Sensitivity assessment of Biomphalaria glabrata (SAY, 1818) using reference substance sodium dodecyl sulfate for ecotoxicological analyzes.

Sodium dodecyl sulfate (SDS) is a surfactant used and recommended by regulatory agencies as a reference substance in ecotoxicological analyzes. In this work, acute toxicity assays were performed with adults and embryos of the freshwater snail Biomphalaria glabrata, an endemic organism with environmental and public health importance, to evaluate the effects of the surfactant and establish a sensitivity control chart. The organisms were exposed to SDS for 24 h to a range of concentrations, as well as a control group. Six assays were performed to establish the control chart for adults (with a median LC50 of 36.87 mg L-1) and differential sensitivity was observed at each embryonic stage (EC50 = blastulae 33.03, gastrulae 35.03, trochophore 39.71 and veliger 72.55 mg L-1). The following behavioral responses were observed in exposed adult snails: release of hemolymph and mucus, body outside the shell, and penile overexposure. Embryos at the blastulae and gastrulae stages were more sensitive, and teratogenic effects were accentuated in the trochophore stage. The difference in results obtained between adults and embryos underscore the importance of carrying out analyzes at different developmental stages. The serial assays established with SDS for B. glabrata demonstrated efficiency and constancy conditions in the assays with good laboratory practice standards. The wide distribution of Biomphalaria species in several countries, their easy maintenance and cultivation in the laboratory, in addition to ecological importance, make them economical alternatives for ecotoxicological assays.

Long non-coding RNAs are essential for Schistosoma mansoni pairing-dependent adult worm homeostasis and fertility.

The trematode parasite Schistosoma mansoni causes schistosomiasis, which affects over 200 million people worldwide. Schistosomes are dioecious, with egg laying depending on the females' obligatory pairing with males. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with low or no protein-coding potential that have been involved in other species with reproduction, stem cell maintenance, and drug resistance. In S. mansoni, we recently showed that the knockdown of one lncRNA affects the pairing status of these parasites. Here, we re-analyzed public RNA-Seq data from paired and unpaired adult male and female worms and their gonads, obtained from mixed-sex or single-sex cercariae infections, and found thousands of differentially expressed pairing-dependent lncRNAs among the 23 biological samples that were compared. The expression levels of selected lncRNAs were validated by RT-qPCR using an in vitro unpairing model. In addition, the in vitro silencing of three selected lncRNAs showed that knockdown of these pairing-dependent lncRNAs reduced cell proliferation in adult worms and their gonads, and are essential for female vitellaria maintenance, reproduction, and/or egg development. Remarkably, in vivo silencing of each of the three selected lncRNAs significantly reduced worm burden in infected mice by 26 to 35%. Whole mount in situ hybridization experiments showed that these pairing-dependent lncRNAs are expressed in reproductive tissues. These results show that lncRNAs are key components intervening in S. mansoni adult worm homeostasis, which affects pairing status and survival in the mammalian host, thus presenting great potential as new therapeutic target candidates.

Algal-Derived Halogenated Sesquiterpenes from Laurencia dendroidea as Lead Compounds in Schistosomiasis Environmental Control.

Schistosomiasis has been controlled for more than 40 years with a single drug, praziquantel, and only one molluscicide, niclosamide, raising concern of the possibility of the emergence of resistant strains. However, the molecular targets for both agents are thus far unknown. Consequently, the search for lead compounds from natural sources has been encouraged due to their diverse structure and function. Our search for natural compounds with potential use in schistosomiasis control led to the identification of an algal species, Laurencia dendroidea, whose extracts demonstrated significant activity toward both Schistosoma mansoni parasites and their intermediate host snails Biomphalaria glabrata. In the present study, three seaweed-derived halogenated sesquiterpenes, (-)-elatol, rogiolol, and obtusol are proposed as potential lead compounds for the development of anthelminthic drugs for the treatment of and pesticides for the environmental control of schistosomiasis. The three compounds were screened for their antischistosomal and molluscicidal activities. The screening revealed that rogiolol exhibits significant activity toward the survival of adult worms, and that all three compounds showed activity against S. mansoni cercariae and B. glabrata embryos. Biomonitored fractioning of L. dendroidea extracts indicated elatol as the most active compound toward cercariae larvae and snail embryos.

