Female Schistosoma Japonicum Research Articles

Molecular characterization of miR-31 for regulating egg production in female Schistosoma japonicum

Schistosomiasis is caused by Schistosoma infection and affects more than 200 million people worldwide. A large number of eggs produced by adult Schistosoma play central the role in host pathology and subsequent disease dissemination. However, the underlying mechanisms of egg production in Schistosoma still need to be further elucidated. Previously, we found that miR-31 was highly enriched in the female reproductive organs of Schistosoma japonicum (S. japonicum), which was shown to be associated with ovarian development. In the present study, we analyzed the potential targets of miR-31 including mRNA and long noncoding RNAs (lncRNAs) in S. japonicum by RNA seq combined with bioinformatics. Then, six putative targets of miR-31 including three mRNAs such as EWB00_000918, EWB00_004242, and EWB00_009323 and three lncRNAs such as LncSJG_010465, LncSJG_015374 and LncSJG_013128 were further analyzed their expressions in the parasites treated with miR-31 inhibitor by qPCR to confirm their potential regulations. Whole mount in suit hybridization (WISH) analysis of some miR-31 targets were carried out to determine their colocalizations with miR-31. Furthermore, we selected EWB00_009323, which is an eggshell synthetic protein and also a target of miR-31, to inhibit its functions by small interfering RNA. The results indicated that inhibition of EB00_009323 led to decreased oviposition and defective ovarian morphology. Overall, the potential targets of miR-31 including mRNA and lncRNAs were identified in female S. japonicum and the results indicated that miR-31 coordinates with its targets, at least EWB00_009323, play an important role in ovarian development and egg production.

Artemisitene shows superiority over artemisinin in preventing Schistosoma japonica-induced liver disease

BackgroundArtemisinin (ART) analogs, such as dihydroartemisinin, arteether, artemether, and artesunate, all featuring an endoperoxide bridge, have demonstrated efficacy against schistosomiasis. Artemisitene (ATT), which contains an additional α, β-unsaturated carbonyl structure, has shown enhanced biological activities. This study aims to evaluate the anti-schistosomaiasis japonica activity of ATT and compare it with ART.MethodsWe assessed liver inflammation and fibrosis in mice using hematoxylin and eosin staining and Sirius red staining, respectively. RNA sequencing analyzed transcriptomics in female and male Schistosoma japonicum (S. japonicum) adult worms and mice livers, with cytokine profiling and flow cytometry to study immune responses under ART or ATT treatment.ResultsATT exhibits a marked reduction in female S. japonicum adult worms and egg numbers, damaging the adult worms’ surface. It also influences the transcription of genes related to cellular anatomical structures. Notably, ATT treatment resulted in significant reductions in liver granuloma size and collagen area, alongside lowering serum levels of glutamic pyruvic and glutamic oxaloacetic transaminase more effectively than ART. Both ART and ATT markedly decreased neutrophil frequency in the liver and elevated eosinophil counts. However, only ATT treatment significantly reduced the M1/M2 and Th1/Th2 indices, indicating a pronounced shift in immune response profiles. ATT-affected host immunity correlated with the extent of liver fibrosis and the count of single males more strongly than ART.ConclusionATT, as a novel preventive strategy for schistosomiasis japonica in mice, significantly outperforms ART.Graphical

An improved medium for in vitro studies of female reproduction and oviposition in Schistosoma japonicum

