Chemical composition of calcareous corpuscles in Echinococcus multilocularis protoscoleces

To systematically identify and characterize the elemental makeup and crystalline phase of the calcareous corpuscles within Echinococcus multilocularis (E. multilocularis) protoscoleces, and to infer their putative biomineralization pathway-thereby laying a mechanistic foundation for clarifying their biological role and harnessing them in diagnostics. Protoscoleces were harvested from Mongolian gerbils with established peritoneal infections. After corpuscle isolation, an integrated analytical suite-combining scanning electron microscopy with energy-dispersive X-ray spectroscopy, transmission electron microscopy (TEM), Raman micro-spectroscopy, X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD)-was employed to interrogate morphology, ultrastructure, elemental composition, valence states and crystallographic identity. Abundant ellipsoidal corpuscles displaying concentric laminations were observed. EDS revealed Ca, C and O as dominant elements with trace Mg and P. Raman spectroscopy exhibited an intense band at ∼1086.7 cm^-1, consistent with the ν1 symmetric stretch of carbonate (CO₃^2-), whereas the characteristic phosphate ν1 mode at ∼960 cm^-1 was absent. XPS corroborated carbonate-associated C 1s (∼288 eV) and Ca 2p peaks, while XRD exclusively indexed reflections attributable to calcite (CaCO₃) without evidence of a stable calcium-phosphate phase. Collectively, the corpuscles may be composed primarily of calcitic CaCO₃ with minimal incorporation of Mg and P. The calcareous corpuscles of E. multilocularis protoscoleces are predominantly calcite-type CaCO₃ that contains only trace Mg and P without forming a discrete calcium-phosphate phase. Cross-validated multimodal data not only establish the mineralogical identity of these inclusions but also point toward an "organic matrix-enzyme-mediated amorphous calcium carbonate (ACC) crystallization" mechanism. These findings provide an indispensable compositional fingerprint for non-destructive diagnostics and open new avenues for functional dissection and targeted intervention against alveolar echinococcosis.