Rhesus macaques self-curing from a schistosome infection can display complete immunity to challenge.

The rhesus macaque provides a unique model of acquired immunity against schistosomes, which afflict >200 million people worldwide. By monitoring bloodstream levels of parasite-gut-derived antigen, we show that from week 10 onwards an established infection with Schistosoma mansoni is cleared in an exponential manner, eliciting resistance to reinfection. Secondary challenge at week 42 demonstrates that protection is strong in all animals and complete in some. Antibody profiles suggest that antigens mediating protection are the released products of developing schistosomula. In culture they are killed by addition of rhesus plasma, collected from week 8 post-infection onwards, and even more efficiently with post-challenge plasma. Furthermore, cultured schistosomula lose chromatin activating marks at the transcription start site of genes related to worm development and show decreased expression of genes related to lysosomes and lytic vacuoles involved with autophagy. Overall, our results indicate that enhanced antibody responses against the challenge migrating larvae mediate the naturally acquired protective immunity and will inform the route to an effective vaccine.

Assessment of reference genes at six different developmental stages of Schistosoma mansoni for quantitative RT-PCR.

Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most used, fast, and reproducible method to confirm large-scale gene expression data. The use of stable reference genes for the normalization of RT-qPCR assays is recognized worldwide. No systematic study for selecting appropriate reference genes for usage in RT-qPCR experiments comparing gene expression levels at different Schistosoma mansoni life-cycle stages has been performed. Most studies rely on genes commonly used in other organisms, such as actin, tubulin, and GAPDH. Therefore, the present study focused on identifying reference genes suitable for RT-qPCR assays across six S. mansoni developmental stages. The expression levels of 25 novel candidates that we selected based on the analysis of public RNA-Seq datasets, along with eight commonly used reference genes, were systematically tested by RT-qPCR across six developmental stages of S. mansoni (eggs, miracidia, cercariae, schistosomula, adult males and adult females). The stability of genes was evaluated with geNorm, NormFinder and RefFinder algorithms. The least stable candidate reference genes tested were actin, tubulin and GAPDH. The two most stable reference genes suitable for RT-qPCR normalization were Smp_101310 (Histone H4 transcription factor) and Smp_196510 (Ubiquitin recognition factor in ER-associated degradation protein 1). Performance of these two genes as normalizers was successfully evaluated with females maintained unpaired or paired to males in culture for 8 days, or with worm pairs exposed for 16 days to double-stranded RNAs to silence a protein-coding gene. This study provides reliable reference genes for RT-qPCR analysis using samples from six different S. mansoni life-cycle stages.

Multiple adverse effects of textile effluents and reactive Red 239 dye to aquatic organisms.

Textile dyeing consumes high volumes of water, generating proportional number of colored effluents which contain several hazardous chemical. These contaminants can implicate in significant changes in aquatic environmental, including several adverse effects to organisms in different trophic levels. The present study was developed to assess the ecotoxicological effects of textile effluent samples and reactive Red 239 dye (used in cotton dyeing) to aquatic organisms Vibrio fischeri bacteria, Daphnia similis crustacean, and Biomphalaria glabrata snail (adults and embryos). Chronic assays with lethal and sublethal effects for Daphnia similis were included and performed only for textile effluents samples. The mutagenicity was also evaluated with Salmonella/microsome assay (TA98, TA100, and YG1041 strains). V. fischeri bacteria was the most sensitive to reactive Red 239 dye (EC50 = 10.14 mg L-1) followed by mollusk embryos at all stages (EC50 = 116.41 to 124.14 mg L-1), D. similis (EC50= 389.42 mg L-1), and less sensitive to adult snails (LC50= 517.19 mg L-1). The textile effluent was toxic for all exposed organisms [E(L)C50 < 15%] and B. glabrata embryos showed different responses in the early stages of blastulae and gastrulae (EC50 = 7.60 and 7.08%) compared to advanced development stages trochophore and veliger (EC50 = 21.56 and 29.32%). Developmental and sublethal effects in B. glabrata embryos and D. similis were evidenced. In the chronic assay with effluent, the EC10/NOEC = 3% was obtained. Mutagenic effects were not detected for dye aqueous solutions neither for effluents samples. These data confirmed the importance of evaluating the effects in aquatic organisms from different trophic levels and reinforce the need for environmental aquatic protection.

