BACKGROUND Biomphalaria glabrata snails are widely distributed in schistosomiasis endemic areas like America and Caribe, displaying high susceptibility to infection by Schistosoma mansoni. After the availability of B. glabrata genome and transcriptome data, studies focusing on genetic markers and small non-coding RNAs have become more relevant. The small RNAs have been considered important through their ability to finely regulate the gene expression in several organisms, thus controlling the functions like cell growth, metabolism, and susceptibility/resistance to infection. OBJECTIVE The present study aims on identification and characterisation of the repertoire of small non-coding RNAs in B. glabrata (Bgl-small RNAs). METHODS By using small RNA sequencing, bioinformatics tools and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we identified, characterised, and validated the presence of small RNAs in B. glabrata. FINDINGS 89 mature miRNAs were identified and five of them were classified as Mollusk-specific. When compared to model organisms, sequences of B. glabrata miRNAs showed a high degree of conservation. In addition, several target genes were predicted for all the mature miRNAs identified. Furthermore, piRNAs were identified in the genome of B. glabrata for the first time. The B. glabrata piRNAs showed strong conservation of uridine as first nucleotide at 5’ end, besides adenine at 10th position. Our results showed that B. glabrata has diverse repertoire of circulating ncRNAs, several which might be involved in mollusk susceptibility to infection, due to their potential roles in the regulation of S. mansoni development. MAIN CONCLUSIONS Further studies are necessary in order to confirm the role of the Bgl-small RNAs in the parasite/host relationship thus opening new perspectives on interference of small RNAs in the organism development and susceptibility to infection.

BACKGROUND Biomphalaria glabrata is the major species used for the study of schistosomiasis-related parasite-host relationships, and understanding its gene regulation may aid in this endeavor. The ubiquitin-proteasome system (UPS) performs post-translational regulation in order to maintain cellular protein homeostasis and is related to several mechanisms, including immune responses. OBJECTIVE The aims of this work were to identify and characterise the putative genes and proteins involved in UPS using bioinformatic tools and also their expression on different tissues of B. glabrata. METHODS The putative genes and proteins of UPS in B. glabrata were predicted using BLASTp and as queries reference proteins from model organism. We characterised these putative proteins using PFAM and CDD software describing the conserved domains and active sites. The phylogenetic analysis was performed using ClustalX2 and MEGA5.2. Expression evaluation was performed from 12 snail tissues using RPKM. FINDINGS 119 sequences involved in the UPS in B. glabrata were identified, which 86 have been related to the ubiquitination pathway and 33 to proteasome. In addition, the conserved domains found were associated with the ubiquitin family, UQ_con, HECT, U-box and proteasome. The main active sites were lysine and cysteine residues. Lysines are responsible and the starting point for the formation of polyubiquitin chains, while the cysteine residues of the enzymes are responsible for binding to ubiquitin. The phylogenetic analysis showed an organised distribution between the organisms and the clades of the sequences, corresponding to the tree of life of the animals, for all groups of sequences analysed. The ubiquitin sequence was the only one with a high expression profile found in all libraries, inferring its wide range of performance. MAIN CONCLUSIONS Our results show the presence, conservation and expression profile of the UPS in this mollusk, providing a basis and new knowledge for other studies involving this system. Due to the importance of the UPS and B. glabrata, this work may influence the search for new methodologies for the control of schistosomiasis.

ABSTRACT The wide eco-bio-social intervention generated by the SaoFrancisco River Integration Project (PISF) may contribute to the dispersion or introduction of schistosomiasis intermediate hosts in areas without prior recording. The objective was to characterize the limnic malacofauna and its distribution along watersheds involved in the PISF. A cross-sectional study based on the collection of mollusks from 33 water bodies, from Aurora, Brejo Santo, Jaguaretama, Jaguaribara, Jati e Mauriti municipalities in the Ceara (CE) State was developed. The conchological characteristics were used to identify snails at the genus level. The snails of the genus Biomphalaria were analyzed for the presence of Schistosoma mansoni cercariae and the molecular identification (only mollusks from Brejo Santo-CE) for differentiation between species. The following species were found: Biomphalaria sp.; Drepanotrema sp.; Melanoides sp.; Physa sp.; and Pomacea sp. Pomacea sp. (75.8%) and Biomphalaria sp. (72.7%) were the most prevalent species. All municipalities showed Biomphalaria sp. Biomphalaria straminea (Porcos Stream) and Biomphalaria kuhniana was identified in the Boi 1 and Cipo reservoirs (Brejo Santo). The evaluated municipalities under the influence of the PISF present areas with potential for schistosomiasis transmission. It is necessary to intensify control actions and health surveillance in these areas.

