Caracterização morfológica, ultraestrutural e modelagem 3D de ovos embrionados de Trichuris muris

Abstract

Soil-transmitted helminthiasis are neglected diseases developed by nematodes that depend on the soil for their complete development. According to WHO data (2018), it is estimated that in the world, 1.5 billion people are infected by soil-transmitted helminths. These are diseases associated with social and climatic characteristics, mainly affecting school-age children. Data from the last national prevalence inquiry of schistosomiasis and Soil-transmitted helminthiasis show a proportion of 5.41% positivity for trichuriasis in Brazil. Trichuriasis is a parasitosis that affects several mammals, having an impact on human and veterinary medicine. The species Trichuris muris is used as an experimental model for trichuriasis, the life cycle begins with the ingestion of embryonated eggs by the host. This egg presents three layers identified by light microscopy. The aim of this work is the ultrastructural characterization of the embryonated egg and the L1 larva of Trichuris muris fixed by high-pressure freezing followed by freeze-substitution and analyzed by electron microscopy. Chemically fixed samples were stained with calcein and DAPI for fluorescence microscopy, confocal and super-resolution microscopy resolution experiments. The results obtained by light microscopy (LM) showed the three layers forming the trilaminar egg shell (vitelline, chitin and lipid layers). The larva esophagus and germ cells concentrated in the posterior region of the larva was also observed. Based on fluorescence microscopy, the Calcein showed high affinity for the eggshell and of the polar plug, while DAPI allowed the characterization of the L1 larvae cells. Through the series of images obtained by confocal, we performed the reconstruction and modeling of the germ cells, which allowed to count 151 cells on average per larvae. Using TEM, it was possible to characterize the sublayers of the eggshell, showing that the vitelline layer is present as a thin outer layer, while the chitin layer is a dense structure. Lipid layer is the most internal and presents high electron density, linear for the entire length of the egg, with 14 sublayers. The plugs are present as a continuation of the chitin layer, with a distinct conformation, besides presenting more internally. It was concluded that the physical fixation and the use of the different microscopy techniques were important to characterize in detail the morphology and morphometry of the different layers of the eggshell, besides of the characterization of the structural and morphological details, and the germ cells in the L1 larva inside of the T. muris eggs.