Characterization of South American Snails of the Genus Biomphalaria (Basommatophora: Planorbidae) and Schistosoma mansoni (Platyhelminthes: Trematoda) in Molluscs by PCR-RFLP.

The identification of snails of the genus Biomphalaria can be done using morphological characteristics which depends on the size of the snails and skill and knowledge of researcher. These methods sometimes are not adequate for identification of species. The PCR-RFLP, using the ITS region of the rDNA, has been used to identify Brazilian species of the genus Biomphalaria. Nevertheless, there is a lack of information about snails from other Latin American countries. In addition, some snails may be infected by Schistosoma mansoni and when submitted to PCR-RFLP they show molecular profiles different from those previously standardized for the other mollusc species. In this work the molecular profiles of 15 species and the subspecies were established by PCR-RFLP of ITS-rDNA with the enzyme DdeI. Moreover, the molecular profiles of host species, B. glabrata, B. straminea, B. tenagophila, and B. prona, infected by S. mansoni were also established. The molluscs were dissected to permit morphological identification. These results contribute to a correct identification of snails of the genus Biomphalaria and detection of these snails infected by S. mansoni.

Hybridism between Biomphalaria cousini and Biomphalaria amazonica and its susceptibility to Schistosoma mansoni.

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.

Hybridism between Biomphalaria cousini and Biomphalaria amazonica and its susceptibility to Schistosoma mansoni

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 percent, 5.6 percent and 2.2 percent, respectively). These results indicate that there is a risk of introducing schistosomiasis mansoni into new areas.

Preliminary studies investigating the occurrence of Biomphalaria cousini in Brazil.

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.

Preliminary studies investigating the occurrence of Biomphalaria cousini in Brazil

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.

Occurrence of Biomphalaria cousini (Mollusca: Gastropoda) in Brazil and its susceptibility to Schistosoma mansoni (Platyhelminths: Trematoda).

In Brazil, there are three intermediate snail vectors and two potential hosts of Schistosoma mansoni. Previous studies showed three variant molecular profiles to B. amazonica and evidenced intraspecific variations using sequence data. In this context, the aim of this study was to verify whether such differences would correspond to either B. amazonica or B. cousini. The snails were morphologically identified; PCR-RFLP and sequencing were carried out. Besides, B. cousini were submitted to susceptibility experiments to S. mansoni. Noteworthy, morphological data of Brazilian specimens predominantly showed the morphology described for B. amazonica. Nevertheless, PCR-RFLP results exhibited three variant molecular profiles for the specimens previously identified as B. amazonica and the phylogenetic analyses showed two groups one to B. amazonica and another to B. cousini. Furthermore, B. cousini showed to be susceptible to S. mansoni. These results confirm the occurrence of B. cousini in Brazil and points to the risk of introduction of schistosomiasis mansoni into new areas.

Investigação da ocorrência de Biomphalaria cousini no Brasil e sua suscetibilidade ao Schistosoma mansoni

No Brasil existem dez espécies e uma subespécie de moluscos do gênero Biomphalaria sendo três hospedeiras intermediárias do Schistosoma mansoni, B. glabrata, B. tenagophila e B. straminea. As espécies B. peregrina e B. amazonica são hospedeiras em potencial deste parasito. A identificação morfológica destes moluscos pode ser dificultada pela semelhança e extensa variação intraespecífica. observada nos caracteres utilizados na identificação. Nestes casos, técnicas moleculares auxiliam a classificação. Estudos prévios utilizando a reação em cadeia da polimerase associada ao polimorfismo de tamanho de fragmentos de restrição (PCR-RFLP) detectaram três perfis distintos para B. amazonica. Além disso, outros autores observaram variação intraespecífica entre espécimes de B. amazonica do. Brasil e da Bolívia a partir da análise de sequências das regiões ITS do rDNA e parte da região 16S do rDNA mitocondrial. Neste contexto, o objetivo desse trabalho foi verificar se as diferenças observadas correspondem às espécies B. amazonica e B. cousini. Neste estudo foram utilizadas populações de Biomphalaria oriundas do Brasil (Amazonas e Mato Grosso), Colômbia (Letícia) e Bolívia (Santa Cruz).

