Schistosoma mansoni: Developmental arrest of miracidia treated with histone deacetylase inhibitors

In the present study, we examined the effect of the histone deacetylase (HDAC) inhibitors trichostatin A (TSA), valproic acid (VA) and sodium-butyrate on the metamorphosis of larvae of the human blood-fluke Schistosoma mansoni from the free-swimming miracidia into the intramolluskal sporocyst. We show that HDAC inhibitors block transformation in concentration dependant manner. TSA reversibly blocks this developmental process: only 13 ± 11% of TSA treated miracidia transform into sporocysts in-vitro, compared to 92 ± 3% in the mock-treated control. Other enzyme inhibitors such as cycloheximide or hydroxyurea had no effect on metamorphosis. For treatment of up to 4 h, the effect of TSA was completely reversible. Our data indicates that HDAC activity is necessary for the transformation of S. mansoni miracidia during infection of the snail host.     

Excreted/secreted Schistosoma mansoni venom allergen-like 9 (SmVAL9) modulates host extracellular matrix remodelling gene expression

The Schistosoma mansoni venom allergen-like (SmVAL) protein family consists of 29 members, each possessing a conserved α-β-α sandwich tertiary feature called the Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain. While the SmVALs have been found in both excretory/secretory (E/S) products and in intra/sub-tegumental (non-E/S) fractions, the role(s) of this family in host/parasite relationships or schistosome developmental processes remains poorly resolved. In order to begin quantifying SmVAL functional diversity or redundancy, dissecting the specific activity (ies) of individual family members is necessary. Towards this end, we present the characterisation of SmVAL9; a protein previously found enriched in both miracidia/sporocyst larval transformation proteins and in egg secretions. While our study confirms that SmVAL9 is indeed found in soluble egg products and miracidia/sporocyst larval transformation proteins, we find it to be maximally transcribed/translated in miracidia and subsequently down-regulated during in vitro sporocyst development. SmVAL9 localisation within sporocysts appears concentrated in parenchymal cells/vesicles as well as associated with larval germinal cells. Furthermore, we demonstrate that egg-derived SmVAL9 carries an N-linked glycan containing a schistosome-specific difucosyl element and is an immunogenic target during chronic murine schistosomiasis. Finally, we demonstrate that recombinant SmVAL9 affects the expression of extracellular matrix, remodelling matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP) gene products in both Biomphalaria glabrata embryonic cell (BgMMP1) and Mus musculus bone marrow-derived macrophage (MmMMP2, MmMMP9, MmMMP12, MmMMP13, MmMMP14, MmMMP28, TIMP1 and TIMP2) in vitro cultures. These findings importantly suggest that excreted/secreted SmVAL9 participates in tissue reorganisation/extracellular matrix remodelling during intra-mammalian egg translocation, miracidia infection and intra-molluscan sporocyst development/migration.     

The invasion of Biomphalaria pfeifferi by Schistosoma mansoni miracidia and the development of daughter sporocysts

Laboratory experiments have been carried out to determine the susceptibility of Gezira Biomphalaria pfeifferi snails to S. mansoni miracidia and the relationship between miracidia and daughter sporocyst production at the 10–17 day development stage. The relationship between snail numbers, miracidia numbers and water volume has also been studied. Two non susceptible snails, Bulinus truncatus and Cleopatra bulimoides, both of which occur naturally in Gezira canals, were tested to see if they act as decoys for S. mansoni miracidia.The results showed that the B. pfeifferi are 100% susceptible to S. mansoni invasion, at least to the daughter sporocyst development stage. The more miracidia that penetrated the more daughter sporocysts were produced, however individual variation and overlap were great. When one miracidium was released to find one snail it succeeded in low water volumes (5 m, 50 ml), but failed in 5 litres. When 100 miracidia were released mortality of snails was high suggesting superinfection particularly when only one or five snails were available. Among survivors daughter sporocyst counts were very high. Cleopatra and Bulinus snails do have a decoy effect when present in large numbers. In their presence the number of infected snails was marginally reduced and the number of daughter sporocysts greatly reduced. However, if superinfection is reduced by decoy effect, it is conceivable that Biomphalaria may be protected by decoy snails in circumstances where miracidia counts are high.     

