BACKGROUND Schistosomiasis is a disease caused by Schistosoma. Due to its complex life cycle, evolutionary position and sexual dimorphism, schistosomes have several mechanisms of gene regulation. MicroRNAs (miRNAs) are short endogenous RNAs that regulate gene expression at the post-transcriptional level by targeting mRNA transcripts. OBJECTIVES Here, we tested 12 miRNAs and identified their putative targets using a computational approach. METHODS We performed the expression profiles of a set of miRNAs and their putative targets during the parasite’s life cycle by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). FINDINGS Our results showed differential expression patterns of the mature miRNAs sma-miR-250; sma-miR-92a; sma-miR-new_4-3p; sma-miR-new_4-5p; sma-miR-new_5-5p; sma-miR-new_12-5p; sma-miR-new_13-3p and sma-miR-new_13-5p. Interestingly, many of the putative target genes are linked to oxidative phosphorylation and are up-regulated in adult-worms, which led us to suggest that miRNAs might play important roles in the post-transcriptional regulation of genes related to energetic metabolism inversion during parasite development. It is noteworthy that the expression of sma-miR-new_13-3p exhibited a negative correlation on SmNADH:ubiquinone oxidoreductase complex I. MAIN CONCLUSIONS Our analysis revealed putative miRNA genes related to important biological processes, such as transforming growth factor beta (TGF-β) signaling, proteasome regulation, glucose and lipid metabolism, immune system evasion and transcriptional regulation.

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) using successive drug treatments of Biomphalaria glabrata snails infected with S. mansoni. Infected B. glabrata were treated three times with 100 mg/kg PZQ for five consecutive days with a one-week interval between them. After the treatment, the cercariae (LE-PZQ) produced from these snails and the LE strains (susceptible) were used to infect mice. Forty-five days after infection, mice were treated with 200, 400 or 800 mg/kg PZQ. Thirty days post-treatment, we observed that the mean number of worms recovered by perfusion was significantly higher in the group of mice infected with the LE-PZQ isolate treated with 200 and 400 mg/kg in comparison to the LE strain with the same treatment. Moreover, there was a significant difference between the ED50 (effective dose required to kill 50% of the worms) of the LE-PZQ isolate (362 mg/kg) and the LE strain (68 mg/kg). In the in vitro assays, the worms of the LE-PZQ isolate were also less susceptible to PZQ. Thus, the use of infected snails as an experimental model for development of resistance to S. mansoni is effective, fast, simple and cheap.

Mitochondrial DNA of Biomphalaria tenagophila, a mollusc intermediate host of Schistosoma mansoni in Brazil, was sequenced and characterised. The genome size found for B. tenagophila was 13,722 bp and contained 13 messenger RNAs, 22 transfer RNAs (tRNA) and two ribosomal RNAs (rRNA). In addition to sequencing, the mitochondrial DNA (mtDNA) genome organization of B. tenagophila was analysed based on its content and localization of both coding and non-coding regions, regions of gene overlap and tRNA nucleotide sequences. Sequences of protein, rRNA 12S and rRNA 16S nucleotides as well as gene organization were compared between B. tenagophila and Biomphalaria glabrata, as the latter is the most important S. mansoni intermediate host in Brazil. Differences between such species were observed regarding rRNA composition. The complete sequence of the B. tenagophila mitochondrial genome was deposited in GenBank (accession EF433576). Furthermore, phylogenetic relationships were estimated among 28 mollusc species, which had their complete mitochondrial genome deposited in GenBank, using the neighbour-joining method, maximum parsimony and maximum likelihood bootstrap. B. tenagophila was positioned at a branch close to B. glabrata and Pulmonata molluscs, collectively comprising a paraphyletic group, contrary to Opistobranchia, which was positioned at a single branch and constituted a monophyletic group.

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.

