Female Schistosomes Research Articles

PLoS Neglected Tropical Diseases Issue Image | Vol. 2(9) September 2008

The ovaries of a starved female schistosome from a rhesus macaque (left) and a normal female (right). The infection becomes patent in the macaque and eggs are produced, but the host immune system slowly overwhelms the worms, they cease feeding, and eventually starve to death, suggesting a route to a human therapeutic vaccine (see Wilson et al., doi:10.1371/journal.pntd.0000290). Image Credit: Joanne Marrison, Gary Dillon, and Alan Wilson

A bone morphogenetic protein homologue in the parasitic flatworm, Schistosoma mansoni

Members of the bone morphogenetic protein (BMP) subfamily of cytokines control many aspects of metazoan development including patterning and organogenesis. Despite the recognition that schistosomes possess key components of a BMP signaling pathway, a BMP-like ligand in the parasitic flatworm Schistosoma mansoni remained elusive. Here, we describe the cloning and characterisation of an S. mansoni BMP ( SmBMP). SmBMP is most closely related to BMP homologues from the free-living flatworms Schmidtea mediterranea and Dugesia japonica, with 51% and 47% identity at the amino acid level, respectively. Based on reverse transcription-PCR, SmBMP is expressed throughout the mammalian life-cycle of the parasite in both male and female schistosomes. In support of these results, antibodies to SmBMP successfully immunoprecipitated the protein in adult male and female antigen preparations with more protein detected in male parasites. Immunofluorescent studies localised SmBMP to the protonephridia of adult parasites, and SmBMP was identified in the excretory/secretory products of adult male parasites via immunoprecipitation. With the previous description of a TGF-β subfamily homologue in S. mansoni, ligands representing both arms of the TGF-β superfamily have now been described in this trematode.

A proteomic approach to the identification of tegumental proteins of male and female Schistosoma bovis worms

Schistosoma bovis, a parasite of ruminants, can live for years in the bloodstream in spite of the immune response of its host. The parasite tegument covers the entire surface of the worm and plays a key role in the host–parasite relationship. The parasite molecules involved in host immune response evasion mechanisms must be expressed on the tegument surface and are potential targets for immune or drug intervention. The purpose of the present work was to identify the tegumental proteomes of male and female S. bovis worms, in particular the proteins expressed on the outermost layers of the tegument structure. Adult worms of each sex were treated separately with trypsin in order to digest their tegumental proteins, after which the peptides released were analysed by LC–MS/MS for identification. This experimental approach afforded valuable information about the protein composition of the tegument of adult S. bovis worms. A range of tegumental proteins was identified, most of which had not been identified previously in this species. Although an absolute purification of the proteins expressed on the outermost layers of the tegument structure was not achieved, it is likely that present among the proteins identified are some of the molecules most closely associated with the tegument surface. Our study also suggests that there may be differences in the protein composition of the tegument of male and female schistosomes. Finally, the presence of actin and GAPDH on the surface of male and female worms and the presence of enolase exclusively on the surface of male worms were verified by confocal microscopy.

Microarray analysis of gene expression induced by sexual contact in Schistosoma mansoni

BackgroundThe parasitic trematode Schistosoma mansoni is one of the major causative agents of Schistosomiasis, a disease that affects approximately 200 million people, mostly in developing countries. Since much of the pathology is associated with eggs laid by the female worm, understanding the mechanisms involved in oogenesis and sexual maturation is an important step towards the discovery of new targets for effective drug therapy. It is known that the adult female worm only develops fully in the presence of a male worm and that the rates of oviposition and maturation of eggs are significantly increased by mating. In order to study gene transcripts associated with sexual maturation and oviposition, we compared the gene expression profiles of sexually mature and immature parasites using DNA microarrays.ResultsFor each experiment, three amplified RNA microarray hybridizations and their dye swaps were analyzed. Our results show that 265 transcripts are differentially expressed in adult females and 53 in adult males when mature and immature worms are compared. Of the genes differentially expressed, 55% are expressed at higher levels in paired females while the remaining 45% are more expressed in unpaired ones and 56.6% are expressed at higher levels in paired male worms while the remaining 43.4% are more expressed in immature parasites. Real-time RT-PCR analysis validated the microarray results. Several new maturation associated transcripts were identified. Genes that were up-regulated in single-sex females were mostly related to energy generation (i.e. carbohydrate and protein metabolism, generation of precursor metabolites and energy, cellular catabolism, and organelle organization and biogenesis) while genes that were down-regulated related to RNA metabolism, reactive oxygen species metabolism, electron transport, organelle organization and biogenesis and protein biosynthesis.ConclusionOur results confirm previous observations related to gene expression induced by sexual maturation in female schistosome worms. They also increase the list of S. mansoni maturation associated transcripts considerably, therefore opening new and exciting avenues for the study of the conjugal biology and development of new drugs against schistosomes.