In vitro cultivation of Schistosoma japonicum-parasites and cells

Schistosomiasis is a serious parasitic zoonosis caused by blood-dwelling flukes of the genus Schistosoma. Understanding functions of genes and proteins of this parasite is important for uncovering this pathogen's complex biology, which will provide valuable information to design new strategies for schistosomiasis control. Effective applications of molecular tools reported to investigate schistosome gene function, such as inhibitor studies and transgenesis, rely on the developments of in vitro cultivation system of this parasite and cells. Besides the in vitro culture studies dealing with Schistosoma mansoni, there are also numerous excellent studies about the in vitro cultivation of Schistosoma japonicum, which were performed by Chinese researchers and published in Chinese journals. Nearly every stage of the life-cycle of S. japonicum, including miracidia, mother sporocysts, cercariae, schistosomula, and egg-laying adult worms, was employed for developing in vitro cultivation methods, being accompanied by the introduction of several media and supplements that helped to improve culture conditions. It was not only possible to generate mother sporocysts from miracidia in vitro, but also to obtain adult worms from cercariae through in vitro cultivation. The main obstacles to complete the life cycle of S. japonicum in the lab are the transition from mother sporocysts to cercariae, and the production of fertilized and completely developed eggs by adult worms generated in vitro. With regard to cells from S. japonicum, besides established isolation protocols and morphological observations, media optimizations were conducted by using different chemical reagents, biological supplements and physical treatment. Among these, mutagens like N-methyl-N-nitro-N-nitrosoguanidine and the addition of extracellular matrix were found to be able to induce mitogenic activities. Although enzyme activities or the level of silver-stained nucleolar region associated protein in cultured cells indicated still suboptimal conditions, the achievements made point to the possibility of reaching the aim of establishing cell lines for S. japonicum. Both the improvements of the in vitro culture of larval and adult worms of S. japonicum as well as the access of cells of this parasite provide excellent advances for research on this important parasite in the future.     

Protein kinase C signalling during miracidium to mother sporocyst development in the helminth parasite, Schistosoma mansoni

For schistosomes, development of the miracidium to mother sporocyst within a compatible molluscan host requires considerable physiological and morphological changes by the parasite. The molecular mechanisms controlling such development have not been explored extensively. To begin to elucidate the importance of kinase-mediated signal transduction to this process, the phosphorylation (activation) of protein kinase C (PKC) in larval stages of Schistosoma mansoni undergoing in vitro transformation was explored. Mining of the S. mansoni genomic database revealed two S. mansoni PKC proteins with high homology to human PKCβ and containing the conserved autophosphorylation (activation) site represented by serine 660 of human PKCβ_II. Western blotting with anti-phosphospecific antibodies directed to this site demonstrated that miracidia freshly-hatched from eggs possessed PKC (78 kDa) which was phosphorylated (activated) when miracidia were exposed to phorbol ester, and dephosphorylated (inhibited) following exposure to the PKC inhibitor GF109203X. Miracidia treated with the phospholipase C (PLC) inhibitor U73122 also displayed decreased PKC phosphorylation. S. mansoni PKC was phosphorylated during the initial 24 h development of miracidia into mother sporocysts; after 31 h and 48 h development, phosphorylation was reduced by 72% and 86%, respectively. Confocal microscopy of miracidia revealed phosphorylated PKC associated with the neural mass, excretory vesicle, tegument, ciliated plates, terebratorium and germinal cells; in larvae undergoing transformation for 31 h, phosphorylated PKC was only occasionally detected, being present in regions likely corresponding to the ridge cyton. Inhibition of PKC in miracidia by GF109230X resulted in accelerated transformation, particularly to the postmiracidium stage; ciliated plates were also shed from developing larvae more rapidly. These results highlight the dynamic nature of PKC signalling during S. mansoni postembryonic development and support a role for active PKC in restricting transformation of S. mansoni miracidia into mother sporocysts.     

Intermediate host specificity in Schistosoma mansoni

Miracidia of Schistosoma mansoni penetrate into many kinds of snails, but development of normal sporocysts takes place only in certain species of Biomphalaria. Different populations of this snail vary greatly in laboratory infection rates with S. mansoni originating from diverse geographic localities. Cross-exposure experiments show that compatibility factors exist in both snails and parasites. Susceptibility of stocks of Biomphalaria to particular strains of S. mansoni is genetically determined and may be modified by selection in the laboratory. In a compatible snail, the sporocyst develops without host tissue reaction; in incompatible snails the early larvae are rapidly surrounded by amebocytes and fibroblasts, and destroyed. This reaction resembles the generalized host cellular response elicited by any foreign body. An individual snail exposed to many miracidia may have both developing and encapsulated sporocysts side by side within its tissues. The weight of current evidence suggests that elicitation or absence of this cellular response resides in the recognition or nonrecognition of the sporocyst as a foreign body. The sporocyst tegument surface, which forms within a few hours after miracidial penetration, may have a molecular conformation identical with that of the snail, or may be able to bind specific host molecules, so that detection and subsequent encapsulation by host cells are averted. Presuming genetic determination of the sporocyst surface structure and of the host cell detection capability, differing infection rates would result from the particular frequencies of relevant genes in the populations concerned.     