Resumo Objetivo: descrever a distribuição geográfica dos hospedeiros intermediários do Schistosoma mansoni em cinco estados brasileiros. Métodos: estudo transversal; foram selecionados municípios dos estados do Paraná (78), Minas Gerais (120), Bahia (82), Pernambuco (51) e Rio Grande do Norte (98), nos anos de 2012 a 2014; esses municípios foram escolhidos por não possuírem registros atualizados da presença de caramujos hospedeiros intermediários de S. mansoni; moluscos foram capturados, taxonomicamente identificados e examinados para verificação de cercárias de S. mansoni. Resultados: os trabalhos foram realizados em 427 municípios (99,5% dos 429 selecionados); foi registrada presença de moluscos em 300 (70,2%) municípios e a ocorrência de Biomphalaria glabrata em 62 (21%) municípios, B. straminea em 181 (60%), B. tenagophila em três (1%); associação de B. glabrata/B. straminea foi encontrada em 53 (18%), e de B. glabrata/B. tenagophila em um (0,3%). Conclusão: os registros de B. glabrata, B. straminea e B. tenagophila estão de acordo com a distribuição conhecida.

Abstract Objective: to describe the geographical distribution of intermediate hosts of Schistosoma mansoni in five Brazilian states. Methods: this was a descriptive cross-sectional study; municipalities were selected in the states of Paraná (78), Minas Gerais (120), Bahia (82), Pernambuco (51) , and Rio Grande do Norte (98), for the period 2012 to 2014; these municipalities were chosen because they did not have current records of the presence of snails vectores de S. mansoni. The molluscs were captured and taxonomically identified and examined for S. mansoni cercariae. Results: the work was carried out in 427 municipalities (99.5% of the 429 selected); the presence of mollusks was registered in 300 (70.2%) municipalities; Biomphalaria glabrata were found in 62 (21%) municipalities, B. straminea in 181 (60%), B. tenagophila in three (1%); B. glabrata/B. straminea association was found in 53 municipalities (18%) and B. glabrata/B. tenagophila association in one (0.3%) municipality. Conclusion: B. glabrata, B. straminea and B. tenagophila distribution records obtained in this study are consistent with previously known distribution.

Resumen Objetivo: describir la distribución geográfica de los hospedadores intermediarios de Schistosoma mansoni en cinco estados brasileños. Métodos: estudio epidemiológico transversal; el estudio fue realizado en municipios de los estados de Paraná (78), Minas Gerais (120), Bahia (82), Pernambuco (51) y Rio Grande do Norte (98), entre 2012 y 2014; estos municipios fueron elegidos por no poseer registros actualizados de la presencia de caracoles vectores de S. mansoni; los moluscos fueron capturados, taxonómicamente identificados y examinados para la verificación de cercarias de S. mansoni. Resultados: los trabajos fueron realizados en 427 municipios (99,5% de 429 municipios seleccionados); fue registrada presencia de moluscos en 300 (70,2%) municípios; la presencia de Biomphalaria glabrata fue registrada en 62 (21%) municipios, B. straminea en 181 (60%) y B. tenagophila en três (1%); se observó asociación de B. glabrata con B. straminea en 53 (18%) y de B. glabrata con B. tenagophila en uno (0,3%). Conclusión: los registros de Biomphalaria están de acuerdo con la distribución conocida.