BackgroundSchistosomiasis is a disease primarily caused by eggs laid by pathogens called schistosomes. Among the schistosome species infecting humans, Schistosoma japonicum possesses the largest fecundity; each adult female produces an average of 3500 eggs per day. The lack of proper culture conditions supporting continuous oviposition in vitro has precluded detailed investigation of mechanisms regulating sexual maturation and egg production in Schistosoma japonicum.MethodsWe optimized in vitro culture conditions by replacing reagents that are part of the classical ABC169 medium. Fast Blue BB staining and 4′,6-diamidino-2-phenylindole (DAPI) labeling were applied to observe the sexual development status of the females. In vitro RNA interference (RNAi) technology was used to validate the capability of the modified medium. The detection of male β-alanyl-tryptamine (BATT) was conducted using liquid chromatography–mass spectrometry (LC–MS).ResultsBoth m-AB169 (1640) and AB169 (1640) media are capable of facilitating the sexual development of paired virgin female S. japonicum, as well as sustaining the mature reproductive organs and egg production of adult S. japonicum for at least 22 days in vitro. M-AB169 (1640) provided a more stable condition for supporting the sexual maturity of female S. japonicum, as evidenced by the consistent initiation of egg production compared with AB169 (1640). Through a comparative analysis of S. japonicum and S. mansoni in diverse media, we demonstrated that these closely related species display distinct demands for their sexual development and egg production, suggesting a potential influence of nutritional factors on the observed variations in host ranges among different schistosome species. Importantly, we successfully identified the presence of the pheromone β-alanyl-tryptamine (BATT) in S. japonicum, previously identified in S. mansoni, highlighting its conserved role in schistosome reproductive development. Through the employment of double-stranded RNA (dsRNA) treatment to silence two genes that are involved in either the male (gli1, glioma-associated oncogene homolog 1) or female (vf1, vitellogenic factor 1) side in male-induced female reproductive development of S. mansoni, we confirmed that the combination of m-AB169 (1640) and RNAi technology has the capacity to facilitate in vitro studies of S. japonicum’s reproductive and oviposition processes.ConclusionsWe developed a novel medium, m-AB169 (1640), that not only maintains the mature reproductive organs and continuous oviposition of adult female Schistosoma japonicum for up to 22 days but also supports the reproductive development and subsequent egg-laying of virgin females after pairing with male worms. This study provides a valuable in vitro platform for functional studies of the mechanisms underlying the fascinating biology of the female sexual development and egg production of S. japonicum, which may accelerate the development of new strategies targeting schistosome egg production.Graphical

MicroRNA-1 targets ribosomal protein genes to regulate the growth, development and reproduction of Schistosoma japonicum

Eggs laid by mature female schistosomes are primarily responsible for the pathogenesis of schistosomiasis and critical for transmission. Consequently, elucidating the mechanism of sexual maturation as well as egg production may lead to new strategies for the control of schistosomiasis. MicroRNAs (miRNAs) are involved in multiple biological processes including reproduction in many organisms, yet their roles have not been well characterized in schistosomes. Here, we investigated microRNA-1 (miR-1), which was downregulated gradually in both male and female Schistosoma japonicum after they reached sexually maturity. The expression of miR-1, as shown with quantitative reverse transcription PCR (qRT-PCR), was lower in the reproductive organs of adult females compared with the somatic tissues. Overexpression of miR-1 in adult worms destroyed the morphological architecture of reproductive organs and reduced the subsequent oviposition, which may be due to the activation of apoptosis pathways. Through in silico analysis, 34 potential target genes of miR-1 were identified, including five ribosomal protein genes, called rp-s13, rp-l7ae, rp-l14, rp-l11 and rp-s24e. In vitro dual-luciferase reporter gene assays and miRNA overexpression experiments further validated that these ribosomal protein genes were directly regulated by miR-1. In contrast to the gene expression of miR-1, qRT-PCR and in situ hybridization experiments demonstrated these ribosomal protein genes were enriched in the sexual organs of adult females. Using RNA interference to silence the ribosomal protein genes in different developmental stages in a mouse model system, we demonstrated that these miR-1 target genes not only participated in the reproductive development of S. japonicum, but also were required for the growth and survival of the parasite in the early developmental stages. Taken together, our data suggested that miR-1 may affect the growth, reproduction and oviposition of S. japonicum by targeting the ribosomal protein genes, which provides insights for exploration of new anti-schistosome strategies.

