The unusual cytoarchitecture of “vitelline follicles” in freshwater blood flukes of the genus Sanguinicola (Digenea, Aporocotylidae)

This is the first study assessing the cytoarchitecture of the vitellarium of members of the freshwater, teleost-infecting lineage of blood-flukes (Aporocotylidae). The vitelline cytoarchitecture of two innominate species of Sanguinicola from freshwater fishes in Russia showed that vitelline cells at different stages of maturation are widely distributed throughout much of the body and are mixed with other cell types. The latter feature indicates that use of the term “follicular vitellarium” is inappropriate for species of this genus. An additional characteristic of the vitelline cells in these Sanguinicola spp. is their ability to form long, pseudopodia-like extensions of the peripheral cytoplasm that contact neighbouring vitelline cells and sarcoplasmic extensions, forming both heterologous and homologous intercellular junctions. Within the vitelline duct lumen, the cytoplasm of mature vitelline cells is filled with regular clusters (0.5–1.0 μm in diameter), comprising 10–30 vitelline globules, which have heterogeneous contents and electron-lucent lipid droplets (1.1–1.7 μm in diameter), but no apparent modifications of vitelline globules occur within the vitelline duct. The flattened, ciliated, epithelial lining of the common vitelline duct contains intra-epithelial nuclei, its luminal surface bears shallow lamellae and adjacent cells are adjoined by apical septate junctions. All of these observations, when compared to the marine Aporocotyle simplex, likely represent additional characteristics supporting the divergent evolutionary lineages of marine and freshwater aporocotylids.

The repositioning of epigenetic probes/inhibitors identifies new anti-schistosomal lead compounds and chemotherapeutic targets

BackgroundPraziquantel represents the frontline chemotherapy used to treat schistosomiasis, a neglected tropical disease (NTD) caused by infection with macro-parasitic blood fluke schistosomes. While this drug is safe, its inability to kill all schistosome lifecycle stages within the human host often requires repeat treatments. This limitation, amongst others, has led to the search for novel anti-schistosome replacement or combinatorial chemotherapies. Here, we describe a repositioning strategy to assess the anthelmintic activity of epigenetic probes/inhibitors obtained from the Structural Genomics Consortium.Methodology/Principle findingsThirty-seven epigenetic probes/inhibitors targeting histone readers, writers and erasers were initially screened against Schistosoma mansoni schistosomula using the high-throughput Roboworm platform. At 10 µM, 14 of these 37 compounds (38%) negatively affected schistosomula motility and phenotype after 72 hours of continuous co-incubation. Subsequent dose-response titrations against schistosomula and adult worms revealed epigenetic probes targeting one reader (NVS-CECR2-1), one writer (LLY-507 and BAY-598) and one eraser (GSK-J4) to be particularly active. As LLY-507/BAY-598 (SMYD2 histone methyltransferase inhibitors) and GSK-J4 (a JMJD3 histone demethylase inhibitor) regulate an epigenetic process (protein methylation) known to be critical for schistosome development, further characterisation of these compounds/putative targets was performed. RNA interference (RNAi) of one putative LLY-507/BAY-598 S. mansoni target (Smp_000700) in adult worms replicated the compound-mediated motility and egg production defects. Furthermore, H3K36me2, a known product catalysed by SMYD2 activity, was also reduced by LLY-507 (25%), BAY-598 (23%) and siSmp_000700 (15%) treatment of adult worms. Oviposition and packaging of vitelline cells into in vitro laid eggs was also significantly affected by GSK-J4 (putative cell permeable prodrug inhibitor of Smp_034000), but not by the related structural analogue GSK-J1 (non-permeable inhibitor).Conclusion/SignificanceCollectively, these results provide further support for the development of next-generation drugs targeting schistosome epigenetic pathway components. In particular, the progression of histone methylation/demethylation modulators presents a tractable strategy for anti-schistosomal control.Author SummaryHuman schistosomiasis is caused by infection with parasitic blood fluke worms. Global control of this NTD is currently facilitated by administration of a single drug, praziquantel (PZQ). This mono-chemotherapeutic strategy of schistosomiasis control presents challenges as PZQ is not active against all human-dwelling schistosome lifecycle stages and the evolution of PZQ resistant parasites remains a theat. Therefore, new drugs to be used in combination with or in replacement of PZQ are urgently needed. Here, continuing our studies on Schistosoma mansoni epigenetic processes, we performed anthelmintic screening of 37 epigenetic probes/epigenetic inhibitors obtained from the Structural Genomics Consortium (SGC). The results of these studies highlighted that schistosome methylation/demethylation processes are acutely vulnerable. In particular, compounds affecting schistosome SMYD (LLY-507, BAY-598) or JMJD (GSK-J4) homologues are especially active on schistosomula and adult worms during in vitro phenotypic drug screens. The active epigenetic probes identified here as well as their corresponding S. mansoni protein targets offers new starting points for the development of next-generation anti-schistosomals.