Ribeiroia marini: Irradiated miracidia and induction of acquired resistance in Biomphalaria glabrata

Biomphalaria glabrata snails sensitized by exposure to X-irradiated miracidia of the trematode, Ribeiroia marini, acquired resistance to challenge with nonirradiated R. marini miracidia. Resistance was acquired within 1 day of sensitization; was strongest at 1 week, when infection rates of sensitized snails were 15% of the controls (i.e., $\text{S}\text{C}\text{= 0.15}$); and persisted for at least 3 weeks. By 30 days the difference between the infection rates of sensitized and control snails was no longer statistically significant. As in previous studies with echinostomes, acquired resistance to R. marini was characterized histologically by the destruction of irradiated sporocysts by host amoebocytes. Following destruction of all irradiated sporocysts, snails became resistant and encapsulated and destroyed nonirradiated challenge sporocysts within 1 day postchallenge. Associated with sporocyst destruction was an enlargement of the amoebocyte-producing organ, which showed intense mitotic activity. A proportion of the nonirradiated challenge sporocysts were also destroyed in most nonsensitized control snails, which consequently had a temporarily enlarged amoebocyte-producing organ. In contrast to acquired resistance reported to echinotomes, which is quite specific, acquired resistance to R. marini was associated with nonsusceptibility to both Echinostoma paraensei ($\text{S}\text{C}\text{= 0.19}$) and Schistosoma mansoni ($\text{S}\text{C}\text{= 0.81}$).     

Glycotope analysis in miracidia and primary sporocysts of Schistosoma mansoni: Differential expression during the miracidium-to-sporocyst transformation

Fucosylated carbohydrate epitopes (glycotopes) expressed by larval and adult schistosomes are thought to modulate the host immune response and possibly mediate parasite evasion in intermediate and definitive hosts. While previous studies showed glycotope expression is developmentally and stage-specifically regulated, relatively little is known regarding their occurrence in miracidia and primary sporocysts. In this study, previously defined monoclonal antibodies were used in confocal laser scanning microscopy, standard epifluorescence microscopy and Western blot analyses to investigate the developmental expression of the following glycotopes in miracidia and primary sporocysts of Schistosoma mansoni: GalNAcβ1-4GlcNAc (LDN), GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F), Fucα1-3GalNAcβ1-4GlcNAc (F-LDN), Fucα1-3GalNAcβ1-4(Fucα1-3)GlcNAc (F-LDN-F), GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF), Fucα1-2Fucα1-3GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (DF-LDN-DF), Galβ1-4(Fucα1-3)GlcNAc (Lewis X) and the truncated trimannosyl N-glycan Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (TriMan). All but Lewis X were variously expressed by miracidia and sporocysts of S. mansoni. Most notably, α3-fucosylated LDN (F-LDN, F-LDN-F, LDN-F) was prominently expressed on the larval surface and amongst glycoproteins released during larval transformation and early sporocyst development, possibly implying a role for these glycotopes in snail–schistosome interactions. Interestingly, Fucα2Fucα3-subsituted LDN (LDN-DF, DF-LDN-DF) and LDN-F were heterogeneously surface-expressed on individuals of a given larval population, particularly amongst miracidia. In contrast, LDN and TriMan primarily localised in internal somatic tissues and exhibited only minor surface expression. Immunoblots indicate that glycotopes occur on overlapping but distinct protein sets in both larval stages, further demonstrating the underlying complexity of schistosome glycosylation. Additionally, sharing of specific larval glycotopes with Biomphalaria glabrata suggests an evolutionary convergence of carbohydrate expression between schistosomes and their snail host.     

