Differential proteomic analysis of laser-microdissected penetration glands of avian schistosome cercariae with a focus on proteins involved in host invasion

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

Functional ultrastructure of eggs and cellular organization of hexacanths of the cyclophyllidean cestode Thysanotaenia congolensis: a phylogenetic implication of obtained results

SUMMARYThe functional ultrastructure of eggs and cellular organization of hexacanths from gravid proglottids of Thysanotaenia congolensis, from black rats from Cape Verde, were examined by transmission electron microscopy. Mature eggs with fully formed hexacanths are grouped within parenchymatous capsules of gravid proglottids. Oncospheral envelopes surrounding mature hexacanths are reduced to a very thin membranous embryophore as their protective function is taken over by the parenchymatous capsules originating from the medullary parenchyma of immature proglottids and composed of three layers. Six major cell types are present: a bi-nucleate medullary centre; a six-nucleate U-shaped penetration gland; a second type of penetration gland; two neurosecretory-type nerve cells; about 30 somatic cells; and about 12 germinative cells. Present results on the functional ultrastructure of eggs and cellular organization of hexacanths support the phylogenetic distinction between T. congolensis and cestodes of the subfamily Anoplocephalinae.

Oncospheral Penetration Glands and Secretory Blebs Are the Sources of Taenia ovis Vaccine Antigens

ABSTRACT Taenia ovis is a cestode parasite infecting primarily sheep as intermediate hosts and dogs as definitive hosts. The first highly effective, recombinant vaccine against a parasitic organism was developed against T. ovis infection in sheep. Three separate host-protective antigens (To16, To18, and To45W) have been cloned from the oncosphere of the parasite. We localize these antigens in the oncosphere by using quantitative immunogold labeling and transmission electron microscopy. The three antigens were uniquely associated with penetration gland cells. The cytoplasm and secretory granules of both penetration gland type 1 and type 2 cells exhibited statistically significant levels of staining for each of the three antigens. The intensity of labeling of the penetration gland type 1 cell was approximately three to five times greater (P < 0.01) compared to the level of staining intensity seen in the penetration gland type 2 cell. In activated oncospheres, secretory blebs were found to contain granules with a structure similar to those observed in the penetration gland cells. The granules within the secretory blebs were shown to stain specifically for the presence of each of the three host-protective antigens. The absence of surface location of the T. ovis antigens suggests that the parasite may not be susceptible to vaccine-induced antibody- and complement-mediated attack until some postoncospheral development has occurred after infection of the intermediate host.

The miracidium of Transversotrema patialense (Soparkar, 1924)

The miracidium of Transversotrema patialense (Digenea: Transversotrematidae) has 21 epidermal cells arranged in four tiers with an epidermal formula of 6, 9, 4, 2. The penetration gland is large, conspicuous, contains a posterior globular part with 6 prominent nuclei and opens out through 6 pores arranged in two lateral groups at the tip of the apical papilla. The miracidium lacks lateral papillae, possesses a well developed subepithelium, a pair of flame cells and a germ ball.

Ultrastructure of the tegument and penetration glands of developing procercoids of Haplobothrium globuliforme Cooper, 1914 (Cestoda: Haplobothrioidea)

Developing procercoids of Haplobothrium globuliforme were examined using transmission electron microscopy. Procercoids require 12–20 days, at approximately 20 °C, to develop to the infective stage. Six-day-old procercoids have a microvillar tegument and numerous undifferentiated subtegumental cells. In 9-day-old procercoids the cercomer is visible as a distinct appendage at the posterior end. Developing microtriches are evident on the tegument of the larval body. These are long and slender, and bear small electron-dense tips. No degenerating microvilli were evident and the microtriches were never seen emerging de novo from the tegument. Fully developed procercoids, 15 days postinfection, have both robust and slender microtriches at the anterior end of the larva, and only long slender microtriches over the remainder of the body. The cercomer retains a microvillar tegument until the larva is fully developed, at which time the tegumental projections on the anterior-most part of the cercomer bear small electron-dense tips. Penetration glands in the anterior part of the larva contain electron-dense secretory granules. The penetration gland ducts, which extend to the tegument, are lined with microtubules.