The Prospective Use of Brazilian Marine Macroalgae in Schistosomiasis Control.

Schistosomiasis is a parasitic disease that affects more than 250 million people. The treatment is limited to praziquantel and the control of the intermediate host with the highly toxic molluscicidal niclosamide. Marine algae are a poorly explored and promising alternative that can provide lead compounds, and the use of multivariate analysis could contribute to quicker discovery. As part of our search for new natural compounds with which to control schistosomiasis, we screened 45 crude extracts obtained from 37 Brazilian seaweed species for their molluscicidal activity against Biomphalaria glabrata embryos and schistosomicidal activities against Schistosoma mansoni. Two sets of extracts were taxonomically grouped for metabolomic analysis. The extracts were analyzed by GC-MS, and the data were subjected to Pattern Hunter and Pearson correlation tests. Overall, 22 species (60%) showed activity in at least one of the two models. Multivariate analysis pointed towards 3 hits against B. glabrata veliger embryos in the Laurencia/Laurenciella set, 5 hits against B. glabrata blastula embryos, and 31 against S. mansoni in the Ochrophyta set. Preliminary annotations suggested some compounds such as triquinane alcohols, prenylated guaianes, dichotomanes, and xenianes. Despite the putative identification, this work presents potential candidates and can guide future isolation and identification.

Systems Biology Analysis of the Radiation-Attenuated Schistosome Vaccine Reveals a Role for Growth Factors in Protection and Hemostasis Inhibition in Parasite Survival.

In spite of several decades of research, an effective vaccine against schistosomiasis remains elusive. The radiation-attenuated (RA) cercarial vaccine is still the best model eliciting high protection levels, although the immune mechanisms have not yet been fully characterized. In order to identify genes and pathways underlying protection we investigated patterns of gene expression in PBMC and skin draining Lymph Nodes (LN) from mice using two exposure comparisons: vaccination with 500 attenuated cercariae versus infection with 500 normal cercariae; one versus three doses. Vaccinated mice were challenged with 120 normal parasites. Integration of PBMC and LN data from the infected group revealed early up-regulation of pathways associated with Th2 skewing and polarization of IgG antibody profiles. Additionally, hemostasis pathways were downregulated in infected mice, correlating with platelet reduction, potentially a mechanism to assist parasite migration through capillary beds. Conversely, up regulation of such mechanisms after vaccination may explain parasite blockade in the lungs. In contrast, a single exposure to attenuated parasites revealed early establishment of a Th1 bias (signaling of IL-1, IFN-γ; and Leishmania infection). Genes encoding chemokines and their receptors were more prominent in vaccinated mice, indicating an enhanced capacity for inflammation, potentially augmenting the inhibition of intravascular migration. Increasing the vaccinations from one to three did not dramatically elevate protection, but there was a clear shift towards antibody-mediated effectors. However, elements of the Th1 bias were still evident. Notable features after three vaccinations were markers of cytotoxicity (including IL-6 and NK cells) together with growth factors and their receptors (FGFR/VEGF/EGF) and the apoptosis pathway. Indeed, there is evidence for the development of anergy after three vaccinations, borne out by the limited responses detected in samples after challenge. We infer that persistence of a Th1 response puts a limit on expression of antibody-mediated mechanisms. This feature may explain the failure of multiple doses to drive protection towards sterile immunity. We suggest that the secretions of lung stage parasites would make a novel cohort of antigens for testing in protection experiments.

Long non-coding RNA levels can be modulated by 5-azacytidine in Schistosoma mansoni.