Extended survival and reproductive potential of single-sex male and female Schistosoma japonicum within definitive hosts

Schistosomiasis is caused by dioecious helminths of the genus Schistosoma. Recent work indicated that unpaired female and male schistosomes can survive within their definitive host for at least 1 year, although the viability or fertility of these worms after subsequent pairing remained untested. We performed two experiments on laboratory mice, one with female Schistosoma japonicum exposure first and male schistosomes second and another vice versa. After surviving as single-sex unpaired forms for up to 1 year, 58.5% of male and 70% of female schistosomes were able to mate and produce viable eggs. This highlights an additional biological challenge in achieving elimination of schistosomiasis.

Proteomic and deep sequencing analysis of extracellular vesicles isolated from adult male and female Schistosoma japonicum.

Schistosomes are the causative agent of schistosomiasis, which affects more than 200 million people worldwide. Unlike other trematode parasites, schistosomes (along with the Didymozoidae) have evolved separate sexes. Pairing of males and females is a prerequisite for female sexual development and subsequent egg production. However, the mechanisms underlying these processes remain poorly understood. Extracellular vesicles (EVs) have been shown to play important roles in many biological processes. In the present study, we characterized EVs isolated from adult male and female Schistosoma japonicum. Proteomic analyses of the isolated EVs revealed that some proteins are significantly enriched in male or female EVs. RNA-sequencing analysis of a small RNA population associated with EVs identified 18 miRNAs enriched in male and female S. japonicum EVs. Among these, miR-750 was specifically enriched in female EVs. Additionally, the inhibition of miR-750 by a miRNA inhibitor led to decreased egg production in female schistosomes cultured in vitro. Collectively, our results suggest that miR-750 within female EV cargo may be involved in regulating ovary development and egg production in S. japonicum females.

Comparative Metabonomic Investigations of Schistosoma japonicum From SCID Mice and BALB/c Mice: Clues to Developmental Abnormality of Schistosome in the Immunodeficient Host.

The growth and development of schistosome has been affected in the immunodeficient hosts. But it remains unresolved about the molecular mechanisms involved in the development and reproduction regulation of schistosomes. This study tested and compared the metabolic profiles of the male and female Schistosoma japonicum worms collected from SCID mice and BALB/c mice at 5 weeks post infection using liquid chromatography tandem mass spectrometry (LC-MS/MS) platform, in which the worms from SCID mice were the investigated organisms and the worms from BALB/c mice were used as the controls. There were 1015 ion features in ESI+ mode and 342 ion features in ESI- mode were identified after filtration by false discovery rate. Distinct metabolic profiles were found to clearly differentiate both male and female worms in SCID mice from those in BALB/c mice using multivariate modeling methods including the Principal Component Analysis (PCA), Partial Least Squares Discriminant Analysis (PLS-DA), and Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA). There were more differential metabolites in female worms than in male worms between SCID mice and BALB/c mice. And common and uniquely perturbed metabolites and pathways were identified among male and female worms from SCID mice when compared with BALB/c mice. The enriched metabolite sets of the differential metabolites in male worms between SCID mice and BALB/c mice included bile acid biosynthesis, taurine and hypotaurine metabolism, sphingolipid metabolism, retinol metabolism, purine metabolism, etc. And the enriched metabolite sets of differential metabolites in female worms included retinol metabolism, alpha linolenic acid and linoleic acid metabolism, purine metabolism, sphingolipid metabolism, glutamate metabolism, etc. Further detection and comparison in transcript abundance of genes of the perturbed retinol metabolism and its associated meiosis process in worms identified clues suggesting accumulated retinyl ester and perturbed meiotic process. These findings suggested an association between the schistosome with retarded growth and development in SCID mice and their perturbed metabolites and metabolic pathways, and provided a new insight into the growth and development regulation of S. japonicum worms from the metabolic level, which indicated great clues for discovery of drugs or vaccines against the parasites and disease with more researches.

