Fernanda Pires Ohlweiler

Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni

Schistosoma mansoni is the primary causative agent of schistosomiasis, which affects 200 million individuals in 74 countries. We generated 163,000 expressed-sequence tags (ESTs) from normalized cDNA libraries from six selected developmental stages of the parasite, resulting in 31,000 assembled sequences and 92% sampling of an estimated 14,000 gene complement. By analyzing automated Gene Ontology assignments, we provide a detailed view of important S. mansoni biological systems, including characterization of metazoa-specific and eukarya-conserved genes. Phylogenetic analysis suggests an early divergence from other metazoa. The data set provides insights into the molecular mechanisms of tissue organization, development, signaling, sexual dimorphism, host interactions and immune evasion and identifies novel proteins to be investigated as vaccine candidates and potential drug targets.

Schistosoma mansoni and other larval trematodes in Biomphalaria tenagophila (Planorbidae) from Guarulhos, São Paulo State, Brazil

A total of 909 Biomphalaria tenagophila were collected from two areas in Guarulhos (Metropolitan area of São Paulo, São Paulo State, Brazil) to assess larval trematode infections. In all collection sites, only this species was found and 183 (20.13%) harbored trematode infections. In these collections, four morphologically distinguishable types of cercariae were identified by confocal microscopy. Xiphidiocercaria (Cercaria lutzi) was the most common type of cercaria recovered, contributing 76.5% of all infections. Schistosoma mansoni cercariae were recovered and comprised the total of 13.11%. Strigea cercaria (Cercaria caratinguensis) and Brevifurcate pharyngeate Clinostomatoide cercaria (Cercaria ocellifera) contributed 8.33% and 2.22% of all infections, respectively. Double infections (S. mansoni and C. lutzi) were found in twelve snails, contributing 6.55% of all infections. In all sites studied, small vertebrates were found in snail habitats and it was observed human contact with the water. The presence of trematode infected snails in large cities has public health implications. It further provides a starting point for some comprehensive studies on snail-related aspects of transmission and biology of trematode of medical and veterinary importance.

Establishment of the dominant lethal test in the freshwater mollusk Biomphalaria glabrata (Say, 1818)

Mutagens in the environment may represent a long-term risk for ecosystems. The reproductive potential of populations can be affected by alterations in the fecundity and offspring viability caused by germ cell mutations. Despite the ecological relevance of these effects, there are few studies on germ cell mutagenicity in natural populations. Biomphalaria glabrata was chosen for this study because of the scarcity of data on freshwater invertebrates and the ecological importance of this group. The aim of this study was to establish a germ cell mutagenicity test in B. glabrata by using a similar approach to that used in the dominant lethal test in rodents. Mitomycin C was used as a direct mutagen and cyclophosphamide as a mutagen that requires metabolic activation. Wild-type snails were exposed for 10 days to three concentrations of each agent and crossed with non-exposed albino snails at the end of the treatment. The total frequencies of malformations were analyzed in the offspring of wild-type snails; among the offspring of albino snails, only the heterozygous wild-type embryos were analyzed for malformations. Both agents induced germ cell mutations. The analysis of the offspring of the wild-type snails showed an effect of the exposure up to approximately 5 days after the end of the treatment with cyclophosphamide; the effect of mitomycin C was observed until 45 days after the end of the exposure. There was an increase in the frequencies of malformations in the wild-type offspring of the non-exposed albino snails crossed with the wild-type snails exposed to both agents. The dominant lethal test in B. glabrata proposed in this work is easy to perform, efficient, specific and sensitive in the evaluation of germ cell mutations induced by reference mutagens. The possibility of expanding its use to environmental biomonitoring studies seems very promising and worth trying.

Schistosomiasis Control Using Piplartine against Biomphalaria glabrata at Different Developmental Stages

Background Schistosomiasis is one of the most significant diseases in tropical countries and affects almost 200 million people worldwide. The application of molluscicides to eliminate the parasites intermediate host, Biomphalaria glabrata, from infected water supplies is one strategy currently being used to control the disease. Previous studies have shown a potent molluscicidal activity of crude extracts from Piper species, with extracts from Piper tuberculatum being among the most active. Methods and Findings The molluscicidal activity of P. tuberculatum was monitored on methanolic extracts from different organs (roots, leaves, fruit and stems). The compounds responsible for the molluscicidal activity were identified using 1H NMR and ESIMS data and multivariate analyses, including principal component analysis and partial least squares. These results indicated that the high molluscicidal activity displayed by root extracts (LC50 20.28 µg/ml) was due to the presence of piplartine, a well-known biologically-active amide. Piplartine was isolated from P. tuberculatum root extracts, and the molluscicidal activity of this compound on adults and embryos of B. glabrata was determined. The compound displayed potent activity against all developmental stages of B. glabrata. Next, the environmental toxicity of piplartine was evaluated using the microcrustacean Daphnia similis (LC50 7.32 µg/ml) and the fish Danio rerio (1.69 µg/ml). The toxicity to these organisms was less compared with the toxicity of niclosamide, a commercial molluscicide. Conclusions The development of a new, natural molluscicide is highly desirable, particularly because the commercially available molluscicide niclosamide is highly toxic to some organisms in the environment (LC50 0.25 µg/ml to D. similis and 0.12 µg/ml to D. rerio). Thus, piplartine is a potential candidate for a natural molluscicide that has been extracted from a tropical plant species and showed less toxic to environment.

