Schistosomiasis is a water-borne parasitic illness caused by neoophoran trematodes of the genus Schistosoma. Using classical histological techniques and whole-mount preparations, the present work describes the embryonic development of Schistosoma mansoni eggs in the murine host and compares it with eggs maintained under in vitro conditions. Two pre-embryonic stages occur inside the female worm: the prezygotic stage is characterized by the release of mature oocytes from the female ovary until its fertilization. The zygotic stage encompasses the migration of the zygote through the ootype, where the eggshell is formed, to the uterus. Fully formed eggs are laid still undeveloped, without having suffered any cleavage. In the outside environment, eight embryonic stages can be defined: stage 1 refers to early cleavages and the beginning of yolk fusion. Stage 2 represents late cleavage, with the formation of a stereoblastula and the onset of outer envelope differentiation. Stage 3 is defined by the elongation of the embryonic primordium and the onset of inner envelope formation. At stage 4, the first organ primordia arise. During stages 5 to 7, tissue and organ differentiation occurs (neural mass, epidermis, terebratorium, musculature, and miracidial glands). Stage 7 is characterized by the nuclear condensation of neurons of the central neural mass. Stage 8 refers to the fully formed larva, presenting muscular contraction, cilia, and flame-cell beating. This staging system was compared to a previous classification and could underlie further studies on egg histoproteomics (morphological localizome). The differentiation of embryonic structures and their probable roles in granulomatogenesis are discussed herein.

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...

Mass treatment with praziquantel (PZQ) monotherapy is the mainstay for schistosome treatment. This drug shows imperfect cure rates in the field and parasites showing reduced response to PZQ can be selected in the laboratory, but the extent of resistance in Schistosoma mansoni populations is unknown. We examined the genetic basis of variation in PZQ response in a S. mansoni population (SmLE-PZQ-R) selected with PZQ in the laboratory: 35% of these worms survive high dose (73 µg/mL) PZQ treatment. We used genome wide association to map loci underlying PZQ response. The major chr. 3 peak shows recessive inheritance and contains a transient receptor potential (Sm.TRPMPZQ) channel (Smp_246790), activated by nanomoles of PZQ. Marker-assisted selection of parasites at a single Sm.TRPMPZQ SNP enriched populations of PZQ-R and PZQ-S parasites showing >377 fold difference in PZQ response. The PZQ-R parasites survived treatment in rodents better than PZQ-S. Resistant parasites show 2.25-fold lower expression of Sm.TRPMPZQ than sensitive parasites. Specific chemical blockers of Sm.TRPMPZQ enhanced PZQ resistance, while Sm.TRPMPZQ activators increased sensitivity. A single SNP in Sm.TRPMPZQ differentiated PZQ-ER and PZQ-ES lines, but mutagenesis showed this was not involved in PZQ-R, suggesting linked regulatory changes. We surveyed Sm.TRPMPZQ sequence variation in 259 individual parasites from the Newand Old World revealing one nonsense mutation, that results in a truncated protein with no PZQ binding site. Our results demonstrate that Sm.TRPMPZQ underlies variation in PZQ response in S. mansoni and provides an approach for monitoring emerging PZQ-resistance alleles in schistosome elimination programs..

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.

Eukaryotic protein kinases (ePKs) are good medical targets for drug development in different biological systems. ePKs participate in many cellular processes, including the p38 MAPK regulation of homeostasis upon oxidative stress. We propose to assess the role of Smp38 MAPK signaling pathway in Schistosoma mansoni development and protection against oxidative stress, parasite survival, and also to elucidate which target genes have their expression regulated by Smp38 MAPK. After a significant reduction of up to 84% in the transcription level by Smp38 MAPK gene knockdown, no visible phenotypic changes were reported in schistosomula in culture. The development of adult worms was tested in vivo in mice infected with the Smp38 knocked-down schistosomula. It was observed that Smp38 MAPK has an essential role in the transformation and survival of the parasites as a low number of adult worms was recovered. Smp38 knockdown also resulted in decreased egg production, damaged adult worm tegument, and underdeveloped ovaries in females. Furthermore, only ~13% of the eggs produced developed into mature eggs. Our results suggest that inhibition of the Smp38 MAPK activity interfere in parasites protection against reactive oxygen species. Smp38 knockdown in adult worms resulted in 80% reduction in transcription levels on the 10th day, with consequent reduction of 94.4% in oviposition in vitro. In order to search for Smp38 MAPK pathway regulated genes, we used an RNASeq approach and identified 1,154 DEGs in Smp38 knockdown schistosomula. A substantial proportion of DEGs encode proteins with unknown function. The results indicate that Smp38 regulates essential signaling pathways for the establishment of parasite homeostasis, including genes related to antioxidant defense, structural composition of ribosomes, spliceosomes, cytoskeleton, as well as, purine and pyrimidine metabolism pathways. Our data show that the Smp38 MAPK signaling pathway is a critical route for parasite development and may present attractive therapeutic targets for the treatment and control of schistosomiasis.