Dynamic transcriptomes identify biogenic amines and insect-like hormonal regulation for mediating reproduction in Schistosoma japonicum

Eggs produced by the mature female parasite are responsible for the pathogenesis and transmission of schistosomiasis. Female schistosomes rely on a unique male-induced strategy to accomplish reproductive development, a process that is incompletely understood. Here we map detailed transcriptomic profiles of male and female Schistosoma japonicum across eight time points throughout the sexual developmental process from pairing to maturation. The dynamic gene expression pattern data reveal clear sex-related characteristics, indicative of an unambiguous functional division between males and females during their interplay. Cluster analysis, in situ hybridization and RNAi assays indicate that males likely use biogenic amine neurotransmitters through the nervous system to control and maintain pairing with females. In addition, the analyses indicate that reproductive development of females involves an insect-like hormonal regulation. These data sets and analyses serve as a foundation for deeper study of sexual development in this pathogen and identification of novel anti-schistosomal interventions.

The Up-Regulation of Ribosomal Proteins Further Regulates Protein Expression Profile in Female Schistosoma japonicum after Pairing

BackgroundPairing of Schistosoma males and females leads to and maintains female sexual maturation. However, the mechanism by which pairing facilitates sexual maturation of females is not clear. An increasing body of evidence suggests that ribosomal proteins have regulatory rather than constitutive roles in protein translation.Methodology/Principal FindingsTo investigate the effect of ribosome regulation on female sex maturation, Solexa and iTRAQ techniques were used to analyze the relationship between ribosomal gene or protein expression and sexual development of Schistosoma females. In the present study, considerably higher number of ribosomal genes or proteins were found to be differentially expressed in paired 23-day-old females. Moreover, mature female-specific proteins associated with egg production, such as ferritin-1 heavy chain and superoxide dismutase, were selectively highly expressed in paired females, rather than higher level of protein synthesis of all transcripts compared with those in unpaired 23-day-old females. Furthermore, other developmental stages were utilized to investigate different expression pattern of ribosomal proteins in females by analysing 18-day-old female schistosomula from single- or double-sex infections to determine the relationship between ribosomal protein expression pattern and development. Results showed that undeveloped 18-day-old females from single- and double-sex infections, as well as 23-day-old unpaired females, possessed similar ribosomal protein expression patterns, which were distinct from those in 23-day-old paired females.Conclusions/SignificanceOur findings reveal that the pairing of females and males triggers a specialized ribosomal protein expression profile which further regulates the protein profile for sexual maturation in Schistosoma japonicum, based on its gene expression profile.

Intake of Erythrocytes Required for Reproductive Development of Female Schistosoma japonicum.

The reproductive development and maturation of female schistosomes are crucial since their released eggs are responsible for the host immunopathology and transmission of schistosomiasis. However, little is known about the nutrients required by female Schistosoma japonicum during its sexual maturation. We evaluated the promoting effect of several nutrients (calf serum, red blood cells (RBCs), ATP and hypoxanthine) on the reproductive development of pre-adult females at 18 days post infection (dpi) from mixed infections and at 50 dpi from unisexual infections of laboratory mice in basic medium RPMI-1640. We found RBCs, rather than other nutrients, promoted the female sexual maturation and egg production with significant morphological changes. In 27% of females (18 dpi) from mixed infections that paired with males in vitro on day 14, vitelline glands could be positively stained by Fast Blue B; and in 35% of females (50 dpi) from unisexual infections on day 21, mature vitelline cells were observed. Infertile eggs were detected among both groups. To analyze which component of mouse RBCs possesses the stimulating effect, RBCs were fractionated and included in media. However, the RBC fractions failed to stimulate development of the female reproductive organs. In addition, bovine hemoglobin hydrolysate, digested by neutral protease, was found to exhibit the promoting activity instead of untreated bovine hemoglobin. The other protein hydrolysate, lactalbumin hydrolysate, exhibited a similar effect with bovine hemoglobin hydrolysate. Using quantitative RT-PCR, we found the expression levels of four reproduction-related genes were significantly stimulated by RBCs. These data indicate that RBCs provide essential nutrients for the sexual maturation of female S. japonicum and that the protein component of RBCs appeared to constitute the key nutrient. These findings would improve laboratory culture of pre-adult schistosomes to adult worms in medium with well-defined components, which is important to investigate the function of genes related to female sexual maturation.