Reconstruction and in silico analysis of the MAPK signaling pathways in the human blood fluke, Schistosoma japonicum

At present, little is known about signal transduction mechanisms in schistosomes, which cause the disease of schistosomiasis. The mitogen-activated protein kinase (MAPK) signaling pathways, which are evolutionarily conserved from yeast to Homo sapiens, play key roles in multiple cellular processes. Here, we reconstructed the hypothetical MAPK signaling pathways in Schistosoma japonicum and compared the schistosome pathways with those of model eukaryote species. We identified 60 homologous components in the S. japoncium MAPK signaling pathways. Among these, 27 were predicted to be full-length sequences. Phylogenetic analysis of these proteins confirmed the evolutionary conservation of the MAPK signaling pathways. Remarkably, we identified S. japonicum homologues of GTP-binding protein β and α-I subunits in the yeast mating pathway, which might be involved in the regulation of different life stages and female sexual maturation processes as well in schistosomes. In addition, several pathway member genes, including ERK, JNK, Sja-DSP, MRAS and RAS, were determined through quantitative PCR analysis to be expressed in a stage-specific manner, with ERK, JNK and their inhibitor Sja-DSP markedly upregulated in adult female schistosomes.

Genomic organization of the Schistosoma mansoni aspartic protease gene, a platyhelminth orthologue of mammalian lysosomal cathepsin D

Schistosomes are considered the most important of the helminth parasites of humans in terms of morbidity and mortality. Schistosomes employ proteolytic enzymes to digest host hemoglobin from ingested human blood, including a cathepsin D-like, aspartic protease that is overexpressed in the gut of the adult female schistosome. Because of its key role in parasite nutrition, this enzyme represents a potential intervention target. To continue exploration of this potential, here we have determined the sequence, structure and genomic organization of the cathepsin D gene locus of Schistosoma mansoni. Using the cDNA encoding S. mansoni cathepsin D as a probe, we isolated several positive bacterial artificial chromosomes (BAC) from a BAC library that represents an ∼8-fold coverage of the schistosome genome. Sequencing of BAC clone 25_J_24 revealed that the cathepsin D gene locus was ∼13 kb in length, and included seven exons interrupted by six introns. The exons ranged in length from 49 to 294 bp, and the introns from 30 to 5025 bp. The genomic organization of schistosome cathepsin D was similar in sequence, structure and complexity to human cathepsin D, including to a greater or lesser extent the conservation of all six exon/intron boundaries of the schistosome gene. It was less similar to aspartic protease genes of the nematodes Caenorhabditis elegans and Haemonchus contortus, and dissimilar to those of plasmepsins from malarial parasites. Examination of the introns revealed the presence of endogenous mobile genetic elements including SR2, the ASL-associated retrotransposon, and the SINE-like element, SMα. Phylogenetically, schistosome cathepsin D appeared to be more closely related to mammalian cathepsin D than to other sub-families of eukaryotic aspartic proteases known from mammals. Taken together, these features indicated that schistosome cathepsin D is a platyhelminth orthologue of mammalian lysosomal cathepsin D.

Test of pangamy by genetic analysis of Schistosoma mansoni pairs within its natural murine host in Guadeloupe.

Mating system plays a determinant role in the maintenance and distribution of genetic variations. It can be assessed indirectly by analyzing the distribution of the genetic variability within populations or directly by considering how mating pairs are formed. In the present study, 71 pairs of adult Schistosoma mansoni worms sampled from naturally infected rats were genotyped to investigate how male and female schistosomes paired according to their genetic relatedness. Among all samples, pangamy, the random association between males and females, could not be rejected. Whereas the schistosome mating system has been intensively studied under experimental conditions, to the best of our knowledge, our study is the first to attempt to understand the way in which males and females pair in natural conditions.