Schistosoma mansoni is a flatworm that causes schistosomiasis, a neglected tropical disease that affects more than 200 million people worldwide. There is only one drug indicated for treatment, praziquantel, which may lead to parasite resistance emergence. The ribonucleoside analogue 5-azacytidine (5-AzaC) is an epigenetic drug that inhibits S. mansoni oviposition and ovarian development through interference with parasite transcription, translation and stem cell activities. Therefore, studying the downstream pathways affected by 5-AzaC in S. mansoni may contribute to the discovery of new drug targets. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with low or no protein coding potential that have been involved in reproduction, stem cell maintenance and drug resistance. We have recently published a catalog of lncRNAs expressed in S. mansoni life-cycle stages, tissues and single cells. However, it remains largely unknown if lncRNAs are responsive to epigenetic drugs in parasites. Here, we show by RNA-Seq re-analyses that hundreds of lncRNAs are differentially expressed after in vitro 5-AzaC treatment of S. mansoni females, including intergenic, antisense and sense lncRNAs. Many of these lncRNAs belong to co-expression network modules related to male metabolism and are also differentially expressed in unpaired compared with paired females and ovaries. Half of these lncRNAs possess histone marks at their genomic loci, indicating regulation by histone modification. Among a selected set of 8 lncRNAs, half of them were validated by RT-qPCR as differentially expressed in females, and some of them also in males. Interestingly, these lncRNAs are also expressed in other life-cycle stages. This study demonstrates that many lncRNAs potentially involved with S. mansoni reproductive biology are modulated by 5-AzaC and sheds light on the relevance of exploring lncRNAs in response to drug treatments in parasites.

The antischistosomal potential of GSK-J4, an H3K27 demethylase inhibitor: insights from molecular modeling, transcriptomics and in vitro assays.

BACKGROUND: Schistosomiasis chemotherapy is largely based on praziquantel (PZQ). Although PZQ is very safe and tolerable, it does not prevent reinfection and emerging resistance is a primary concern. Recent studies have shown that the targeting of epigenetic machinery in Schistosoma mansoni may result in severe alterations in parasite development, leading to death. This new route for drug discovery in schistosomiasis has focused on classes of histone deacetylases (HDACs) and histone acetyltransferases (HATs) as epigenetic drug targets. Schistosoma histone demethylases also seem to be important in the transition of cercariae into schistosomula, as well as sexual differentiation in adult worms. METHODS: The Target-Pathogen database and molecular docking assays were used to prioritize the druggability of S. mansoni histone demethylases. The transcription profile of Smp_03400 was re-analyzed using available databases. The effect of GSK-J4 inhibitor in schistosomula and adult worms' motility/viability/oviposition was assessed by in vitro assays. Ultrastructural analysis was performed on adult worms exposed to GSK-J4 by scanning electron microscopy, while internal structures and muscle fiber integrity was investigated by confocal microscopy after Langeron's carmine or phalloidin staining. RESULTS: The present evaluation of the potential druggability of 14 annotated S. mansoni demethylase enzymes identified the S. mansoni ortholog of human KDM6A/UTX (Smp_034000) as the most suitable druggable target. In silico analysis and molecular modeling indicated the potential for cofactor displacement by the chemical probe GSK-J4. Our re-analysis of transcriptomic data revealed that Smp_034000 expression peaks at 24 h in newly transformed schistosomula and 5-week-old adult worms. Moreover, this gene was highly expressed in the testes of mature male worms compared to the rest of the parasite body. In in vitro schistosome cultures, treatment with GSK-J4 produced striking effects on schistosomula mortality and adult worm motility and mortality, as well as egg oviposition, in a dose- and time-dependent manner. Unexpectedly, western blot assays did not demonstrate overall modulation of H3K27me3 levels in response to GSK-J4. Confocal and scanning electron microscopy revealed the loss of original features in muscle fibers and alterations in cell-cell contact following GSK-J4 treatment. CONCLUSIONS: GSK-J4 presents promising potential for antischistosomal control; however, the underlying mechanisms warrant further investigation.

Schistosoma mansoni venom allergen-like protein 18 (SmVAL18) is a plasminogen-binding protein secreted during the early stages of mammalian-host infection.

Schistosomiasis is a neglected tropical disease caused by trematodes of the genus Schistosoma which have a complex life cycle characterized by an asexual multiplication phase in the snail intermediate host and a sexual reproduction phase in the mammalian definitive host. The initial steps of the human host infection involve the secretion of proteins contained in the acetabular glands of cercariae that promote parasite adhesion and proteolysis of the skin layers. Herein, we performed a functional analysis of SmVAL18, identified as one of the three SCP/TAPS proteins constituent of cercarial secretions. We evaluated the SmVAL18 binding to immobilized macromolecules of the extracellular matrix (ECM) and to plasma components. Recombinant protein, expressed in E. coli, was found to maintain an ordered secondary structure typical of the SCP/TAPS domain after purification. Expression of native SmVAL18 protein was verified to be restricted to cercariae and 3-h schistosomula stages; furthermore, the protein was observed in the corresponding secretions, confirming that SmVAL18 is secreted during the first 3 h of in vitro culture. rSmVAL18 was able to interact specifically with plasminogen (PLG) and enhance its conversion into plasmin in the presence of the urokinase-type plasminogen activator (uPA). Protein homology modelling suggested that the PLG-rSmVAL18 interaction was mediated by lysine residues of the protein. This was supported by in vitro data using the lysine analogue, 6-aminocaproic acid (ACA), which abolished the interaction. Finally, our results showed that both cercariae and 3-h schistosomula, as well as their corresponding secretions, exhibited the capacity to bind PLG and enhance its conversion into plasmin in vitro in the same way as observed for the recombinant protein. In conclusion, our findings show that SmVAL18 is a novel PLG-binding protein secreted during the early stages of the mammalian-host infection.