ATP synthase: an identified target gene of bantam in paired female Schistosoma japonicum.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that function in transcriptional and post-transcriptional regulation of gene expression. An increasing number of schistosome miRNAs have been identified and are expected possibly involved in differentiation, development, and metabolism. However, limited information is available concerning the target genes of schistosome miRNAs. In the present study, the key target genes of bantam, an abundant miRNA found in paired female Schistosoma japonicum, were predicted by bioinformatics analysis and Solexa technology. Luciferase reporter assay and bantam mimic assay were applied in combination to further verify the targets of bantam. Results showed that ATP synthase (CAX76793.1), one of the three selected predicted targets, was confirmed as the target of bantam; bantam mimic assay results also showed that the two other predicted targets, namely, ataxia telangiectasia mutated (ATM)-related (XP_002571630.1), and ribosomal protein L30 (CAX72575.1), were not confirmed as targets. This research proposed the design and significance of reasonable biological experiments that could be performed to identify miRNA target genes in schistosomes.

Novel expression profiles of microRNAs suggest that specific miRNAs regulate gene expression for the sexual maturation of female Schistosoma japonicum after pairing

BackgroundSchistosoma japonicum is one of the major causative agents of schistosomiasis. The pairing of males and females leads to female sexual maturation and maintains this mature state. However, the mechanisms by which pairing facilitates sexual maturation are yet to be investigated.MethodsParasites isolated from single- and double-sex cercariae-infected mice were analyzed by Solexa to uncover pair-regulated miRNA profiles. To reveal the biological functions of differentially expressed miRNAs among the samples, we predicted the target genes of these differentially expressed miRNAs and compared the gene expression between 23-d-old female schistosomula from double-sex infections (23DSI) and 23-d-old female schistosomula from single-sex infections (23SSI) by analyzing digital gene expression profiling (DGE). KEGG pathway analysis was used to investigate the relevant biological processes of these target genes to understand the significance of differentially expressed miRNAs after pairing.ResultsThe differentially expressed miRNA profiles of female 18- and 23-d post-single- and double-sex infections were analysed by Solexa. Similar miRNA profiles were observed in 18SSI and 18DSI, with the presence of identically expressed high-abundance miRNA, such as miRNA-1, miRNA-71b-5p and let-7. By contrast, in 23DSI and 23SSI, most of these high-abundance miRNAs were down-regulated. Furthermore, among all samples, bantam was distinctly up-regulated in 23 DSI, and miR-1, miR-71, miR-7-5p, and miR-7 were distinctly up-regulated in 23SSI. The transcriptomes of 23DSI and 23SSI revealed that the predicted target genes of miRNA-1, miRNA-71, miRNA-7, and miR-7-5p were associated with the ribonucleoprotein complex assembly and microtubule-based process. Conversely, the predicted target genes of bantam were related to the embryo development, development of primary sexual characteristics and regulation of transcription. KEGG pathway analysis revealed that in unpaired females, the highly-expressed miRNA-1, miRNA-71, miRNA-7, and miR-7-5p only inhibited the limited pathways, such as proteasome and ribosome assembly. Meanwhile, in paired mature females, highly-expressed bantam inhibited more biological pathways, such as the citrate cycle, glycolysis, fatty acid biosynthesis and RNA degradation.ConclusionsThe differentially expressed miRNAs between 23SSI and 23DSI and their different functions indicated that more genes or metabolic pathways in paired mature females were inhibited than those in unpaired ones. The results suggested that after pairing, specific miRNAs regulated gene expression to lead to female sexual maturation.

