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.

To elucidate the mechanisms of antischistosoma resistance, drug-resistant Schistosoma mansoni laboratory isolates are essential. We developed a new method for inducing resistance to praziquantel (PZQ) The current strategy for schistosomiasis control is based on large-scale treatments of populations aimed at reducing disease morbidity (WHO 2002). Currently, praziquantel (PZQ) is the drug of choice (Utzinger & Keiser 2004, Fenwick & Webster 2006, with the main advantages of its use being oral administration, single dose, low toxicity and low cost (Fenwick et al. 2003, Utzinger & Keiser 2004. Despite the advantages of PZQ, there is concern about the development of Schistosoma mansoni resistance to PZQ, both under laboratory and field conditions (Abdul-Ghani et al. 2009). In the laboratory, induction of resistance is based on the treatment of mice infected with S. mansoni, initially using sub-curative doses of PZQ. Afterwards, the dosage is increased for at least seven passages in mice/snails to complete the life cycle of the parasite (Ismail et al. 1994, Fallon et al. 1995.The complete mechanism of action of PZQ is still unclear (Doenhoff et al. 2008). Obtaining resistant strains is important for the evaluation of such mechanisms as well as for the development of alternative drugs for schistosomiasis treatment and control. Studies show that PZQ is effective not only in adult worms, but also in the intramolluscan phase of the parasite (Coelho et al. 1988. We report a novel meth- od for the induction (or selection) of S. mansoni worms resistant to PZQ using successive treatments of infected Biomphalaria glabrata snails. SUBJECTS, MATERIALS AND METHODSParasites and hosts -The S. mansoni (LE strain) life cycle was maintained using B. glabrata (Barreiro de Cima strain) snails as intermediate hosts and Swiss mice as definitive hosts, according to Pellegrino and Katz (1968) and Souza et al. (1995).Perfusion of adult worms from infected mice -Two methods were used. The methodology described by Pellegrino and Siqueira (1956) used a needle attached to a Brewer's automatic pipetter to inject saline solution under pressure into the descendent aorta. Afterwards, saline was injected into the hepatic hilum of mice after sectioning the portal vein, allowing the perfusion of the portal system and mesenteric veins. Worms were recovered and counted. To recover the worms using the methodology described by Smithers and Terry (1965) the portal vein of the mice was sectioned and the culture medium was gently injected into the base of the left ventricle of the infected mice's hearts. It is not possible to recover all the worms using this methodology with a lower pressure injection, but the integrity of the parasite's tegument is preserved. Therefore, this methodology is ideal for the recovery of worms when the goal is to cultivate or evaluate other parameters such as tegumental integrity and/or excretory activity.Induction of resistance to PZQ in the intramolluscan phase -Two-hundred B. glabrata were individually infected with 10 S. man...

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Long noncoding RNAs (lncRNAs) are transcripts generally longer than 200 nucleotides with no or poor protein coding potential, and most of their functions are also poorly characterized. Recently, an increasing number of studies have shown that lncRNAs can be involved in various critical biological processes such as organism development or cancer progression. Little, however, is known about their effects in helminths parasites, such as Schistosoma mansoni. Here, we present a computational pipeline to identify and characterize lncRNAs from RNA-seq data with high confidence from S. mansoni adult worms. Through the utilization of different criteria such as genome localization, exon number, gene length, and stability, we identified 170 new putative lncRNAs. All novel S. mansoni lncRNAs have no conserved synteny including human and mouse. These closest protein coding genes were enriched in 10 significant Gene Ontology terms related to metabolism, transport, and biosynthesis. Fifteen putative lncRNAs showed differential expression, and three displayed sex-specific differential expressions in praziquantel sensitive and resistant adult worm couples. Together, our method can predict a set of novel lncRNAs from the RNA-seq data. Some lncRNAs are shown to be differentially expressed suggesting that those novel lncRNAs can be given high priority in further functional studies focused on praziquantel resistance.