Resumo Objetivo: descrever a distribuição geográfica dos hospedeiros intermediários do Schistosoma mansoni em cinco estados brasileiros. Métodos: estudo transversal; foram selecionados municípios dos estados do Paraná (78), Minas Gerais (120), Bahia (82), Pernambuco (51) e Rio Grande do Norte (98), nos anos de 2012 a 2014; esses municípios foram escolhidos por não possuírem registros atualizados da presença de caramujos hospedeiros intermediários de S. mansoni; moluscos foram capturados, taxonomicamente identificados e examinados para verificação de cercárias de S. mansoni. Resultados: os trabalhos foram realizados em 427 municípios (99,5% dos 429 selecionados); foi registrada presença de moluscos em 300 (70,2%) municípios e a ocorrência de Biomphalaria glabrata em 62 (21%) municípios, B. straminea em 181 (60%), B. tenagophila em três (1%); associação de B. glabrata/B. straminea foi encontrada em 53 (18%), e de B. glabrata/B. tenagophila em um (0,3%). Conclusão: os registros de B. glabrata, B. straminea e B. tenagophila estão de acordo com a distribuição conhecida.

Abstract Objective: to describe the geographical distribution of intermediate hosts of Schistosoma mansoni in five Brazilian states. Methods: this was a descriptive cross-sectional study; municipalities were selected in the states of Paraná (78), Minas Gerais (120), Bahia (82), Pernambuco (51) , and Rio Grande do Norte (98), for the period 2012 to 2014; these municipalities were chosen because they did not have current records of the presence of snails vectores de S. mansoni. The molluscs were captured and taxonomically identified and examined for S. mansoni cercariae. Results: the work was carried out in 427 municipalities (99.5% of the 429 selected); the presence of mollusks was registered in 300 (70.2%) municipalities; Biomphalaria glabrata were found in 62 (21%) municipalities, B. straminea in 181 (60%), B. tenagophila in three (1%); B. glabrata/B. straminea association was found in 53 municipalities (18%) and B. glabrata/B. tenagophila association in one (0.3%) municipality. Conclusion: B. glabrata, B. straminea and B. tenagophila distribution records obtained in this study are consistent with previously known distribution.

Resumen Objetivo: describir la distribución geográfica de los hospedadores intermediarios de Schistosoma mansoni en cinco estados brasileños. Métodos: estudio epidemiológico transversal; el estudio fue realizado en municipios de los estados de Paraná (78), Minas Gerais (120), Bahia (82), Pernambuco (51) y Rio Grande do Norte (98), entre 2012 y 2014; estos municipios fueron elegidos por no poseer registros actualizados de la presencia de caracoles vectores de S. mansoni; los moluscos fueron capturados, taxonómicamente identificados y examinados para la verificación de cercarias de S. mansoni. Resultados: los trabajos fueron realizados en 427 municipios (99,5% de 429 municipios seleccionados); fue registrada presencia de moluscos en 300 (70,2%) municípios; la presencia de Biomphalaria glabrata fue registrada en 62 (21%) municipios, B. straminea en 181 (60%) y B. tenagophila en três (1%); se observó asociación de B. glabrata con B. straminea en 53 (18%) y de B. glabrata con B. tenagophila en uno (0,3%). Conclusión: los registros de Biomphalaria están de acuerdo con la distribución conocida.

ABSTRACT INTRODUCTION Angiostrongylus cantonensis is causes eosinophilic meningoencephalitis in humans. Worldwide expansion of this nematode is linked to the dispersion of their hosts. This study aimed to determine the prevalence of A. cantonensis infection in Achatina fulica in the nine municipalities that make up Baixada Santista, São Paulo, Brazil. METHODS Angiostrongylus cantonensis larvae were analyzed using optical microscopy. We performed polymerase chain reaction and restriction fragment length polymorphism using restriction endonuclease ClaI, directed to the internal transcribed spacer region 2 of A. cantonensis larval DNA. RESULTS Of the 540 snails analyzed, 117 (21.7%) were infected by A. cantonensis. For morphological and morphometric analyses, 60 larvae were used. Second-stage larvae were, on average, 358.2µm long and 26.4µm wide, while third-stage larvae were, on average, 450µm long and 21.12µm wide. The tails of the larvae ended in a fine tip. CONCLUSIONS All municipalities comprising Baixada Santista had A. fulica that were naturally infected with A. cantonensis. All of the observed characteristics were typical of the species.