Stage and tissue expression patterns of Schistosoma mansoni venom allergen-like proteins SmVAL 4, 13, 16 and 24.

BACKGROUND: Schistosoma mansoni venom allergen-like protein (SmVAL) is a gene family composed of 29 members divided into group 1 encoding proteins potentially secreted, and group 2 encoding intracellular components. Some members were found to be upregulated in the transition of germ ball - cercariae - day 3 schistosomula, suggesting that group 1 SmVAL proteins are associated with the invasion of the human host, although their functions are not completely established. Recently, we have described the localization of SmVAL7 (group 1) and SmVAL6 (group 2) transcripts in the oesophageal gland and in the oral and ventral suckers of adult parasites, respectively. The expression patterns of the two genes suggest that SmVAL7 protein plays a role in the blood-feeding process while SmVAL6 is associated with the parasite attachment and movement in the vasculature. In this way, searching for additional secreted SmVAL proteins that could be involved in key processes from skin penetration to the beginning of blood-feeding, we investigated the tissue localization of SmVAL4, 13, 16 and 24 by whole-mount in situ hybridization (WISH). RESULTS: We report here the localization of group 1 SmVAL4 and 24 transcripts in the pre-acetabular glands of developing germ balls. Time course experiments of in vitro cultured schistosomula after cercariae transformation demonstrated that SmVAL4 protein is secreted during the first 3 h of in vitro culture, correlating with the emptying of acetabular glands as documented by confocal microscopy. In addition, the localization of SmVAL13 transcripts in adult male anterior oesophageal gland suggests that the respective protein may be involved in the first steps of the blood-feeding process. SmVAL16 was localized close to the neural ganglia and requires further investigation. CONCLUSIONS: Our findings demonstrate that SmVAL proteins have localizations that place them in strategic positions to be considered as potential vaccine candidates as some members are exposed to interaction with the immune system and may participate in key processes of mammalian invasion and parasitism establishment.

HSP70 expression in Biomphalaria glabrata snails exposed to cadmium.

In this study, the effects of the heavy metal cadmium on the stress protein HSP70 are investigated in freshwater mollusks Biomphalaria glabrata. Adult snails were exposed for 96h to CdCl2 at concentrations ranging from 0.09 to 0.7mgL-1 (LC50/96h=0.34 (0.30-0.37). Time and concentration-dependent increases in the expression of HSP70 were observed at sub-lethal levels in the immunoblotting assay. Further, an increased survival to a lethal heat shock was observed in animals pre-exposed to a nonlethal concentration of cadmium, evidencing the induction of acquired tolerance. The present study demonstrated the inducibility of B. glabrata HSP70 by cadmium, a relevant environmental contaminant, at non-lethal levels, providing evidences that the assessment of HSP70 in B. glabrata can be regarded as a suitable biomarker for ecotoxicological studies.

Antischistosomal activity from Brazilian marine algae

Abstract Schistosomiasis may be caused by six different species of the genus Schistosoma. Current treatment is based only on two drugs: oxamniquine, which is only effective against the Schistosoma mansoni species, and praziquantel, which is ineffective against young parasites. Therefore, research on new drugs and their targets for the treatment of this disease is urgently needed. In the present work, the efficacies of several seaweeds extracts against S. mansoni were tested. Worm couples were incubated with different concentration of seaweed extracts for 120 h and monitored after the first 2 h and then every 24 h to evaluate death, mobility reduction and couple detachment. The extracts of 13 different seaweed species were tested in a first trial and the active extracts were further evaluated in lower concentrations. The extracts of Gracilaria ornata and species belonging to the genera Dictyota and Laurencia showed activity at relatively low concentrations. The active extracts were analyzed by LC–MS, and possible candidates are proposed.

