Morphological and Histochemical Analysis of Amanita muscaria (L.) Lam. (Agaricales, Basidiomycota) Using Scanning Electron Microscopy

Abstract

Amanita muscaria (L.) Lam. 1783 is a macroscopic mushroom that belongs to the group of basidiomycetes; this group of fungi produces basidiospores in structures called basidia. There are some varieties, mainly described to distinguish the different color forms of the cap, such as A. muscaria var. muscaria (L.: Fr.) Hooker (red cap, white stem and warts), A. muscaria var. alba Peck (cap, warts, and stem from white to tan), A. muscaria var. flavivolvata (Singer) Jenkins (cap from orange to red, warts yellowish tan, stem from white to cream), A. muscaria var. formosa (Pers.: Fr.) Bertillon in DeChambre (cap from orange to yellow, warts and stem yellowish to tan), A. muscaria var. persicina Jenkins (melon-colored cap, warts from tan to yellowish), and A. muscaria var. regalis (Fr.) Bertillon in DeChambre (brown cap, warts from cinnamon to yellowish) [1]. Longitudinal sections were made of the lamellae and scales of fresh specimens of A. muscaria, collected from August to October 2025 in the Pinus-Quercus forest of Rancho Santa Elena, in the municipality of Huasca de Ocampo, Hidalgo State, Mexico. Sections were obtained from dehydrated specimens; the obtained sections were placed on slides that had previously been fitted with copper tape with adhesive on both sides. Once the samples were prepared, they underwent a process of coating with a gold film using the sputtering method. The coated samples were placed in the scanning electron microscope for the observation of cellular structures, shape, size, ornamentation, wall thickness, and texture. For the histochemical analysis of the specimens, longitudinal sections of the mushroom plates were made and placed in a 0.04% bromophenol blue solution for 10 minutes. Afterward, three washes with distilled water were performed to remove the excess dye. Finally, the structures were differentiated with a 10% acetic acid solution and observed under an optical microscope to identify the structures stained from blue to reddish-purple. The anatomical regions stained in these colorations show peptides, which for this genus would correspond to deposits of toxic peptide macrocyles such as amanitins. Results showed that the taxonomy of the samples included Agaricomycetes, Agaricales, and Amanitaceae. Amanita muscaria is characterized by the presence of remnants of the universal veil on the surface of the pileus, forming pyramidal scales with a texture ranging from inflata to intricate with irregular cells, elongated hyphae that swell, with thin and smooth walls. In SEM images, they appear collapsed. This is the first work that shows the shape and arrangement of this anatomical region of the mushroom. In the hymenium region, globose cells with thickened and smooth walls are observed; these cells will give rise to basidia and basidiospores. These cells are surrounded by an abundant gelatinous extracellular matrix, and the subhymenium is formed by globose cells with thin, smooth walls. The hymenophoral trama is formed by elongated intertwined hyphae with globose cells. The genus Amanita is one of the most macroscopically prominent and taxonomically diverse groups of fungi worldwide, consisting of hundreds of species with a wide variety of forms, ecological tendencies, and both macroscopic and microscopic morphologies. Although Amanita is best known for emblematic species such as Amanita muscaria (the “fly agaric”) and Amanita phalloides (the death cap), in Mexico this genus also represents an important ecological component of temperate and tropical forests, associated with ectomycorrhizal communities of pines, oaks, and other conifers. [2]. Amanita muscaria is an ectomycorrhizal species of medical importance. The presence of toxic metabolites of a peptidic nature contained in the cells of the gills, in the hymenium region, was identified. It was found that the scales are formed by elongated hyphae interspersed with globose cells. In the hymenium, globose cells with thickened walls surrounded by a gelatinous matrix were identified. Additionally, the subhymenium is formed by larger globose cells with thin and smooth walls, in contrast to the hymenophoral trama, which contains elongated hyphae interspersed with globose cells. Scanning electron microscopy of Amanita muscaria (L.) Lam. A-B, general view of the gill section. C, Detail of the hymenium cells. D, Detail of the subhymenium cells. Scanning electron microscopy of Amanita muscaria (L.) Lam. A, General view of a pileus scale. B, Detail of the hymenophoral trama cells. C, Detail of the pileus scale cells. Histochemistry of Amanita muscaria (L.) Lam. A, Hymenophoral tissue with a positive reaction to bromophenol blue. B, Hymenium with a positive reaction to bromophenol blue. C, Set of subhymenal cells with a positive reaction to bromophenol blue.