Moluscos da família Lymnaeidae são hospedeiros intermediários no ciclo biológico de Fasciola hepatica, agente etiológico da fasciolose, doença parasitária de importância médica para humanos e animais. O presente trabalho teve como objetivo datar e mapear a distribuição espacial dos hospedeiros intermediários de F. hepatica no Brasil. Os dados de distribuição das espécies de limneídeos foram obtidos das Coleções de Malacologia Medica (Fiocruz-CMM, CPqRR) e de Malacologia do Museu de Zoologia da Universidade de São Paulo (MZUSP), rede “SpeciesLink” (CRIA) e através de pesquisas sistemáticas na literatura. Os mapas de distribuição dos limneideos mostram que Pseudosuccinea columella é a espécie mais comum e distribuída nas regiões Sul e Sudeste com poucos registros nas regiões Centro-Oeste, Norte e Nordeste. Além disso, na região Sul os registros para G. viatrix são escassos e existe apenas um único relato de ocorrência para L. rupestris. Os dados obtidos resultaram no primeiro mapa de distribuição espacial das espécies de Lymnaeidae no Brasil, informação básica e relevante para estruturação de políticas de controle da fasciolose humana e animal.

Snails of the family Lymnaeidae act as intermediate hosts in the biological cycle of Fasciola hepatica, which is a biological agent of fasciolosis, a parasitic disease of medical importance for humans and animals. The present work aimed to update and map the spatial distribution of the intermediate host snails of F. hepatica in Brazil. Data on the distribution of lymnaeids species were compiled from the Collection of Medical Malacology (Fiocruz-CMM, CPqRR), Collection of Malacology (MZUSP), “SpeciesLink” (CRIA) network and through systematic surveys in the literature. Our maps of the distribution of lymnaeids show that Pseudosuccinea columella is the most common species and it is widespread in the South and Southeast with few records in the Midwest, North and Northeast regions. The distribution of the Galba viatrix, G. cubensis and G. truncatula showed a few records in the South and Southeast regions, they were not reported for the Midwest, North and Northeast. In addition, in the South region there are a few records for G. viatrix and one occurrence of Lymnaea rupestris. Our findings resulted in the first map of the spatial distribution of Lymnaeidae species in Brazil which might be useful to better understand the fasciolosis distribution and delineate priority areas for control interventions.

Angiostrongylus cantonensis is the most common aetiological agent of human eosinophilic meningoencephalitis. Following a report indicating the presence of this parasite in Brazil in 2007, the present study was undertaken to investigate the presence of A. cantonensis in the surrounding Brazilian port areas. In total, 30 ports were investigated and the following molluscs were identified: Achatina fulica, Belocaulus sp., Bradybaena similaris sp., Cyclodontina sp., Helix sp., Leptinaria sp., Melampus sp., Melanoides tuberculata, Phyllocaulis sp., Pomacea sp., Pseudoxychona sp., Rhinus sp., Sarasinula marginata, Streptaxis sp., Subulina octona, Succinea sp., Tomigerus sp., Wayampia sp. and specimens belonging to Limacidae and Orthalicinae. Digestion and sedimentation processes were performed and the sediments were examined. DNA was extracted from the obtained larvae and the internal transcribed spacer region 2 was analysed by polymerase chain reaction-restriction fragment length polymorphism after digestion with the endonuclease ClaI. Of the 30 ports investigated in this study, 11 contained molluscs infected with A. cantonensis larvae. The set of infected species consisted of S. octona, S. marginata, A. fulica and B. similaris. A total of 36.6% of the investigated ports were positive for A. cantonensis, indicating a wide distribution of this worm. It remains uncertain when and how A. cantonensis was introduced into South America.