Synergy of Omeprazole and Praziquantel In Vitro Treatment against Schistosoma mansoni Adult Worms.

BACKGROUND: Treatment and morbidity control of schistosomiasis relies on a single drug, praziquantel (PZQ), and the selection of resistant worms under repeated treatment is a concern. Therefore, there is a pressing need to understand the molecular effects of PZQ on schistosomes and to investigate alternative or synergistic drugs against schistosomiasis. METHODOLOGY: We used a custom-designed Schistosoma mansoni expression microarray to explore the effects of sublethal doses of PZQ on large-scale gene expression of adult paired males and females and unpaired mature females. We also assessed the efficacy of PZQ, omeprazole (OMP) or their combination against S. mansoni adult worms with a survival in vitro assay. PRINCIPAL FINDINGS: We identified sets of genes that were affected by PZQ in paired and unpaired mature females, however with opposite gene expression patterns (up-regulated in paired and down-regulated in unpaired mature females), indicating that PZQ effects are heavily influenced by the mating status. We also identified genes that were similarly affected by PZQ in males and females. Functional analyses of gene interaction networks were performed with parasite genes that were differentially expressed upon PZQ treatment, searching for proteins encoded by these genes whose human homologs are targets of different drugs used for other diseases. Based on these results, OMP, a widely prescribed proton pump inhibitor known to target the ATP1A2 gene product, was chosen and tested. Sublethal doses of PZQ combined with OMP significantly increased worm mortality in vitro when compared with PZQ or OMP alone, thus evidencing a synergistic effect. CONCLUSIONS: Functional analysis of gene interaction networks is an important approach that can point to possible novel synergistic drug candidates. We demonstrated the potential of this strategy by showing that PZQ in combination with OMP displayed increased efficiency against S. mansoni adult worms in vitro when compared with either drug alone.

Characterization of α-L-fucosidase and other digestive hydrolases from Biomphalaria glabrata.

Schistosoma mansoni is one of the major agents of the disease Schistosomiasis, which is one of the major global public health concerns. Biomphalaria glabrata is an obligate intermediate mollusc host of S. mansoni. Although the development of S. mansoni occurs in the snail hepatopancreas, studies that focus on this organ remain limited. In this study, we biochemically identified five distinct carbohydrases (amylase, maltase, α-glucosidase, trehalase, and α-L-fucosidase), lipases, and peptidases in the B. glabrata hepatopancreas and focused on the isolation and characterization of the activity of α-L-fucosidase. The isolated α-L-fucosidase has a molecular mass of 141 kDa, an optimum pH of 5.8, and is inhibited by Tris, fucose, and 1-deoxyfuconojirimycin. B. glabrata α-L-fucosidase is an exoglycosidase that can hydrolyze the natural substrate fucoidan to fucose residues. It presented Km values of 48.4 µM to 4-Methylumbelliferyl α-L-fucopyranoside and 0.55 mM to p-nitrophenyl-α-L-fucopyranoside. Thus, α-L-fucosidase has a high activity in the hepatopancreas of B. glabrata, and the differential expression of this enzyme between susceptible and resistant strains indicates that besides its digestive role, α-L-fucosidase may also be important in host/parasite interactions.

Two SmDLC antigens as potential vaccines against schistosomiasis.

The Schistosoma mansoni transcriptome revealed new members of the dynein light chain family (DLC/LC8). The antigenicity and immunogenicity of these proteins, and their potential as vaccine candidates were investigated. Two DLC genes (DLC12_JI392413.1 and DLC13_JI387686.1) were cloned and the recombinant proteins produced in E. coli. The immunization of mice with the rDLCs, using alhydrogel as adjuvant, resulted in high titers of antibodies, indicated that these proteins are highly immunogenic. The anti-DLCs antibodies presented cross reactivity with both recombinant antigens and also recognized proteins from S. mansoni adult worm extracts. The DLC12 and DLC13 immunized animals were challenged by infection with cercariae and a protective profile was observed in three different assays, with a significant decreased in worm burden, of 43% and 51% respectively, when compared to the non-vaccinated group. The granulomas formation due to egg retention in the hepatic tissues was evaluated 45 days after infection. Smaller granulomas were observed in the liver of DLC immunized animals, up to 70% reduction in comparison to the granulomas size in the non-vaccinated animals. Fifty-five days after infection, the average size of the hepatic granulomas was still 25-35% smaller in the DLCs vaccinated groups. The interference of DLC immunization on the hepatic granuloma formation may reflect the lower worm burden and consequent decrease on the number of eggs retained in the liver, resulting in lower pro-inflammatory level in the tissue. The protective effect of DLCs immunization, decreasing the worm burden and delaying the rate of granuloma formation, suggests that these antigens should be further studied as potential vaccine candidates.