Schistosoma mansonimale–female interactions

Schistosome parasites are muticellular eucaryotic organisms with a complex life cycle that involves mammalian and snail hosts. Unlike other trematode parasites, schistosomes (along with the Didymozoidae) have evolved separate sexes or dioecy. Sex is determined by a chromosomal mechanism. The dioecious state created an opportunity for the sexes to play a role in schistosome evolution that has resulted in an interesting interplay between the sexes. The classical observation, made more than 50 years ago, is that female schistosomes do not develop unless a male worm is present. Studies up through the 1990s focused on dissecting the role of the sexes in mate attraction, mate choice, mating behavior, female growth, female reproductive development, egg production, and other sex-evolved functions. In the mid-1980s, studies began to address the molecular events of male–female interactions. The classic morphological observation that female schistosomes do not complete reproductive development unless a male worm is present has been redefined in molecular terms. The male by an unknown mechanism transduces a signal that regulates female gene expression in a stage-, tissue-, and temporal-specific manner. A number of female-specific genes have been identified, along with signaling pathways and nuclear receptors, that play a role in female reproductive development. In addition, a number of host factors such as cytokines have also been demonstrated to affect adult male and female development and egg production. This review focuses on the biological interactions of the male and female schistosome and the role of parasite and host factors in these interactions as they contribute to the life cycle of Schistosoma mansoni.

Schistosoma mansoni: sex-specific modulation of parasite growth by host immune signals

Development of female schistosomes from infectious cercariae to mature egg-producing adults requires both male schistosomes and an intact adaptive immune system. By examining single sex infections in immunodeficient mice, we provide evidence that female schistosome development is not directly influenced by the adaptive immune system, whereas male development is. Our data are consistent with a sequential model of schistosome development, where the adaptive immune system signals development of mature males, which subsequently stimulate development of mature females. The male schistosome therefore appears to play a central role both in transducing signals from the adaptive immune system and in facilitating female development.

Altered behavior of the snail Biomphalaria glabrata as a result of infection with Schistosoma mansoni.

In this comparative behavioral study, the effect of infection with Schistosoma mansoni on its snail intermediate host Biomphalaria glabrata was investigated. Three groups of snails were compared for their activity: (1) uninfected, (2) infected with male parasites, and (3) infected with female parasites. In solitary movement trials, uninfected snails traveled greater distances at faster rates, explored more surface area, and had shorter rest periods than snails infected with either male or female schistosomes. In Y-maze experiments designed to determine attraction, the uninfected snails more often and more quickly moved toward other snails than the infected individuals. Snails from all 3 groups were more attracted to infected individuals than to uninfected ones. There was no difference in attraction toward snails infected with male or female parasites. These experiments provide evidence that behavioral alterations as a result of infection may lead to aggregation of infected snails in the field. We propose that such an effect may result in enhanced parasite transmission.

Quantification of DNA synthesis in multicellular organisms by a combined DAPI and BrdU technique.

The development of a novel method to detect and quantify mitotic activity in multicellular organisms is reported. The method is based on the combinatorial use of 4',6-diamidino-2-phenylindole (DAPI) as a dye for the specific staining of DNA and the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) as a marker for DNA synthesis. It is shown that on nitrocellulose filters, the amount of DNA can be determined by DAPI as a prerequisite for the subsequent quantification of mitotic activity by BrdU. As a model system to prove the applicability of this technique, the blood fluke Schistosoma mansoni has been used. It is demonstrated that the DNA synthesis rate is higher in adult female schistosomes than in adult males. Furthermore, dimethyl sulfoxide, a widely used solvent for many mitogens and inhibitors of mitosis, has no influence on mitotic activity in adult schistosomes.

Presidential address. Sex and schistosomes: an interesting biological interplay with control implications.

Schistosome parasites are muticellular eucaryotic organisms that have a complex life cycle. The egg stage produced by the worm pairs is important in transmission of the parasite and responsible for pathogenesis. For 1 species, Schistosoma mansoni, the female worm produces approximately 300 eggs per day (Moore and Sandground, 1956). The eggs, after they are deposited by the female schistosome, incite a granulomatous inflammatory reaction (Damian, 1987). Approximately half of the deposited eggs reach the outside environment by passing through the wall of the intestine and being voided with excreta, to continue the life cycle. The other 50% are swept up in the circulation and filter out in the periportal tracts of the liver, resulting in periportal fibrosis, portal hypertension, and the serious sequelae of intestinal schistosomiasis such as hepatosplenomegaly and esophageal and gastric varicies.