Molecular techniques can aid in the classification of Biomphalaria species because morphological differentiation between these species is difficult. Previous studies using phylogeny, morphological and molecular taxonomy showed that some populations studied were Biomphalaria cousini instead of Biomphalaria amazonica. Three different molecular profiles were observed that enabled the separation of B. amazonica from B. cousini. The third profile showed an association between the two and suggested the possibility of hybrids between them. Therefore, the aim of this work was to investigate the hybridism between B. cousini and B. amazonica and to verify if the hybrids are susceptible to Schistosoma mansoni. Crosses using the albinism factor as a genetic marker were performed, with pigmented B. cousini and albino B. amazonica snails identified by polymerase chain reaction-restriction fragment length polymorphism. This procedure was conducted using B. cousini and B. amazonica of the type locality accordingly to Paraense, 1966. In addition, susceptibility studies were performed using snails obtained from the crosses (hybrids) and three S. mansoni strains (LE, SJ, AL). The crosses between B. amazonica and B. cousini confirmed the occurrence of hybrids. Moreover, hybrids can be considered potential hosts of S. mansoni because they are susceptible to LE, SJ and AL strains (4.4%, 5.6% and 2.2%, respectively). These results indicate that there is a risk of introducing schistosomiasis mansoni into new areas.

Specific genetic profiles of Brazilian Biomphalaria species were previously standardized by molecular taxonomy through the analysis of restriction fragments, which were generated by digesting the internal transcribed spacer region of rDNA with the DdeI endonuclease. Biomphalaria amazonica displayed three distinct profiles. To investigate these distinct profiles, the same molecular technique, polymerase chain reaction and restriction fragment length polymorphism, was used with different endonucleases. In addition, morphological data were also used to compare B. amazonica specimens that were collected from Brazil, Colombia and Bolivia. The morphological characters of Bolivian molluscs were similar to B. amazonica, displayed a molecular profile of five restriction fragments and morphological data, whereas the Colombian mollusc population showed morphological characters similar to Biomphalaria cousini and a molecular profile of three restriction fragments, similar to B. cousini. The Brazilian specimens showed the B. amazonica and B. cousini molecular profiles as well as a third profile, which resembled a combination of the Colombian and Bolivian molecular profiles.

The present study was aimed at characterising Biomphalaria species using both morphological and molecular (PCR-RFLP) approaches. The specimens were collected in 15 localities in 12 municipalities of the southern region of the state of Rio Grande do Sul, Brazil. The following species were found and identified: Biomphalaria tenagophila guaibensis, Biomphalaria oligoza and Biomphalaria peregrina. Specimens of the latter species were experimentally challenged with the LE Schistosoma mansoni strain, which showed to be refractory to infection.

Neste trabalho, são discutidos os modos de transmissão da esquistossomose na localidade de São José da Serra, com 500 habitantes, no Município de Jaboticatubas, Minas Gerais, Brasil. A região ao redor recebe, durante todo o ano, milhares de pessoas para a prática de ecoturismo. A ocorrência de esquistossomose aguda em um casal que viajou para essa área, no carnaval de 2007, motivou este estudo. Exames de fezes foram feitos em 268 moradores, 53,6% da população total. Trinta e cinco (13%) estavam eliminando ovos nas fezes. Comparando os resultados coproscópicos com os de um inquérito feito em 2005, observa-se um aumento da proporção de positivos de 9,6% para 12,5%, entre 56 pessoas que participaram em ambos os estudos. Foram coletados 65 exemplares de Biomphalaria glabrata, sendo 1 (1,5%) positivo. No inquérito malacológico realizado em 2005, em 182 biomphalarias, nenhum exemplar foi encontrado positivo. Esses dados indicam a ocorrência de transmissão ativa de esquistossomose na área, revelando a necessidade de planejamento coordenado no desenvolvimento do setor turístico, incluindo a implantação de programas educativos integrados para a comunidade e, principalmente, para os turistas.