Freshwater snails belonging to the genus Biomphalaria act as intermediate hosts for the parasite trematode Schistosoma mansoni in Africa and in the neotropical region. Identification of such molluscs is carried out based on morphological characters and the presence of cercariae is verified through squeezing snails between two glass slides or by exposing them to artificial light. However, sometimes, the material collected includes molluscs with decomposed bodies or, yet, only empty shells, which precludes their identification and S. mansoni detection. Due to these difficulties, we have developed a methodology in which DNA may be extracted from traces of organic material from inside shells in order to identify molluscs through polymerase chain reaction and restriction fragment length polymorphism and to detect S. mansoni into these snails, by using low stringency polymerase chain reaction. Species-specific profiles obtained from B. glabrata, B. straminea, and B. tenagophila snails and their shells, maintained in laboratory for ten years, showed the same profiles. S. mansoni profiles showed to be present in shell specimens as far as the eighth week after being removed from aquarium.

Em Belo Horizonte, Minas Gerais, Brasil, a Biomphalaria straminea é encontrada na região da Pampulha. Recentemente, o molusco foi encontrado em valas da antiga Barragem de Santa Lúcia, foco extinto de esquistossomose transmitida por B. glabrata. Os moluscos foram coletados e examinados para verificar se estavam naturalmente infectados com Schistosoma mansoni. Os exemplares negativos foram usados para criação ou infecção com a cepa LE de S. mansoni, mantida no laboratório, e outra cepa VGS, obtida de ovos de fezes de escolar de Belo Horizonte. Dentre 1.890 moluscos capturados em 1994 e 1995, nenhum estava infectado com S. mansoni. Dentre 87 exemplares coletados no criadouro e expostos à cepa LE, nove (10,3%) eliminaram cercárias; dentre 83 moluscos da F1, dez (12,0%) eliminaram cercárias e dentre 88 exemplares coletados e expostos à cepa VGS, dez (11,3%) eliminaram cercárias. Em Belo Horizonte, a esquistossomose é transmitida por B. glabrata e B. tenagophila. Entretanto, atualmente existe o risco de aparecimento de novo foco, no qual a B. straminea poderá vir a ser a transmissora, se medidas profiláticas adequadas não forem tomadas pelas autoridades responsáveis pela construção de um parque e lago no local.

In Belo Horizonte, Minas Gerais, Brazil, the snail Biomphalaria straminea was found in the Pampulha region. Recently the snail was found in ditches in the old Santa Lúcia Reservoir, a now-extinct focus of schistosomiasis transmission by B. glabrata. The snails were collected and examined to verify whether they were naturally infected with Schistosoma mansoni. Negative specimens were used for breeding or infection with the LE strain of S. mansoni from the laboratory and another strain obtained from eggs found in the feces of a schoolchild (VGS) from Belo Horizonte. Among the 1890 snails collected from 1994 to 1995, none were infected with S. mansoni. Among 87 snails collected and exposed to the LE strain, 9 (10.3%) shed cercariae; among 83 snails from F1, and exposed to LE, 10 (12.0%) shed cercariae. Among 88 snails exposed to VGS, 10 (11.3%) shed cercariae. In Belo Horizonte, schistosomiasis is transmitted by B. glabrata and B. tenagophila. However, currently there exists the risk of appearance of a focus in which B. straminea could be the host if prophylactic measures are not taken by the authorities responsible for the construction of a park and lake at that site.

Em Belo Horizonte, Minas Gerais, Brasil, a Biomphalaria straminea é encontrada na região da Pampulha. Recentemente, o molusco foi encontrado em valas da antiga Barragem de Santa Lúcia, foco extinto de esquistossomose transmitida por B. glabrata. Os moluscos foram coletados e examinados para verificar se estavam naturalmente infectados com Schistosoma mansoni. Os exemplares negativos foram usados para criação ou infecção com a cepa LE de S. mansoni, mantida no laboratório, e outra cepa VGS, obtida de ovos de fezes de escolar de Belo Horizonte. Dentre 1.890 moluscos capturados em 1994 e 1995, nenhum estava infectado com S. mansoni. Dentre 87 exemplares coletados no criadouro e expostos à cepa LE, nove (10,3%) eliminaram cercárias; dentre 83 moluscos da F1, dez (12,0%) eliminaram cercárias e dentre 88 exemplares coletados e expostos à cepa VGS, dez (11,3%) eliminaram cercárias. Em Belo Horizonte, a esquistossomose é transmitida por B. glabrata e B. tenagophila. Entretanto, atualmente existe o risco de aparecimento de novo foco, no qual a B. straminea poderá vir a ser a transmissora, se medidas profiláticas adequadas não forem tomadas pelas autoridades responsáveis pela construção de um parque e lago no local.