Transcriptome profilings of female Schistosoma japonicum reveal significant differential expression of genes after pairing

Pairing of Schistosoma japonicum initiates female development, leads to female sexual maturation, and maintains this mature state. To understand the mechanism involved in these processes, we studied parasites isolated from single- and double-sex cercariae-infected mice using deep-sequencing analysis, Solexa, to uncover pair-regulated transcriptional profiles. In this study, we report the results of high-throughput tag-sequencing (Tag-seq) analysis of the transcriptome of female worms 18 and 23 days postsingle- and double-sex infections. We sequenced over 3 million tags, obtained a total of 14,034, 27,251, 22,755, and 22,555 distinct tags corresponding to 5,773, 9,794, 8,885, and 8,870 tag-mapped genes for 23-day-old female schistosomula from double-sex infections (23DSI), 23-day-old female schistosomula from single-sex infections (23SSI), 18-day-old female schistosomula from double-sex infections (18DSI), and 18-day-old female schistosomula from single-sex infections (18SSI), respectively. Analyses of differentially expressed genes revealed similarities in the gene expression profiles between 18SSI and 18DSI as well as rational differential gene expression between 18SSI and 23SSI. However, fewer upregulated genes were found in 23DSI compared with 18DSI. Of the 3,446 differentially expressed genes between 23DSI and 23SSI, 2,913 genes were upregulated in 23SSI, whereas only 533 genes were upregulated in 23DSI. In these upregulated genes in 23DSI, phosphoglycerate mutase, superoxide dismutase, egg antigen, ribosomal proteins, ferritin-1 heavy chain, and eukaryotic translation initiation factor 2 were detected. Detection of these genes suggests that gene expression in 23DSI is specialized for functions such as promotion and maintenance of female sexual maturation and egg production. Quantitative real-time (RT)-PCR analysis confirmed the Solexa results, thereby supporting the reliability of the system. Our results offer new insights into the biological significance of pairing, which directs the expression of genes specific for sexual maturation and egg production.

Proteomic Analysis of Tegument-Exposed Proteins of Female and Male Schistosoma japonicum Worms

The interplay between sexes is a prerequisite for female growth, reproductive maturation, and egg production, and the basis of schistosome pathopoiesis and propagation. The tegument is in direct contact with the host environment and its surface membranes are particularly crucial for schistosome survival in the definitive host. In this study, a streptavidin-biotin affinity purification technique combined with LC-MS/MS was used to analyze putative tegument-exposed proteins in female and male adult Schistosoma japonicum worms. In total, 179 proteins were identified in females and 300 in males, including 119 proteins common to both sexes, and 60 female biased and 181 male biased proteins. Some (e.g., serpin and CD36-like class B scavenger receptor) were involved in host-schistosome interactions, while some (e.g., gynecophoral canal protein) were important in the interplay between sexes. Gene Ontology analysis revealed that proteins involved in protein glycosylation and lysosome were highly expressed in females, while proteins involved in intracellular signal transduction, regulation of actin filament polymerization, and proteasome core complex were highly expressed in males. These results might elucidate physiological differences between the sexes. Our study provides new insights into schistosome growth and sexual maturity in the final host and permits the screening of vaccine candidates or drug targets for schistosomiasis.

Worm morphology of Schistosoma japonicum using confocal laser scanning microscopy

Male and female Schistosoma japonicum worms have dissimilar appearances in their final host. In this study, a morphometric and morphological assessment of whole worms derived from unisexual and mixed infections in mice was conducted using confocal laser scanning microscopy. Worms from mixed infections showed significant morphological changes between 15 and 25 days post-infection (PI). On the fifteenth day PI, 33% of males had formed the conspicuous gynecophoric canal, but only 8% of them had testicular lobes containing a few germinative cells; 13% of females had incipient ovaries with a few immature ovarian cells inside. On the twentieth day PI, the testicular lobes contained more germinative cells in all male worms, while female worms presented vitelline glands. On the twenty-fifth day PI, more germinative cells were observed in the male testicular lobes, and differentiated cells were present in the female ovaries. All worms had fully developed reproductive organs from 30 days PI onwards. Morphometric analysis showed significant differences between mixed and unisexual infections at 35 days PI. Ovaries of worms from unisexual infections contained cells in one stage of maturation and vitelline glands had undifferentiated cells. Our study of S. japonicum provides a detailed comparison of different morphological traits from worms of mixed and unisexual infections throughout development.