This paper discusses schistosomiasis transmission in São José da Serra, a village with a population of 500 in the county of Jaboticatubas, Minas Gerais State, Brazil. The area receives thousands of visitors a year for ecotourism. The study was motivated by a case of acute schistosomiasis involving a couple that spent the 2007 Carnival (Mardi Gras) holiday in the area. Stool tests from 268 local residents (53.6% of the population) showed that 35 (13%) were positive for the infection. A comparison with a previous survey (2005) in the same location showed an increase in the schistosomiasis-positive rate from 9.6% to 12.5%, among the 56 individuals who participated in both surveys. A malacological survey of 65 Biomphalaria glabrata snails showed one specimen (1.5%) eliminating cercariae. In a similar survey in 2005, no positive snail specimens were found. The study indicates that active schistosomiasis transmission is occurring in the area, and that integrated educational programs are needed for both the local community and tourists.

Neste trabalho, são discutidos os modos de transmissão da esquistossomose na localidade de São José da Serra, com 500 habitantes, no Município de Jaboticatubas, Minas Gerais, Brasil. A região ao redor recebe, durante todo o ano, milhares de pessoas para a prática de ecoturismo. A ocorrência de esquistossomose aguda em um casal que viajou para essa área, no carnaval de 2007, motivou este estudo. Exames de fezes foram feitos em 268 moradores, 53,6% da população total. Trinta e cinco (13%) estavam eliminando ovos nas fezes. Comparando os resultados coproscópicos com os de um inquérito feito em 2005, observa-se um aumento da proporção de positivos de 9,6% para 12,5%, entre 56 pessoas que participaram em ambos os estudos. Foram coletados 65 exemplares de Biomphalaria glabrata, sendo 1 (1,5%) positivo. No inquérito malacológico realizado em 2005, em 182 biomphalarias, nenhum exemplar foi encontrado positivo. Esses dados indicam a ocorrência de transmissão ativa de esquistossomose na área, revelando a necessidade de planejamento coordenado no desenvolvimento do setor turístico, incluindo a implantação de programas educativos integrados para a comunidade e, principalmente, para os turistas.

This paper discusses schistosomiasis transmission in São José da Serra, a village with a population of 500 in the county of Jaboticatubas, Minas Gerais State, Brazil. The area receives thousands of visitors a year for ecotourism. The study was motivated by a case of acute schistosomiasis involving a couple that spent the 2007 Carnival (Mardi Gras) holiday in the area. Stool tests from 268 local residents (53.6% of the population) showed that 35 (13%) were positive for the infection. A comparison with a previous survey (2005) in the same location showed an increase in the schistosomiasis-positive rate from 9.6% to 12.5%, among the 56 individuals who participated in both surveys. A malacological survey of 65 Biomphalaria glabrata snails showed one specimen (1.5%) eliminating cercariae. In a similar survey in 2005, no positive snail specimens were found. The study indicates that active schistosomiasis transmission is occurring in the area, and that integrated educational programs are needed for both the local community and tourists.

Seeking the identification of Angiostrongylus cantonensis as a potential etiological agent of three clinical cases of eosinophilic meningitis, mollusc specimens were collected in the state of Espírito Santo, Brazil. The snails were identified as Sarasinula marginata (45 specimens), Subulina octona (157), Achatina fulica (45) and Bradybaena similaris (23). Larvae obtained were submitted to polymerase chain reaction and restriction fragment length polymorphism diagnosis. Their genetic profile were corresponded to A. cantonensis. Rattus norvegicus experimentally infected with third-stage larvae, developed menigoencephalitis, and parasites became sexually mature in the lungs. Additionally, larvae obtained from A. fulica snails, from São Vicente, state of São Paulo, also showed genetic profiles of this nematode. This is the first record of Brazilian molluscs infected with this nematode species.

To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (<A HREF="http://biology.unm.edu/biomphalaria-genome.html">biology.unm.edu/biomphalaria-genome.html</A>), working with the Arizona Genomics Institute (AGI) and supported by the National Human Genome Research Institute (NHGRI), produced a high quality bacterial artificial chromosome (BAC) library. The BB02 strain B. glabrata, a field isolate (Belo Horizonte, Minas Gerais, Brasil) that is susceptible to several strains of Schistosoma mansoni, was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa) consists of 61824 clones (136.3 kb average insert size) and provides 9.05 × coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt) indicated that the genome of B. glabrata contains ~ 63% AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI.