Structural insights into Trichuris muris eggs through 3D modeling, Cryo-SEM, and TEM of samples prepared with HPF-FS

Trichuriasis - a disease caused by Trichuris trichiura - affects underserved communities. Infection occurs by the ingestion of embryonated eggs, a resilient structure against environmental fluctuations - an essential feature for the survival and transmission of trichurids. This study aims to enhance our comprehension of the trichurid eggs by providing a characterization of the ultrastructure of eggshell and first-stage (L1) larvae of T. muris, a key experimental model for trichuriasis. We employed the following microscopy techniques: light, fluorescence, confocal, Cryo-SEM, and Transmission electron microscopy (TEM), analyzing unfixed, chemical and cryofixed samples. Light microscopy revealed the structure of the eggshell, consisting of three main layers: Pellicula ovi (PO), chitinous layer (CHI), and the electron-dense parietal coating (EdPC). Fluorescence microscopy showed the calcein's high affinity for the eggshell and polar plugs, while the DAPI distinctly stained the L1 larval cells. Using confocal microscopy and 3D modeling, we quantified an average of 151 larval cells. TEM of high-pressure frozen and freeze-substituted samples revealed that the PO and the EdPC layers lacked sublayers, while the CHI layer was composed of 12-14 sublayers. The CHI also contained continuous distinct organization structure forming the polar plugs. The combination of different sample fixation methods and advanced imaging techniques was crucial for revealing structural details of both the eggshell and L1 larva, including the arrangement of cells, cuticle, and an anterior pointed structure. These findings provide deeper insights into the structural biology of T. muris and offer valuable information for advancing parasite control strategies in both human and veterinary context.