Differential gene expression, including Sjfs800, in Schistosoma japonicum femalesbefore, during, and after male-female pairing

Schistosomiasis is a prevalent but neglected tropical disease caused by parasitic trematodes of the genus Schistosoma, with the primary disease-causing species being S. haematobium, S. mansoni, and S. japonicum. Male-female pairing of schistosomes is necessary for sexual maturity and the production of a large number of eggs, which are primarily responsible for schistosomiasis dissemination and pathology. Here, we used microarray hybridization, bioinformatics, quantitative PCR, in situ hybridization, and gene silencing assays to identify genes that play critical roles in S. japonicum reproduction biology, particularly in vitellarium development, a process that affects male-female pairing, sexual maturation, and subsequent egg production. Microarray hybridization analyses generated a comprehensive set of genes differentially transcribed before and after male-female pairing. Although the transcript profiles of females were similar 16 and 18 days after host infection, marked gene expression changes were observed at 24 days. The 30 most abundantly transcribed genes on day 24 included those associated with vitellarium development. Among these, genes for female-specific 800 (fs800), eggshell precursor protein, and superoxide dismutase (cu-zn-SOD) were substantially upregulated. Our in situ hybridization results in female S. japonicum indicated that cu-zn-SOD mRNA was highest in the ovary and vitellarium, eggshell precursor protein mRNA was expressed in the ovary, ootype, and vitellarium, and Sjfs800 mRNA was observed only in the vitellarium, localized in mature vitelline cells. Knocking down the Sjfs800 gene in female S. japonicum by approximately 60% reduced the number of mature vitelline cells, decreased rates of pairing and oviposition, and decreased the number of eggs produced in each male-female pairing by about 50%. These results indicate that Sjfs800 is essential for vitellarium development and egg production in S. japonicum and suggest that Sjfs800 regulation may provide a novel approach for the prevention or treatment of schistosomiasis.Author SummarySchistosomiasis is a common but largely unstudied tropical disease caused by parasitic trematodes of the genus Schistosoma. The eggs of schistosomes are responsible for schistosomiasis transmission and pathology, and the production of these eggs is dependent on the pairing of females and males. In this study, we determined which genes in Schistosoma japonicum females were differentially expressed before and after pairing with males, identifying the 30 most abundantly expressed of these genes. Among these 30 genes, we further characterized those in female S. japonicum that were upregulated after pairing and that were related to reproduction and vitellarium development, a process that affects male-female pairing, sexual maturation, and subsequent egg production. We identified three such genes, S. japonicum female-specific 800 (Sjfs800), eggshell precursor protein, and superoxide dismutase, and confirmed that the mRNAs for these genes were primarily localized in reproductive structures. By using gene silencing techniques to reduce the amount of Sjfs800 mRNA in females by about 60%, we determined that Sjfs800 plays a key role in development of the vitellarium and egg production. This finding suggests that regulation of Sjfs800 may provide a novel approach to reduce egg counts and thus aid in the prevention or treatment of schistosomiasis.

Disruption of vitellogenesis and spermatogenesis by triclabendazole (TCBZ) in a TCBZ-resistant isolate of Fasciola hepatica following incubation in vitro with a P-glycoprotein inhibitor