Relationship between worm burden and male proportion in Schistosomamansoni experimentally infected rodents and primates. A meta-analytical approach

This paper investigates the relationship between the worm burden and the male proportion of adult Schistosoma mansoni. This relationship was tested using data from the literature on S. mansoni experimentally infected rodents and primates. The analyses were performed within each animal model (rat, mouse, hamster and monkey) and showed positive linear relationships between worm burden and male proportion. An analysis between the four animal models showed the same positive linear relationship. The role of the genetic diversity of male and female schistosomes in this relationship is discussed.

Schistosome male-female interaction: induction of germ-cell differentiation.

Male and female schistosomes are permanently paired while they live in the bloodstream of their vertebrate hosts. Female schistosomes produce eggs only when they are in intimate association with a male. Here, I combine classical cytological knowledge about the cellular processes in the female that are affected by the male with recent molecular results that are beginning to allow speculation about the signalling events involved.

PCR effectiveness for sexing Schistosoma mansoni cercariae: application for sexing clonal cercarial populations

Schistosoma mansoni is a digenean parasite known for its importance as a human pathogen. Compared with the other genera belonging to the digenean families, the Schistosoma genus is characterized by having acquired gonochorism from hermaphroditic ancestors [1]. Sex is genetically determined in the egg [2] and female schistosome is heterogametic (ZW) such as lepidoptera, reptiles and birds. A marked sexual dimorphism exists between male and female worms in the definitive host. However, no clear evidence for sexual dimorphism exists in the free larval stages (miracidia or cercariae) and in the intramolluscan stages (sporocysts). Morphological, histological and molecular methods have been developed in order to discriminate between male and female schistosome larvae. Differences in the number and pattern of the argentophilic papillae (chaetotaxy) were observed between male and female cercariae [3]. Histological studies were based on the fact that the female exhibits heterochromatin of the W chromosome in the interphase nuclei. Staining methods were applied on cercariae [4] and C-banding on miracidia, cercariae and sporocysts of S. mansoni [5]. Since 10 years, molecular methods have emerged in order to sex schistosome larvae and are always based on the heterosexual female-specific W chromosome. Three DNA repeated sequences were isolated and characterized from female W chromosome, D9 [6], W1 [7] and W2 [8], whose genomic sizes are ,respectively, 339, 476 and 715 bp. The W1 repeated sequence was shown to be female-specific in each stage of the life cycle in a Puerto Rican strain of the parasite [9]. These authors used a PCR technique with W1-specific primers on extracted DNA. The female specificity was confirmed by Southern blot. However, Grevelding [10] contested the female specificity of W sequences. This author showed, using extracted adult DNA, that the femalespecific W1 sequence may occur in both genders of a Liberian strain, but reminds female-specific in a Puerto Rican strain of S. mansoni. The variability of the W gender-specificity was studied for many strains by Quack et al. [11]. This study revealed that W1 and W2 elements exist in both genders in numerous strains, Liberian, Old Kenya, New Kenya, Senegal, Campinas and an isolate of Puerto Rican strain. But, the Old Brazilian strain remained W1 and W2 female-specific. The presence of W sequences in males comes from genomic instability, probably originating from DNA recombinations during sporocyst development [12]. The PCR technique commonly uses extracted DNA. However, a PCR technique without DNA extraction (direct PCR) was developed for individual S. mansoni cercariae [13]. This paper aims to test the effectiveness of the direct PCR on cercariae from a W1 female-specific strain and to propose an optimized protocol in order to determine the sex of mollusc infection. * Corresponding author. Tel: +33-4-68662050; fax: +33-468662281. E-mail address: mone@univ-perp.fr (H. Mone).

A confocal microscopical study of the musculature of adult Schistosoma mansoni.

Using the filamentous actin marker, FITC-conjugated phalloidin, the major muscle systems of adult male and female schistosomes have been examined. The body wall musculature comprises an outer sheath of circular fibres, within which there is a compact layer of short, spindle-shaped longitudinal fibres and a lattice-like arrangement of inner diagonal fibres. Within the oral sucker and acetabulum 3 fibre types, circular, radial and longitudinal can be distinguished. The wall of the oesophagus is lined by a grid-like array of circular and longitudinal fibres, whereas the walls of the intestinal caeca contain only comparably broad circular fibres. Within the female reproductive system, only circular fibres are present in the oviduct, vitelline duct and uterus. In contrast, the wall of the ootype displays closely arranged circular and longitudinal muscle fibres. Antisera to previously identified myoactive compounds (serotonin [5-hydroxytryptamine, 5-HT], neuropeptide F [Moniezia expansa] and GYIRFamide [Bdelloura candida, Dugesia tigrina]) were used as neuronal markers in a preliminary study of the spatial inter-relationships of specific nerve fibres and various muscle systems. Serotoninergic fibres innervate both suckers and also constitute a subtegumental nerve net. In males they provide innervation to the dorso-ventral muscle fibres of the gynaecophoric canal, and in females they innervate the circular and longitudinal muscle fibres of the ootype. Neuropeptide F and the FMRFamide-related peptide, GYIRFamide are both localized within nerve plexuses associated with the dorso-ventral fibres of the gynaecophoric canal, and are evident in the innervation of the ventral and oral sucker.