Schistosoma infection in Biomphalaria glabrata can be detected by either exposing snails to light to induce cercarial shedding or by squeezing them between glass slides to detect parasites in the digestive gland and other regions. The methods available are inefficient for identification of prepatent infections and do not allow the diagnosis of infection in snails that die before arriving in the laboratory. Furthermore, infection is undetectable after migration of sporocysts from the head-foot region of the snail. We examined the use of polymerase chain reaction (PCR) amplification of minisatellite repeats from Schistosoma mansoni mitochondrial DNA (mtDNA) to identify snail infection. We found that amplification of mtDNA under low stringency conditions (LS-PCR) allowed for the identification of specific S. mansoni infection in Biomphalaria snails. To confirm these results, specific amplification reactions were performed using 2 sets of primers that allowed for the diagnosis of infection and an internal control of the reaction (multiplex PCR). Results obtained using multiplex PCR demonstrated the ability of the assay to detect S. mansoni-specific infection. Thus, LS-PCR as well as specific multiplex PCR allow for the detection of prepatent infections and show high specificity for S. mansoni in comparison with other trematode infections in either living or dead snails.

To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (biology.unm.edu/biomphalaria-genome.html Key words: genomics -gene discovery -fingerprinting -schistosomiasis -medical malacologyThe application of molecular approaches continues to contribute novel insights into the biology, including genomics of molluscs . To date, several mitochondrial genomes of molluscs have been sequenced (DeJong et al. 2004, Mizi et al. 2005, but the nuclear genome of a representative of the Phylum Mollusca remains to be fully characterized. In fact, lophotrochozoan protostomes of which mollusca represent the largest group (Rouse 1999), are underrepresented among the animals from which the current assembly of fully sequenced genomes has been obtained. Thus, genomic data from a mollusc will help fill a gap in the information on the evolutionary history of animal life (Collins et al. 2003). Molluscs are a highly diverse group that includes some of the largest, longest living, and most intelligent invertebrates. Genome information will instruct on several remarkable properties of molluscs such as shell formation (biomineralization; Milet et al. 2004), the evolution of body asymmetry (Schilthuizen & Davison 2005), and hermaphrodism (Paraense & Corrêa 1988). Molluscs are also being used to study pharmo-toxicology (Terlau & Olivera 2004); neuroendocrinology (Altelaar et al. 2005); parthenogenesis (Jokela et al. 2003); and the molecular basis of behavior and learning (Williamson & Chrachri 2004, Zhurov et al. 2005. Molluscs serve as bioindicators for monitoring of the environment (Zhao et al. 2005), and (snails especially) are useful for understanding how natural selection operates (Vermeij 2002). Furthermore, molluscs are economically important as a major source of food, can destroy crops, colonize and impact new habitats as invasive species (Pointier et al. 2005), and transmit medically important pathogens.The latter applies to the freshwater gastropod Biomphalaria glabrata (Planorbidae, Basommatophora). This snail serves as one of the most important intermediate hosts for a widespread pathogen of humans, the digenetic trematode Schistosoma mansoni (Paraense & Corrêa 1963, Morgan et al. 2001). This parasite causes intestinal schistosomiasis, a debilitating disease that afflicts over 50 million humans (Chitsulo et al. 2004). To a large extent, the geographic distribution of B. glabrata defines the et al. 2004). B. glabrata also hosts a variety of other digenetic trematodes and has been adopted as the most commonly used model host to study the basic biology of digeneansnail interactions (Lie 1982, Adema & Loker 1997, Vergote et al. 2005. As one example, B. glabrata has been found to produce after exposure to digeneans a unique family of hemolymph molecules termed FREPs (fibrinogen-related proteins). FREPs consist of a juxtaposition of fibrinogen and immunoglobulin superfamily domains, and have proven to be remarkably diverse in their composition...

Transplantation of the haematopoietic organ from Biomphalaria tenagophila (Taim strain, RS, Brazil), resistant to Schistosoma mansoni, to a highly susceptible strain (Cabo Frio, RJ, Brazil) of the same species, showed in the recipient snails resistance against the trematode, when a successful transplant occurred. The success of transplantation could be confirmed by a typical molecular marker of the Taim strain in haemocytes of the recipients (350 bp detected by PCR-RFLP). The recipient snails which did not present the donor marker in haemocytes (unsuccessful transplantation) were infected with the parasite. The use of an atoxic modelling clay for closing the hole in the transplantation site reduced significantly the mortality caused by bleeding after transplantation procedures.

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