Current distribution of Achatina fulica, in the State of São Paulo including records of Aelurostrongylus abstrusus (Nematoda) larvae infestation

The currently known distribution range of Achatina fulica Bowdich, 1822, in the state of São Paulo, Brazil, is presented. The record of A. fulica naturally infested with Aelurostrongylus abstrusus larvae (Railliet, 1898) (Nematoda: Metastrongylidae) can be found in the city of Guaratinguetá. It was found A. fulica with Metastrongylidae larvae without known medical and veterinary importance in the cities of Carapicuíba, Embu-Guaçu, Itapevi, São Caetano do Sul, São Paulo and Taboão da Serra.

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.

Biomphalaria tenagophila (Orbigny, 1835) (Mollusca): adaptation to desiccation and susceptibility to infection with Schistosoma mansoni Sambon, 1907

Experiments were carried out to test the susceptibility of Biomphalaria tenagophila to the infection with strain SJ of Schistosoma mansoni in the F1, F2 and non-selected parental generation. The potential adaptation of B. tenagophila to desiccation, in healthy mollusks and those exposed to the larvae of S. mansoni of the F1, F2 and non-selected parental generations was also studied. The presence of mucus and soil, at the shell opening, protected the snails against desiccation, favoring survival. The healthy mollusks performed more attempts against desiccation than those exposed to the larvae of the parasite. The mortality rate, during desiccation, was higher among mollusks that remained buried and with the shell opening unobstructed. During the desiccation period the stage of development of the parasite was influenced by the weight loss and the survival of the snails. The longer the period of desiccation, the greater was the weight loss observed, abbreviating survival. The non-selected parental generation was more sensitive to desiccation than the F1 and F2 generations, both in healthy mollusks and in those exposed to S. mansoni larvae. Healthy mollusks were more resistant to desiccation than those exposed to the larvae of the S. mansoni. Desiccation did not interrupt the development of S. mansoni larvae in mollusks, causing a delay in the cercariae elimination. The susceptibility of B. tenagophila to the SJ strain of S. mansoni, in mollusks maintained in water during the larvae incubation period, was similar in all three generations.

Pattern of Genetic Divergence of Mitochondrial DNA Sequences in Biomphalaria tenagophila Complex Species Based on Barcode and Morphological Analysis

Neotropical region has a high diversity of Biomphalaria (Pulmonata: Basommatophora: Planorbidae) fauna with at least twenty-six of the world‘s estimated thirtyseven species recorded (Carvalho,2008). Nearly one –third of these species occur in freshwater ecosystems related to the main drainage river basins in Brazil (Estrada et al.,2006; Teodoro,2010),where Biomphalaria glabrata (Say,1818),Biomphalaria tenagophila (Orbigny,1835) and Biomphalaria straminea (Dunker,1848) have serious impact on human health for these species host Schistosoma mansoni Sambon 1907 (Figure 1).

Molecular and morphological identification of Biomphalaria species from the state of São Paulo, Brazil

Abstract DNA barcoding and morphological characters were used to identify adult snails belonging to the genus Biomphalaria from 17 municipalities in the state of São Paulo, Brazil. The DNA barcode analysis also included twenty-nine sequences retrieved from GenBank. The final data set of 104 sequences of the mitochondrial cytochrome oxidase I (COI) gene was analyzed for K2P intraspecific and interspecific divergences, through tree-reconstruction methods (Neighbor-Joining, Maximum Likelihood and Bayesian inference), and by applying different models (ABGD, bPTP, GMYC) to partition the sequences according to the pattern of genetic variation. Twenty-seven morphological parameters of internal organs were used to identify specimens. The molecular taxonomy of Biomphalaria agreed with the morphological identification of specimens from the same collection locality. DNA barcoding may therefore be a useful supporting tool for identifying Biomphalaria snails in areas at risk for schistosomiasis.

Larvas de trematódeos associadas a moluscos de água doce em municípios da Região Metropolitana de São Paulo, Estado de São Paulo, Brasil

This study found trematode larvae as parasites in freshwater mollusks in municipalities of Metropolitan Region of São Paulo, São Paulo State, Brazil. Malacological search was made from November, 2008 to October, 2011. The following mollusks were identified and had parasitological examination: Ampullariidae, Ancylidae, Lymnaeidae, Physidae, Planorbidae, and Thiaridae families. Twenty-one trematode larvae were observed parasitizing mollusks, but seven were not found in literature search: cercaria I, echinocercaria I, echinocercaria IV, strigeocercaria I, strigeocercaria II, strigeocercaria III and strigeocercaria IV. Freshwater mollusks as intermediate hosts of trematodes can be a risk of transmission of infectious agents causing diseases of great medical and veterinarian concern in Metropolitan Region of São Paulo. Therefore monitoring such areas is extremely important.