Acid phosphatase in granulocytic capsules formed in strains of Biomphalaria glabrata totally and partially resistant to Schistosoma mansoni

Cheng T. C. and Garrabrant T. A. 1977. Acid phosphatase in granulocytic capsules formed in strains of Biomphalaria glabrata totally and partially resistant to Schistosoma mansoni. International Journal for Parasitology7: 467–472. Acid phosphatase (EC 3.1.3.2, orthophosphoric monoester phosphohydrolase) has been demonstrated cytochemically in isolated granulocytes from the hemolymph of three strains of Biomphalaria glabrata. This enzyme was not detected in hyalinocytes. By employing acid phosphatase as a marker, it was determined that the cells comprising the capsule surrounding Schislosoma mansoni mother sporocysts in a totally and partially resistant strain of B. glabrata are granulocytes.The process of encapsulation of S. mansoni mother sporocysts in resistant B. glabrata was traced for 72 h post-penetration by miracidia and has been ascertained to involve two stages: (1) enlargement of the granuloma around intact sporocysts, followed by (2) disintegration of the parasite and a decrease in the size of the granuloma. There is an increase in the level of acid phosphatase activity within granulocytes comprising the granuloma during the second stage.Host cellular responses to S. mansoni mother sporocysts does not occur in susceptible snails.     

Bursts of transposition from non-long terminal repeat retrotransposon families of the RTE clade in Schistosoma mansoni

The genus Schistosoma is composed of blood flukes that infect vertebrates, from which three species are major causative agents of human schistosomiasis, a tropical disease that affects more than 200 million people. Current models of the recent evolution of Schistosoma indicate multiple events of migration and speciation from an Asian ancestral species. Transposable elements are important drivers of genome evolution and have been hypothesised to have an important role in speciation. In this work, we describe a comprehensive inventory of Schistosoma mansoni and Schistosoma japonicum retrotransposons, based on their recently published genomic data. We find a considerable difference in retrotransposon representation between the two species (22% and 13%, respectively). A large part of this difference can be attributed to higher representation of two previously described families of S. mansoni retrotransposons (SR2 and Perere-3/SR3), compared with the representation of their closest relative families in S. japonicum. A more detailed analysis suggests that these two S. mansoni families were the subject of recent bursts of transposition that were not paralleled by their S. japonicum counterparts. We hypothesise that these bursts could be a consequence of the evolutionary pressure resulting from migration of Schistosoma from Asia to Africa and their establishment in this new environment, helping both speciation and adaptation.     

Tissue responses exhibited by Biomphalaria alexandrina snails from different Egyptian localities following Schistosoma mansoni exposure

Snails’ susceptibilities to infection with Schistosoma mansoni were determined through observation of infection rates, total cercarial production and tissue responses of the first generation (F1) of Biomphalaria alexandrina snails, originally collected from different Egyptian governorates (Giza, Fayoum, Kafr El-Sheikh, Ismailia and Damietta) and responses were compared between groups. The emergence of cercariae for a 3-month period and the calculation of survival and infection rates, in control (Schistosome Biological Supply Center; SBSC) and infected snails were evaluated. SBSC and Giza snails showed greater susceptibilities to infection and lower mortality rates. In addition, at 6 and 72 h post-exposure to miracidia all the snail groups showed no difference in the anatomical locations of sporocysts. The larvae were found in the head-foot, the mantle collar and the tentacles of the snails. Sporocysts showed normal development with low tissue reactions in SBSC and Giza snail groups infected with S. mansoni miracidia (SBSC). However, in Fayoum, Kafr El-Sheikh, Ismailia and Damietta snail groups, variable tissue responses were observed in which numerous hemocytes made direct contact with S. mansoni larvae forming capsules. The results suggested that, different responses of B. alexandrina snail’s hemocytes towards S. mansoni are related to the degree of susceptibility of these snails. So this is important in planning the strategy of schistosomiasis control.     

Female biased sex-ratio in Schistosoma mansoni after exposure to an allopatric intermediate host strain of Biomphalaria glabrata

For parasites that require multiple hosts to complete their development, the interaction with the intermediate host may have an impact on parasite transmission and development in the definitive host. The human parasite Schistosoma mansoni needs two different hosts to complete its life cycle: the freshwater snail Biomphalaria glabrata (in South America) as intermediate host and a human or rodents as final host. To investigate the influence of the host environment on life history traits in the absence of selection, we performed experimental infections of two B. glabrata strains of different geographic origin with the same clonal population of S. mansoni. One B. glabrata strain is the sympatric host and the other one the allopatric host. We measured prevalence in the snail, the cercarial infectivity, sex-ratio, immunopathology in the final host and microsatellite frequencies of individual larvae in three successive generations.     