Schistosoma mansoni: the structure and elemental composition of pre-acetabular penetration gland cell secretion in pre-emergent cercariae

SUMMARYThe structure and elemental composition of the secretory products of the pre-acetabular penetration gland cells of pre-emergent cercariae of Schistosoma mansoni have been investigated at an ultrastructural level using X-ray probe microanalysis. The secretion contains 2 types of bodies, one homogeneous and one heterogeneous with electron-lucent areas. Using a variety of fixation and sectioning techniques it has been shown that the apparent electron density of the homogeneous bodies (Type B) is the result of interaction with osmium tetroxide and stain. The small electron-lucent areas of the other type of secretory body (Type A) represent regions where granules have been leached out by processing media. X-ray analysis of cercariae still within sporocysts has indicated that these granules are very rich in calcium, and it is suggested that the calcium is absorbed by the developing cercariae through the sporocyst wall from the molluscan haemolymph.

Hymenolepis nana: the fine structure of the ‘penetration gland’ and nerve cells within the oncosphere

SUMMARYThe fine structure of the oncosphere of Hymenolepis nana has been investigated by transmission and scanning electron microscopy, together with light microscope observations of JB–4 embedded material. The outer surface of the oncosphere is covered by an epithelial layer, termed the embryonic epithelium. Cell types present within the oncosphere include the penetration gland cell, oncoblast, or hook-forming cells, nerve cells, muscle cells (both somatic and hook), and undifferentiated ‘stem’ cells. The penetration gland is a large, U-shaped structure, situated in the anterior region of the oncosphere, and filled with secretory granules of 2 distinct morphological types. Histochemically, the secretory material yields reactions characteristic of an acid mucopolysaccharide. A proteinaceous-substance and small amounts of glycogen are also present. Up to 4 pairs of ducts from the penetration gland have been observed. They pass through the basal lamina and the epithelial layer to open against the polar filament layer at the anterior end of the oncosphere. Nerve cells are described in a cestode oncosphere for the first time. The cells are paraldehyde-fuchsin-positive and show a high level of secretory activity, as evidenced by the large numbers of dense-cored vesicles produced by the Golgi apparatus in the perikarya; consequently, they are tentatively regarded as possible neurosecretory cells. The vesicles are transported down the axon to be stored in specialized swollen axon terminals, which form definite junctions with the muscle cells.

Penetration gland secretion by hexacanths of Hymenolepis diminuta

The rate of expulsion of penetration gland material was determined from photomicrographs of Hymenolepis diminuta hexacanths stained with neutral red dye at intervals after hatching in vitro. Identical secretory rates were recorded for hexacanths incubated in either Tyrode's solution or Tyrode's solution plus an extract of the midgut of Tenebrio molitor. In both media the reduction in stained material observed in the glands after 135 min incubations was equivalent to that observed for hexacanths that had had opportunity to penetrate in vivo during the same time period. These results were interpreted as indicating that the in vivo secretory pattern was similar to that observed in vitro, and that chemical stimuli from the tissue extracts were not required to initiate secretion. Microdensitometric readings also demonstrated a quantitative decrease in dye within the glands as incubation time increased. Ultrastructural examination of the glands showed that their secretory inclusions were exported via ducts to the epithelial cytoplasm where they accumulated in discrete blebs enclosed by the surface membrane. These inclusion-filled, membrane-bounded blebs were apparently formed and released continuously until, after approximately 2 h, only a few inclusions remained in the penetration glands.

The metacercaria of a pleurolophocerca cercaria parasitizingPeringia ulvae(Pennant, 1777)

The metacercaria shown in Fig. 1 was obtained in the laboratory from Gobies (Gobius ruthensparriEuphras.) experimentally infected with a Pleurolophocerca cercaria fromPeringia ulvae. This cercaria is regarded as the most interesting of all the Heterophyid larvae infecting this snail. It has been referred to briefly in a previous publication (Rothschild, 1938). It differs from typical Opisthorchid cercariae in the following characteristics:(1) The lateral fin-folds are continuous and extend the whole length of the tail. The dorso-ventral fin-fold is reduced.(2) The oesophagus and intestinal caeca (in addition to the pharynx) can be clearly made out.(3) The cuticle is more heavily spined.(4) The ventral sucker is better developed.(5) The penetration gland ducts (fourteen in number) are not arranged in such definite bundles.(6) Three acicular boring spines are present in the oral sucker.In other respects the cercaria possesses the usual characters of the group.