Gonad RNA-specific qRT-PCR analyses identify genes with potential functions in schistosome reproduction such as SmFz1 and SmFGFRs.

In the search for new strategies to fight schistosomiasis, the unique reproductive biology of Schistosoma mansoni has come into the focus of research. The development of the gonads and the ability of egg production are fundamental not only for continuing the life cycle but also for pathogenicity. Previous studies of schistosome biology demonstrated an influence of pairing on gonad development of the female and on gene expression profiles in both genders. Due to the limited access to specific tissues, however, most of these studies were done at the level of whole worms neglecting individual tissues that may be targets of pairing-dependent processes. Recently, we established a protocol allowing the isolation of testes and ovaries from adult S. mansoni. Here, we describe tissue-specific qRT-PCR analyses comparing transcript levels of selected genes on the basis of RNA from gonads and whole worms. Gene expression in ovary and testes was in some cases found to be significantly influenced by pairing, which was not traceable in whole worms. Among the candidate genes identified as regulated by pairing in gonads were the frizzled homolog SmFz1 and the two fibroblast growth factor receptor homologs SmFGFR-A and SmFGFR-B. First functional characterizations were done, including comparative qRT-PCR analyses, in situ-localization experiments, heterologous expression in Xenopus oocytes (SmFGFR-A/B), and inhibitor studies using the Fz/Dvl-pathway inhibitor 3289-8625, or BIBF1120 blocking FGFR-signaling. Besides confirming gonad localization and receptor functions, inhibitor-induced phenotypes were observed in vitro such as decreased egg production as well as drastic effects on gonad differentiation, morphology, embryogenesis, and survival of adult worms. In summary, these results emphasise the usefulness of tissue-specific qRT-PCRs for selection of candidate genes with important roles in reproduction, allowing subsequent studies to determine their suitability as drug targets.

Tissue expression patterns of Schistosoma mansoni Venom Allergen-Like proteins 6 and 7.

The Schistosoma mansoni Venom Allergen-Like proteins (SmVALs) are members of the SCP/TAPS (Sperm-Coating Protein/Tpx-1/Ag5/PR-1/Sc7) protein superfamily, which may be important in host-pathogen interactions. Whole mount in situ hybridisation demonstrated a distinct expression pattern in oral and ventral suckers of adult worms for SmVAL6 and in the oesophageal gland for SmVAL7 transcripts, respectively. Additionally, immunocytochemistry analysis corroborated SmVAL7 expression in the oesophageal gland. Analysis of protein expression across the parasite's life cycle revealed that the SmVAL6 protein is upregulated in cercariae and adult male worms. Furthermore, SmVAL6 protein was identified by mass spectrometry in tegument fractions of adult worms. Finally, we speculate on possible functions of these two SmVALs at the host-parasite interface.

A systematically improved high quality genome and transcriptome of the human blood fluke Schistosoma mansoni.

Schistosomiasis is one of the most prevalent parasitic diseases, affecting millions of people in developing countries. Amongst the human-infective species, Schistosoma mansoni is also the most commonly used in the laboratory and here we present the systematic improvement of its draft genome. We used Sanger capillary and deep-coverage Illumina sequencing from clonal worms to upgrade the highly fragmented draft 380 Mb genome to one with only 885 scaffolds and more than 81% of the bases organised into chromosomes. We have also used transcriptome sequencing (RNA-seq) from four time points in the parasite's life cycle to refine gene predictions and profile their expression. More than 45% of predicted genes have been extensively modified and the total number has been reduced from 11,807 to 10,852. Using the new version of the genome, we identified trans-splicing events occurring in at least 11% of genes and identified clear cases where it is used to resolve polycistronic transcripts. We have produced a high-resolution map of temporal changes in expression for 9,535 genes, covering an unprecedented dynamic range for this organism. All of these data have been consolidated into a searchable format within the GeneDB (www.genedb.org) and SchistoDB (www.schistodb.net) databases. With further transcriptional profiling and genome sequencing increasingly accessible, the upgraded genome will form a fundamental dataset to underpin further advances in schistosome research.

Gene expression patterns in larval Schistosoma mansoni associated with infection of the mammalian host.

BACKGROUND: The infective schistosome cercaria develops within the intramolluscan daughter sporocyst from an undifferentiated germ ball, during which synthesis of proteins essential for infection occurs. When the aquatic cercaria locates the mammalian host it rapidly penetrates into the epidermis using glandular secretions. It then undergoes metamorphosis into the schistosomulum, including replacement of its tegument surface membranes, a process taking several days before it exits the skin. Patterns of gene expression underlying this transition have been characterised. METHODS AND PRINCIPAL FINDINGS: All gene models from the S. mansoni genome (www.GeneDB.org) were incorporated into a high-density oligonucleotide array. Double-stranded cDNA from germ balls, cercariae, and day 3 schistosomula was hybridised to the array without amplification. Statistical analysis was performed using Bioconductor to reveal differentially transcribed loci. Genes were categorised on the basis of biological process, tissue association or molecular function to aid understanding of the complex processes occurring. Genes necessary for DNA replication were enriched only in the germ ball, while those involved in translation were up-regulated in the germ ball and/or day 3 schistosomulum. Different sets of developmental genes were up-regulated at each stage. A large number of genes encoding elastases and invadolysins, and some venom allergen-like proteins were up-regulated in the germ ball, those encoding cysteine and aspartic proteases in the cercaria and schistosomulum. Micro exon genes encoding variant secreted proteins were highly up-regulated in the schistosomulum along with tegument and gut-associated genes, coincident with remodelling of the parasite body. Genes encoding membrane proteins were prominently up-regulated in the cercaria and/or day 3 schistosomulum. CONCLUSIONS/SIGNIFICANCE: Our study highlights an expanded number of transcripts encoding proteins potentially involved in skin invasion. It illuminates the process of metamorphosis into the schistosomulum and highlights the very early activation of gut-associated genes whilst revealing little change in the parasite's energy metabolism or stress responses.