Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis.

BackgroundNovel methods to identify anthelmintic drug and vaccine targets are urgently needed, especially for those parasite species currently being controlled by singular, often limited strategies. A clearer understanding of the transcriptional components underpinning helminth development will enable identification of exploitable molecules essential for successful parasite/host interactions. Towards this end, we present a combinatorial, bioinformatics-led approach, employing both statistical and network analyses of transcriptomic data, for identifying new immunoprophylactic and therapeutic lead targets to combat schistosomiasis.Methodology/Principal FindingsUtilisation of a Schistosoma mansoni oligonucleotide DNA microarray consisting of 37,632 elements enabled gene expression profiling from 15 distinct parasite lifecycle stages, spanning three unique ecological niches. Statistical approaches of data analysis revealed differential expression of 973 gene products that minimally describe the three major characteristics of schistosome development: asexual processes within intermediate snail hosts, sexual maturation within definitive vertebrate hosts and sexual dimorphism amongst adult male and female worms. Furthermore, we identified a group of 338 constitutively expressed schistosome gene products (including 41 transcripts sharing no sequence similarity outside the Platyhelminthes), which are likely to be essential for schistosome lifecycle progression. While highly informative, statistics-led bioinformatics mining of the transcriptional dataset has limitations, including the inability to identify higher order relationships between differentially expressed transcripts and lifecycle stages. Network analysis, coupled to Gene Ontology enrichment investigations, facilitated a re-examination of the dataset and identified 387 clusters (containing 12,132 gene products) displaying novel examples of developmentally regulated classes (including 294 schistosomula and/or adult transcripts with no known sequence similarity outside the Platyhelminthes), which were undetectable by the statistical comparisons.Conclusions/SignificanceCollectively, statistical and network-based exploratory analyses of transcriptomic datasets have led to a thorough characterisation of schistosome development. Information obtained from these experiments highlighted key transcriptional programs associated with lifecycle progression and identified numerous anti-schistosomal candidate molecules including G-protein coupled receptors, tetraspanins, Dyp-type peroxidases, fucosyltransferases, leishmanolysins and the netrin/netrin receptor complex.

Free-living miracidia of Schistosoma mansoni, upon penetration of the their snail intermediate host, undergo dramatic morphological and physiological changes as they transform to the parasitic sporocyst stage. During this transformation process, developing larvae release a diverse array of proteins, herein referred to as larval transformation proteins (LTPs), some of which are postulated to serve a parasite protective function. In the present study, nanoLC-tandem MS analysis was performed on all proteins represented in entire 1-dimensional SDS-PAGE-separated samples in order to gain a more comprehensive picture of the protein constituents associated with miracidium-tosporocyst transformation and thus, their potential role in influencing establishment of intramolluscan infections. Of 127 proteins with sufficient peptide/sequence information, specific identifications were made for 99, while 28 represented unknown or hypothetical proteins. Nineteen percent of identified proteins possessed signal peptides constituting a cohort of classical secretory proteins, while 22% were identified as putative nonclassically-secreted leaderless proteins based on SecretomeP analysis. Proteins comprising these groups consisted mainly of proteases/protease inhibitors, small HSPs, redox/antioxidant enzymes, ion-binding proteins including those with antioxidant Fe-binding activities (ferritins, heme-binding protein), and venom allergen-like (VAL) proteins. A polyclonal antibody generated against whole LTPs recognized proteins primarily associated with the cilia, ciliated epidermal plates and intercellular ridges of miracidia and the tegument of fully-transformed sporocysts, identifying these structures as sources of a subset of LTPs. Thus lysis of plates and/or leakage during formation of the sporocyst syncytium likely represent significant contributors to the overall LTP makeup, especially identified nonsecretory proteins. However, as plate release/degradation and tegument formation are part of the normal developmental process, all LTPs regardless of tissue origin, would be expected at the parasite-host interface upon infection. This study significantly expands the repertoire of LTPs associated with larval transformation and identifies several, e.g., those involved in stress responses, proteolysis/inhibition, antioxidant and detoxication, and immune modulation, that may play a parasite protective role during this crucial period of transition.

In vitro production of Schistosoma mansoni daughter sporocysts (DS) from miracidium-derived mother sporocysts (MS) was achieved by synxenic larval cultivation with cells of the Biomphalaria glabrata embryonic (Bge) cell line. The in vitro growth and viability of MS cocultured with Bge cells or in Bge cell-conditioned medium were significantly extended beyond that of larvae cultured in fresh medium alone. However, complete DS development and emergence from MS were achieved only in the presence of Bge cells. Introduction of either miracidia or previously transformed MS onto Bge cell monolayers resulted in an initial attachment of cultured cells to sporocysts, followed by a gradual encapsulation of larvae by multiple layers of Bge cells. Sporocysts and their encapsulating cells eventually formed large cellular aggregates, within which MS increased 4-fold in size during the first 20 days of cultivation. The timing of in vitro DS development was somewhat variable; however, in general, early embryo formation, i.e., germ cell aggregates with surrounding primitive epithelium, was first detected at 15-20 days of culture, whereas motile, intra-MS daughter stages were seen at 25-30 days and thereafter. Mature, first generation DS, measuring 136 +/- 46 microns long by 22 +/- 6 microns wide, emerged from MS starting at approximately 30-45 days of initial cultivation. Although the basic morphology and size of emergent, in vitro-derived DS were comparable to those propagated in vivo, there was a large reduction in the in vitro reproductive capacity of the MS and a delay in DS culture development.(ABSTRACT TRUNCATED AT 250 WORDS)

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