SUMMARYA study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant fluke isolate was used for these experiments and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. In the first experiment, flukes were initially incubated for 2 h in R(+)-VPL (100 μm), then incubated in R(+)-VPL+triclabendazole sulphoxide (TCBZ.SO) (50 μg mL−1, or 133·1 μm) until flukes ceased movement (at 9 h post-treatment). In a second experiment, flukes were incubated in TCBZ.SO alone and removed from the incubation medium following cessation of motility (after 15 h). In the third experiment, flukes were incubated for 24 h in R(+)-VPL on its own. Changes to the testis tubules and vitelline follicles following drug treatment and following Pgp inhibition were assessed by means of light microscope histology and transmission electron microscopy. Incubation of the Sligo isolate in either R(+)-VPL or TCBZ.SO on their own had a limited impact on the morphology of the two tissues. Greater disruption was observed when the drugs were combined, in terms of the block in development of the spermatogenic and vitelline cells and the apoptotic breakdown of the remaining cells. Sperm formation was severely affected and abnormal. Large spaces appeared in the vitelline follicles and synthesis of shell protein was disrupted. The results of this study support the concept of altered drug efflux in TCBZ-resistant flukes and indicate that drug transporters may play a role in the development of drug resistance.

Reinvestigation of vitellogenesis in Caryophyllaeus laticeps (Pallas, 1781) (Cestoda, Caryophyllidea, Caryophyllaeidae), monozoic tapeworm of Abramis brama (Pisces, Teleostei)

Reinvestigation of vitellogenesis in the caryophyllidean cestode Caryophyllaeus laticeps (Pallas, 1781) has been performed using light microscope (LM) and transmission electron microscopy (TEM), and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) for glycogen. Vitellogenesis is generally similar to that reported in the past, however, some new observations were made. The present study reveals the first evidence of: (i) interstitial tissue in the vitelline follicles, (ii) lipid droplets in maturing and mature vitellocytes from vitelline follicles, and (iii) lamellar bodies in vitellocytes from the vitelloduct in C. laticeps. Projections of interstitial tissue surround each vitellocyte and the follicle periphery. The perinuclear cytoplasm of the interstitial tissue contains granular endoplasmic reticulum and vesicles of various size and density. Cytoplasmic osmiophobic lipid droplets and lamellar bodies, previously believed to be absent in most caryophyllid cestodes, are readily apparent in vitellocytes of C. laticeps. The origin and presumed function of these inclusions are discussed. On the other hand, the formation and storage of massive amounts of glycogen in the nucleus and large amounts in the cytoplasm of mature vitelline cells are similar to the condition found in other caryophyllids. Results are compared and contrasted with previous studies on vitellogenesis in other monopleuroid cestodes (Amphilinidea and Gyrocotylidea) as well as polypleuroid cestodes (Spathebothriidea) and the remaining strobilated Eucestoda.

Ultrastructure of the vitellarium in the digeneans Phyllodistomum angulatum (Plagiorchiida, Gorgoderidae) and Azygia lucii (Strigeida, Azygiidae)

Fine structural features of the vitellarium of two digeneans, Phyllodistomum angulatum and Azygia lucii, are documented and compared with those of other digenean species. The cytodifferentiation of immature vitelline cells (vitellocytes) assumes the production and subsequent accumulation in their cytoplasm of several inclusions. Mature vitelline cells of P. angulatum are characterized by the presence of vitelline clusters (∼2.7 μm in diameter, with ∼100 vitelline globules of ∼0.35 μm in diameter) and osmiophobic, saturated lipid droplets (∼2-3 μm in diameter), and in A. lucii vitelline clusters of the same diameter include much fewer vitelline globules (∼50 globules of ∼0.5 μm in diameter), osmiophilic lipid droplets and α-glycogen. In both P. angulatum and A. lucii, interstitial cells are also present within the vitellarium. Two types of contact sites (septate and tight junctions) between adjoining interstitial cells also occur in both digenean species. Judging from the present and previous ultrastructural studies, it is suggested that there are three potential discriminatory characters of the digenean vitellarium (the number of different types of cell components within the vitellarium, the presence and type of junctional complexes between these cells, and the isolation of the vitellarium from the surrounding tissue) which may prove useful for a better understanding of the biology and evolutionary history of the different digenean groups.