Changes of mate occur in Schistosoma mansoni.

Male and female schistosomes are generally assumed to form stable monogamous pairs for the whole span of their long existence in the mammalian host. Recent evidence from mixed infections has shown that Schistosoma mansoni males can displace S. intercalatum males from their homologous partners, but no information exists about the existence of similar phenomena within a single schistosome species. Here, we determine whether male S. mansoni can displace males of the same species from pre-formed pairs in vivo. The availability of clear-cut genetic markers of drug resistance in schistosomes was exploited to show that hycanthone sensitive S. mansoni males can displace homospecific hycanthone resistant males from pre-formed pairs and vice versa. The frequency of changes is dependent on the magnitude of the excess single males competing with paired worms. The possible mechanics and the biological significance of mate changing are discussed.

Developmental expression of cathepsin D aspartic protease in Schistosoma japonicum

Schistosomes utilise haemoglobin from ingested host erythrocytes as their main source of amino acids. Using reverse-transcriptase PCR, we detected transcripts encoding cathepsin D in eggs, miracidia, and adult male, female and mixed-sex Schistosoma japonicum. Using the synthetic fluorogenic peptide, o-aminobenzoyl-isoleucyl-glutamyl-phenylalanyl- p-nitro-phenylalanyl-arginyl-leucine-NH 2, and human haemoglobin as substrates, we detected cathepsin D-like aspartic protease activity at pH 3.6 in extracts of these developmental stages which was completely inhibited by the addition of 10 μM pepstatin. Using immunoblotting with rabbit antibodies raised against recombinant S. japonicum cathepsin D, we detected the aspartic protease in extracts of all developmental stages examined, although it appeared to be expressed at higher levels in the adult female schistosome. These results indicate that (almost) all stages of S. japonicum express an aspartic protease. Moreover, they are consistent with the hypothesis that this enzyme plays a key role in maturing and adult schistosomes in the proteolysis of host haemoglobin from ingested erythrocytes.

The Oral Route as a Potential Way of Transmission of Schistosoma bovis in Goats

The infectivity of Schistosoma bovis cercariae administered orally was evaluated in Sahelian goats. Compared to the percutaneous route, a single massive oral dose resulted in a worm burden and in fecal egg excretion reduced by one-half. Surprisingly, tissue egg counts were increased by more than 4-fold. Fecundity of individual female schistosomes was, therefore, markedly increased. When infective doses were administered weekly for 20 wk, both worm and egg burdens were doubled without modification of the individual worm pair fecundity. Repeated oral infections seem to have induced an acquired tolerance toward parasite antigens. These results confirm the epidemiologic relevance of the oral route in a host species inclined to become infected through drinking water rather than percutaneous exposures.

Murine Schistosoma bovis infection: analysis of parasitic and immune parameters.

Humoral and cellular responses to Schistosoma bovis antigens have been evaluated over a period of 11 weeks in mice exposed to S. bovis cercariae and data analysed in the context of the parasitic parameters (worm and egg loads) recorded at days 30, 60 and 80 of the ongoing infection. Results revealed a decrease of worm burden, particularly marked for female worms, between day 60 and day 80 of infection suggesting a higher susceptibility of female schistosomes to attrition mechanisms. The B-cell response, studied by measuring the production of different isotypes, was directed against different stage specific antigens, with a predominance of IgG1 antibodies associated with a significant increase of IgA and IgE antibodies after egg deposition. The T-cell response, assessed after in vitro stimulation of splenocytes, showed a predominant production of Th-2 cytokines (IL-4, IL-5 and IL-10) occurring after egg laying. Interestingly in contrast to S. mansoni infection the Th-2 polarization did not seem to be exclusively triggered by egg-associated antigens since significant amounts of IL-10 were produced after stimulation with adult worm antigen preparation (SWAP) before the beginning of egg deposition.