Transcriptional profiles of adult male and female Schistosoma japonicum in response to insulin reveal increased expression of genes involved in growth and development

Microarray analysis was used to investigate differential gene regulation in adult male and female Schistosoma japonicum cultured in the presence or absence of insulin in vitro. A total of 1,101 genes were up- or down-regulated in response to insulin, the majority of differential expression occurring 24 h after the addition of insulin to the cultures. Genes differentially expressed in male or female worms were predominantly involved in growth and development, with significant sex-specific differences in transcriptional profiles evident. Insulin appeared to promote protein synthesis and control protein degradation more prominently in male parasites. The study also indicated that insulin plays a more pronounced role in the uptake of glucose in unpaired female parasites, as reflected in the increased stimulation of gene expression of the phosphatidylinositol 3-kinase sub-pathway of insulin signalling. Insulin may also impact on the sexual differentiation and fecundity of female schistosomes by activation of the mitogenic-activated protein kinase sub-pathway.

Tissue Specific Profiling of Females of Schistosoma japonicum by Integrated Laser Microdissection Microscopy and Microarray Analysis

BackgroundThe functions of many schistosome gene products remain to be characterized. A major step towards elucidating function of these genes would be in defining their sites of expression. This goal is rendered difficult to achieve by the generally small size of the parasites and the lack of a body cavity, which precludes analysis of transcriptional profiles of the tissues in isolation.Methodology/Principal FindingsHere, we describe a combined laser microdissection microscopy (LMM) and microarray analysis approach to expedite tissue specific profiling and gene atlasing for tissues of adult female Schistosoma japonicum. This approach helps to solve the gene characterization “bottle-neck” brought about by acoelomy and the size of these parasites. Complementary RNA obtained after isolation from gastrodermis (parasite gut mucosa), vitelline glands and ovary by LMM were subjected to microarray analyses, resulting in identification of 147 genes upregulated in the gastrodermis, 4,149 genes in the ovary and 2,553 in the vitellaria.ConclusionsThis work will help to shed light on the molecular pathobiology of this debilitating human parasite and aid in the discovery of new targets for the development of anti-schistosome vaccines and drugs.

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.

Identification of a putative eggshell precursor protein of the female Schistosoma japonicum

In adult worms of Schistosoma japonicum, a prominent radiolabelied female-specific protein (34 kDa) was demonstrated on fluorography of SDS gels with the pulse incorporation of 14C-tyrosine in vitro, though it was difficult to detect major femalespecific proteins by direct staining methods. This female-specific protein was demonstrated to localize exclusively in the vitelline cells by indirect immunofluorescence using the rabbit anti-34 kDa female protein antiserum. It was shown that 14C-tyrosine was selectively incorporated into the vitelline cells by the pulse labelled autoradiographs. Two days after the exposure of worms to radio-tyrosine, the shells of eggs in the uterus were demonstrated to have become radioactive, indicating that 14C-tyrosine-labelled protein was used as a material for the eggshell. In the fluorograph of proteins extracted from newly laid eggs in vitro, the prominent band was not found at the 34 kDa region, but a lot of radioactivity appeared at higher than 100 kDa. The results suggested that a 34 kDa female protein was a precursor of the eggshell and became a much larger protein molecule as a result of cross-linking during eggshell hardening.

Isoenzymes of phenol oxidase in adult female Schistosoma japonicum

Isoelectric focusing was used to study the phenol oxidase isozymes in adult female worms of Schistosoma japonicum. More than one form of phenol oxidase has been demonstrated in extracts of female worms when incubated with 3,4-dihydroxyphenylalanine (DOPA), catechol or cresol as substrates. DOPA is the best substrate among all of them. The 5–6 bands of phenol oxidase exhibit p I values in the range of 6.0–7.5, the major band is at p I 6.0.