Ultrastructure of the intrauterine eggs of Didymobothrium rudolphii (Monticelli, 1890) (Cestoda, Spathebothriidea, Acrobothriidae) and its phylogenetic implications

The intrauterine polylecithal eggs of the spathebothriidean cestode Didymobothrium rudolphii (Monticelli, 1890) were examined by means of transmission electron microscopy (TEM). Each unembryonated egg is composed of a fertilised oocyte or ovum and several vitelline cells, all surrounded by a newly formed shell. The lumen of the proximal uterus is packed with unutilised vitelline material and eggs at different stages of shell formation. In the proximal region of the uterus, the fertilised oocytes, initially surrounded by dense, discontinuous islands of eggshell material and containing long axonemes of spermatozoa in their cytoplasm, were frequently observed. Sperm axonemes also remain in the oocyte cytoplasm of eggs surrounded by a thick electron-dense shell until the sperm nucleus is transformed into a male pronucleus. Despite the fact that the two-pronuclei stage and cell divisions within the eggs of D. rudolphii were never observed, individual eggs containing several blastomeres of different sizes were seen in the middle and distal regions of the uterus. This provides indirect evidence that at least a few initial cleavage divisions must take place in the intrauterine eggs and direct evidence that the early embryonic development of D. rudolphii starts in utero. The several vitellocytes present in each egg contain nutritive reserves for the developing embryos; these are composed mainly of numerous lipid droplets and a moderate amount of glycogen. In the eggs containing early embryos composed of several blastomeres, the cytoplasm of the degenerating vitellocytes exhibits the presence of so-called ‘foci of cytoplasmic degradation’, which appear to be involved in the autolytic process of the vitellocyte cell components and inclusions, such as a high accumulation of lipids and glycogen. This progressive degeneration of the vitellocytes, considered as an example of programmed cell death or apoptosis, likely contributes towards the resorption of nutritive reserves by the developing embryo. Some of the results of this study are commented upon in relation to the affiliation of the spathebothriideans with other lower cestode groups.

Physiological and morphological effects of genistein against the liver fluke, Fasciola hepatica

SUMMARYA study has been carried out to determine the activity of genistein against adult liver fluke, Fasciola hepatica. Flukes were incubated in vitro in genistein at a concentration of 0·27 mg/ml (=1 mm). They ceased to move after 3 h, at which point the experiment was terminated and the specimens prepared for examination by scanning and transmission electron microscopy. Surface changes to the flukes comprised swelling and blebbing, especially in the posterior region of the flukes, and there was particular disruption to the spines, accompanied by some spine loss. Fine structural changes to the tegumental syncytium indicated an accelerated release of secretory bodies at the surface, but a reduction in their production within the cell bodies. Autophagic activity was evident in the tegumental cells, a phenomenon that was also observed in the gastrodermal cells. Disruption to the testis and vitelline follicles was severe, with an apparent block in the normal developmental sequence of the spermatogenic and vitelline cells, respectively. Shell protein production by the vitelline cells was also disrupted. In separate experiments, somatic muscle strips were exposed to concentrations of genistein ranging from 1 μm to 1 mm. There were statistically significant increases in the frequency and/or amplitude of muscle contractions at concentrations of 10 μm, 100 μm and 1 mm. The results suggest that genistein is capable of causing severe morphological and neuromuscular disruption to adult flukes in vitro over a short time-span.

Ultrastructure of vitellocytes in the cestode Progrillotia pastinacae Dollfus, 1946 (Trypanorhyncha, Progrillotiidae)

The present study describes the ultrastructure of mature vitellocytes of the trypanorhynch cestode Progrillotia pastinacae Dollfus, 1946 (Progrillotiidae), a parasite of the common stingray Dasyatis pastinaca (Linnaeus, 1758) (Dasyatidae). The vitelline cells of this species measure about 24 μm in length and about 20 μm in width. They have small, elongated, slightly lobulated nuclei, about 4–5 μm in length, with large dense elongated nucleoli and numerous irregularly-shaped dense clumps of heterochromatin. The extensive cytoplasm is rich in numerous cell organelles and cell inclusions. While the perinuclear cytoplasm contains numerous long parallel cisternae of GER, ribo-and polyribosomes, several Golgi complexes and mitochondria, the peripheral cytoplasm contains predominantly three types of cell inclusions: a great number of large lipid droplets, several shell globule clusters, and a very small amount of glycogen-like particles. The most characteristic features of vitellocytes in P. pastinacae are having almost no traces of glycogen and the great number of large, highly osmiophobic lipid droplets representing saturated fatty acids. The presence of large amounts of lipids also in two other trypanorhynchs, Grillotia erinaceus (Beneden, 1858) Guiart, 1927 and Dollfusiella spinulifera (Beveridge et Jones, 2000) Beveridge, Neifar et Euzet, 2004, is in strong contrast to the condition in the most evolved cestodes, Cyclophyllidea, that usually show no trace of lipids.