Developmental toxicity, acute toxicity and mutagenicity testing in freshwater snails Biomphalaria glabrata (Mollusca: Gastropoda) exposed to chromium and water samples.

A protocol combining acute toxicity, developmental toxicity and mutagenicity analysis in freshwater snail Biomphalaria glabrata for application in ecotoxicological studies is described. For acute toxicity testing, LC50 and EC50 values were determined; dominant lethal mutations induction was the endpoint for mutagenicity analysis. Reference toxicant potassium dichromate (K2Cr2O7) was used to characterize B. glabrata sensitivity for toxicity and cyclophosphamide to mutagenicity testing purposes. Compared to other relevant freshwater species, B. glabrata showed high sensitivity: the lowest EC50 value was obtained with embryos at veliger stage (5.76mg/L). To assess the model applicability for environmental studies, influent and effluent water samples from a wastewater treatment plant were evaluated. Gastropod sensitivity was assessed in comparison to the standardized bioassay with Daphnia similis exposed to the same water samples. Sampling sites identified as toxic to daphnids were also detected by snails, showing a qualitatively similar sensitivity suggesting that B. glabrata is a suitable test species for freshwater monitoring. Holding procedures and protocols implemented for toxicity and developmental bioassays showed to be in compliance with international standards for intra-laboratory precision. Thereby, we are proposing this system for application in ecotoxicological studies.

A benzoic acid derivative and flavokawains from Piper species as schistosomiasis vector controls.

The search of alternative compounds to control tropical diseases such as schistosomiasis has pointed to secondary metabolites derived from natural sources. Piper species are candidates in strategies to control the transmission of schistosomiasis due to their production of molluscicidal compounds. A new benzoic acid derivative and three flavokawains from Piper diospyrifolium, P. cumanense and P. gaudichaudianum displayed significant activities against Biomphalaria glabrata snails. Additionally, "in silico" studies were performed using docking assays and Molecular Interaction Fields to evaluate the physical-chemical differences among the compounds in order to characterize the observed activities of the test compounds against Biomphalaria glabrata snails.

OVICIDAL EFFECT OF PIPERACEAE SPECIES ON Biomphalaria glabrata, Schistosoma mansoni HOST / Efeito ovicida de espécies de Piperaceae em Biomphalaria glabrata, hospedeiro do Schistosoma mansoni

SUMMARY Schistosomiasis is a neglected disease with public health importance in tropical and subtropical regions. An alternative to the disease control is the use of molluscicides to eliminate or reduce the intermediate host snail population causing a reduction of transmission in endemic regions. In this study nine extracts from eight Piperaceae species were evaluated against Biomphalaria glabrata embryos at blastula stage. The extracts were evaluated in concentrations ranging from 100 to 10 mg/L. Piper crassinervium and Piper tuberculatum extracts were the most active (100% of mortality at 20 mg/L and 30 mg/L respectively). .

RESUMO A esquistossomose é uma doença negligenciada de importância para a saúde pública em regiões tropicais e subtropicais. Uma alternativa para o controle da doença é o uso de moluscicidas para eliminar ou reduzir a população de caramujo hospedeiro, acarretando uma redução da transmissão da doença nas regiões endemicas. Neste estudo, nove extratos vegetais provenientes de oito espécies de Piperaceae foram expostos a embriões de Biomphalaria glabrata no estágio de blástula. Os extratos foram avaliados em concentrações que variaram entre 100 e 10 mg/L, sendo Piper crassinervium e Piper tuberculatum os extratos mais ativos (100% de mortalidade a 20 mg/L e 30 mg/L respectivamente). .