Estes moluscos foram identificados morfologicamente e realizou-se a técnica da PCRRFLP.. Em seguida as regiões 16S rDNAmt e ITS2 rDNA foram seqüenciadas, comparadas entre si e com sequencias de moluscos do gênero Biomphalaria, disponíveis no Genbank. Alem disso, experimentos de cruzamento foram realizados entre essas duas espécies para verificar a presença de híbridos e B. cousini foi. submetida a experimentos de suscetibilidade ao S. mansoni. A morfologia dos exemplares da Bolívia e da Colômbia coincidiu com aquelas descritas para B. amazonica e B. cousini, respectivamente. Os exemplares do Brasil apresentaram morfologia predominantemente de B. amazonica. Os resultados da PCR-RFLP evidenciaram que os exemplares da Bolívia apresentaram perfil distinto daquele apresentado pelos exemplares da Colômbia e os exemplares do Brasil apresentaram três perfis diferentes: o boliviano, o colombiano e o híbrido, contendo a mistura dos fragmentos dos dois perfis anteriores. As análises filogenéticas mostraram que as populações com perfil colombiano formam um grupo distinto com valor significativo de bootstrap. Além disso, B. cousini mostrou ser suscetível ao S. mansoni e o cruzamento entre as duas espécies mostrou que elas produzem híbridos. Esses resultados confirmam a ocorrência de B. cousini no Brasil e apontam para o risco de introdução da esquistossomose mansônica em novas áreas.

Investigação da ocorrência de Biomphalaria cousini no Brasil e sua suscetibilidade ao Schistosoma mansoni / Investigation of the occurrence of Biomphalaria cousini in Brazil and its susceptibility to Schistosoma mansoni

No Brasil existem dez espécies e uma subespécie de moluscos do gênero Biomphalaria sendo três hospedeiras intermediárias do Schistosoma mansoni, B. glabrata, B. tenagophila e B. straminea. As espécies B. peregrina e B. amazonica são hospedeiras em potencial deste parasito. A identificação morfológica destes moluscos pode ser dificultada pela semelhança e extensa variação intraespecífica. observada nos caracteres utilizados na identificação. Nestes casos, técnicas moleculares auxiliam a classificação. Estudos prévios utilizando a reação em cadeia da polimerase associada ao polimorfismo de tamanho de fragmentos de restrição (PCR-RFLP) detectaram três perfis distintos para B. amazonica. Além disso, outros autores observaram variação intraespecífica entre espécimes de B. amazonica do. Brasil e da Bolívia a partir da análise de sequências das regiões ITS do rDNA e parte da região 16S do rDNA mitocondrial. Neste contexto, o objetivo desse trabalho foi verificar se as diferenças observadas correspondem às espécies B. amazonica e B. cousini. Neste estudo foram utilizadas populações de Biomphalaria oriundas do Brasil (Amazonas e Mato Grosso), Colômbia (Letícia) e Bolívia (Santa Cruz).

Estes moluscos foram identificados morfologicamente e realizou-se a técnica da PCRRFLP.. Em seguida as regiões 16S rDNAmt e ITS2 rDNA foram seqüenciadas, comparadas entre si e com sequencias de moluscos do gênero Biomphalaria, disponíveis no Genbank. Alem disso, experimentos de cruzamento foram realizados entre essas duas espécies para verificar a presença de híbridos e B. cousini foi. submetida a experimentos de suscetibilidade ao S. mansoni. A morfologia dos exemplares da Bolívia e da Colômbia coincidiu com aquelas descritas para B. amazonica e B. cousini, respectivamente. Os exemplares do Brasil apresentaram morfologia predominantemente de B. amazonica. Os resultados da PCR-RFLP evidenciaram que os exemplares da Bolívia apresentaram perfil distinto daquele apresentado pelos exemplares da Colômbia e os exemplares do Brasil apresentaram três perfis diferentes: o boliviano, o colombiano e o híbrido, contendo a mistura dos fragmentos dos dois perfis anteriores. As análises filogenéticas mostraram que as populações com perfil colombiano formam um grupo distinto com valor significativo de bootstrap. Além disso, B. cousini mostrou ser suscetível ao S. mansoni e o cruzamento entre as duas espécies mostrou que elas produzem híbridos. Esses resultados confirmam a ocorrência de B. cousini no Brasil e apontam para o risco de introdução da esquistossomose mansônica em novas áreas.