Schistosoma mansoni: Cloning by microsurgical transplantation of sporocysts

Development of a method of infecting of the molluscan host by microsurgical transplantation of the parasite's sporocysts enables the researcher to maintain the host cycle of Schistosoma mansoni exclusively by asexual means and without the participation of a vertebrate host. After transplantation, larval morphogenesis becomes altered to form an additional generation of sporocysts. These invade the digestive gland of the recipient mollusc progressively, producing normally infective cercariae. The maintenance of the life cycle of S. mansoni in the laboratory for 1 year, solely in the mollusc, has been obtained through six successive transplantations. Thus, a true cloning of S. mansoni has been achieved, the original transplant material being derived from a monomiracidial infection. From the practical viewpoint, this transplantation technique is of definite utility in the maintenance of the cycle, the vertebrate stage having been eliminated. From the theoretical viewpoint, unexpected analogies become apparent with the two types of larval demography found in Digenea (Digenea with sporocyst and Digenea with rediae).     

An example of molecular co-evolution: reactive oxygen species (ROS) and ROS scavenger levels in Schistosoma mansoni/Biomphalaria glabrata interactions

The co-evolution between hosts and parasites involves huge reciprocal selective pressures on both protagonists. However, relatively few reports have evaluated the impact of these reciprocal pressures on the molecular determinants at the core of the relevant interaction, such as the factors influencing parasitic virulence and host resistance. Here, we address this question in a host-parasite model that allows co-evolution to be monitored in the field: the interaction between the mollusc, Biomphalaria glabrata, and its trematode parasite, Schistosoma mansoni. Reactive oxygen species (ROS) produced by the haemocytes of B. glabrata are known to play a crucial role in killing S. mansoni. Therefore, the parasite must defend itself against oxidative damage caused by ROS using ROS scavengers in order to survive. In this context, ROS and ROS scavengers are involved in a co-evolutionary arms race, and their respective production levels by sympatric host and parasite could be expected to be closely related. Here, we test this hypothesis by comparing host oxidant and parasite antioxidant capabilities between two S. mansoni/B. glabrata populations that have co-evolved independently. As expected, our findings show a clear link between the oxidant and antioxidant levels, presumably resulting from sympatric co-evolution. We believe this work provides the first supporting evidence of the Red Queen Hypothesis of reciprocal evolution for functional traits at the field-level in a model involving a host and a eukaryotic parasite.     

Schistosoma mansoni: Agglutination of sporocysts,and formation of gels on miracidia transforming in plasma of Biomphalaria glabrata

The resistance or susceptibility of Biomphalaria glabrata strains to strains of Schistosoma mansoni, the human blood fluke, are evidenced by the responses of snail hemocytes to sporocysts of the schistosome, both in vivo and in vitro. It is now reported that living sporocysts of the PR1 strain of S. mansoni agglutinate in the plasma of all tested strains of B. glabrata, in contrast to fixed sporocysts which agglutinate only in plasma from resistant snail strains. The agglutinating activity in resistant plasmas is not divalent cation dependent, and was not inhibited by the 26 carbohydrates and four amino acids tested. In addition, the observation that gelatinous deposits develop on transforming miracidia-sporocysts in B. glabrata plasmas is also reported. Both the agglutination and gel-formation phenomena may facilitate recognition of, and attacks on, sporocysts, thereby contributing to susceptibility and resistance in this host-parasite system.     

Location of Biomphalaria glabrata (Say) by miracidia of Schistosoma mansoni Sambon in natural standing and running waters on the West Indian Island of St. Lucia

Upatham E. S. 1973. Location of Biomphalaria glabrata (Say) by miracidia of Schistosoma mansoni Sambon in natural standing and running waters on the West Indian Island of St. Lucia. International Journal of Parasitology3: 289–297. The ability of S. mansoni miracidia to locate B. glabrata in natural ditches and streams was investigated. Miracidia located and infected snails at distances of 9–14 and 97-54 in horizontally in standing and running waters respectively. In running water, no infection occurred above a velocity of 13.11 $\text{cm}\text{sec}$. In both types of habitat, infection rates in snails increased with increasing levels of miracidia but decreased as the location of caged snails moved away from the miracidial point of entry. Laboratory experi- ments showed that the number of daughter sporocysts was proportional to the number of miracidia. Judging by the number of daughter sporocysts recovered only a small percentage of miracidia succeeded in locating and penetrating snails (6.8–13-7 % and 1.4–6.2 % in standing and running waters respectively). In standing water, infection may be inhibited by the limited ability of miracidia to move horizontally. In running water, the flow extends significantly the effective scanning capacity of the miracidia, giving them a better chance of coming into contact with snails, which is of importance in the epidemiology of schistosomiasis. Owing to a con- siderable wastage of miracidia and the higher relative efficiency of miracidia at lower densities in detecting snails, control measures such as chemotherapy or provision of safe water supplies designed to lower egg output and reduce contamination may not seriously influence transmission unless S. mansoni egg production or contamination is massively reduced.