In vivo double labeling of putative neuronal and excretory structures in Echinococcus granulosus protoscoleces and associated membranes obtained by a lipophilic dye

Abstract

Echinococcus granulosus is a platyhelminth parasite and the causative agent of cystic echinococcosis, a neglected zoonotic disease affecting humans and animals worldwide. In this study, lipophilic tracing techniques were applied to live protoscoleces and associated membranes to evaluate whether neuronal structures could be visualized in vivo. Incubation with the lipophilic tracer DiI produced a staining pattern consistent with both excretory structures and putative neuronal elements. Although DiI is commonly used as a neuronal tracer, it reproducibly labeled a branched tubular structure resembling the excretory system of live protoscoleces. Notably, staining revealed a continuous connection between the protoscolex excretory system and the excretory network within the brood capsule, suggesting anatomical continuity between these structures. In addition, putative neuronal cell bodies and projections were observed mainly in the scolex region, and some putative multipolar neuronal cells were detected in brood capsules. Close spatial localization between neuronal projections and the terminal regions of the excretory canals was also observed. These staining patterns were absent in dead protoscoleces, indicating that DiI can function as a vital stain under these conditions. In contrast, resazurin produced a different staining pattern and did not label defined internal membranous structures. Although DiI fluorescence was retained after paraformaldehyde fixation, the signal did not withstand the permeabilization and antibody-incubation procedures required for immunohistochemistry. This study represents the first application of a neuronal tracer in live cestode larvae and provides a novel approach for visualizing putative neuronal and excretory structures, potentially aiding the identification of new targets for cystic echinococcosis.