from full (G) to partially regnerated (G') to empty (G”). Notice the contracted myoepithelial cells (mec). (D) Mucous gland of L. raniformis releasing mucous through the duct (arrowhead).
Ultrastructural features of gland periphery in Litoria ewingii. (A) Mucous glands are lined with mucocytes and release material (arrow) into the lumen (L). Notice vesicles of various electron densities in the mucocytes (arrowheads). (B) Secretory syncytium with biosynthesis apparatus of granular gland. Notice large amounts of rough endoplasmatic reticulum (rer) surrounding an interstitial nucleus (n). Peripheral granules (g) are relatively electron-diaphanous with a granular substructure.
Ultrastructural features of granules. (A) Granule–cytoplasm interactions in Litoria raniformis. Exchange of material between granules of varying electron density containing diaphanous vesicles (thin arrow). Distinct gaps in the cytoplasm surrounding granules form perigranular compartments (thick arrow). (B) Potential condensational stages and merging processes of heterogenous granules in L. aurea. Exchange of material (arrows) between granules and cytoplasm (arrowhead) at gland periphery. Myoepithelial cells, mec; rough endoplasmic reticulum, rer; nucleus, n.(C) Maturing granule of L. ewingii in the secretory syncytium showing two phases of condensational stages. An aggregation of electron-diaphanous, seemingly membrane-bound vesicles is surrounded by a layer of dense cortex (double arrow). (D) Granule (g) of L. raniformis with repeating, concentric-circular substructure. Notice compartmentalization of cytoplasm (arrowhead). (E) Interactions between granules of L. ewingii (arrowheads). Cytoplasmic processes extending out from granule periphery (white arrow). (F) Condensing serous material in L. raniformis. Granule in gland periphery undergoing maturation in proximity to rough endoplasmic reticulum (rer). The forming granule has a heterogenous outline and tubule-like spaces in the center (arrow).
Discharge of secretory material from excised dorsal skin after mild electric stimulation (A, Litoria ewingii) or exposure to norepinephrine (B, L. aurea).
Appropriate sections from MALDI-MS spectra of (A) Litoria aurea, (B) L. ewingii, and (C) L. raniformis. Peaks are labeled with the corresponding peptides and masses.
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