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Schistosome invasive stages, cercariae, leave intermediate snail hosts, penetrate the skin of definitive hosts, and transform to schistosomula migrating to final localization. During invasion, cercariae employ histolytic and other bioactive products of specialized holocrine secretory cells – postacetabular (PA) and circumacetabular (CA) penetration glands. Although several studies attempted to characterize protein composition of the in vitro induced gland secretions in Schistosoma mansoni and Schistosoma japonicum, the results were inconsistent and dependent on the method of sample collection and processing. Products of both gland types mixed during their secretion did not allow localization of identified proteins to a particular gland. Here we compared proteomes of separately isolated cercarial gland cells of the avian schistosome Trichobilharzia szidati employing laser-assisted microdissection and shotgun LC-MS/MS, thus obtaining the largest dataset so far concerning the representation and localization of cercarial penetration gland proteins. We optimized the methods of sample processing with cercarial bodies (heads) first. Alizarin-pre-stained, chemically non-fixed samples provided optimal results of MS analyses, and enabled distinguishing PA and CA glands for microdissection. Using 7.5 × 106 μm3 sample volume per gland replicate, we identified 3347 peptides assigned to 792 proteins, from which 461 occurred in at least 2 of 3 replicates in either gland type (PA = 455, 40 exclusives; CA = 421, 6 exclusives; 60 proteins differed significantly in their abundance between the glands). Peptidases of 5 catalytic types accounted for ca. 8 % and 6 % of reliably identified proteins in PA and CA glands, respectively. Invadolysin, nardilysin, cathepsins B2 and L3, and elastase 2b orthologs were the major gland endopeptidases. Two cystatins and a serpin were highly abundant peptidase inhibitors in the glands. CA glands were rich in venom allergen-like proteins. The assembled total cercarial body proteome included 1631 identified proteins and revealed additional interesting factors possibly related to tissue invasion.HighlightsProteomes of two penetration gland types in schistosome cercariae greatly differPostacetabular glands possess 40 unique proteins and are abundant in hydrolasesCircumacetabular glands posses 6 unique proteins and are rich in VAL proteinsPeptidases make up 8 % of postacetabular and 6 % of circumacetabular gland proteinsCercarial elastase is unique to circumacetabular glands of Trichobilharzia szidatiNote: Supplementary data associated with this article All supplementary data files can be accessed from the following link: http://www.helminthology.cz/supplementary_files.html