In Belo Horizonte, Minas Gerais, Brazil, the snail Biomphalaria straminea was found in the Pampulha region. Recently the snail was found in ditches in the old Santa Lúcia Reservoir, a now-extinct focus of schistosomiasis transmission by B. glabrata. The snails were collected and examined to verify whether they were naturally infected with Schistosoma mansoni. Negative specimens were used for breeding or infection with the LE strain of S. mansoni from the laboratory and another strain obtained from eggs found in the feces of a schoolchild (VGS) from Belo Horizonte. Among the 1890 snails collected from 1994 to 1995, none were infected with S. mansoni. Among 87 snails collected and exposed to the LE strain, 9 (10.3%) shed cercariae; among 83 snails from F1, and exposed to LE, 10 (12.0%) shed cercariae. Among 88 snails exposed to VGS, 10 (11.3%) shed cercariae. In Belo Horizonte, schistosomiasis is transmitted by B. glabrata and B. tenagophila. However, currently there exists the risk of appearance of a focus in which B. straminea could be the host if prophylactic measures are not taken by the authorities responsible for the construction of a park and lake at that site.

A relação de vermes machos e fêmeas de Schistosoma mansoni foi determinada em camundongos infectados com cercárias das cepas LE, SJ e AL, eliminadas pelos moluscos hospedeiros do parasita no Brasil. Os números de vermes machos e fêmeas recuperados em inoculações de cercárias provenientes de Biomphalaria glabrata e B. tenagophila, foram semelhantes, variando de 1,1:1 a 1,6:1 com LE e AL e de 1:1,1 com SJ. Nos animais inoculados com cercárias provenientes de B. straminea a relação de vermes macho e fêmea foi semelhante à obtida com cercárias provenientes das outras duas espécies com a cepa LE, relação de 1,5:1. Nas inoculações de cercárias de AL e SJ provenientes de B. straminea a relação foi de 3,1:1 e 6:1, respectivamente. A relação normal de vermes macho para fêmea registrada no Brasil, em inoculações de cercárias de B. glabrata e B. tenagophila, é de 1:1 a 2:1. O grande número de vermes machos provenientes de cercárias das cepas AL e SJ eliminadas por B. straminea mostrou a compatibilidade menor do molusco com essas cepas de S. mansoni.

The ratios of male to female worms of Schistosoma mansoni were determined in mice infected with cercariae from LE, SJ and AL strains shed by mollusc hosts of the parasite in Brazil. The sex ratios of worms in the animals were similar with cercariae from Biomphialaria glabrata and B. tenagophila varying from 1.1:1 to 1.6:1 with LE and AL strains and 1:1.1 with SJ. In the animals infected with cercariae from B. straminea the ratio of male to female worms was similar to those obtained using cercariae shed from the other two species of molluscs, 1.5:1 with LE strain. Inoculations by AL and SJ cercariae resulted in sex ratios of 3.1:1 and 6:1 respectively. The normal sex ratios of worms established in Brazil in animals inoculated with cercariae from B. glabrata and B. tenagophila is from 1:1 to 2:1. The higher number of male worms that developed from cercariae of the AL and SJ strains obtained from B. straminea indicate a lower compatibility of the snail concerning these strains of S. mansoni.

Os níveis de infecciosidade de Schistosoma mansoni para as três espécies de Biomphalaria, hospedeiras intermediárias do parasita no Brasil, foram pesquisados após exposições conjuntas dos moluscos a miracídios, no laboratório e no campo. Foram utilizadas as cepas LE e SJ de S. mansoni, mantidas no laboratório e WVS e RFS obtidas de fezes de escolares de Belo Horizonte, MG. Os resultados mostraram a infecciosidade maior de S. mansoni para B. glabrata, com taxas de infecção de 4, 7 a 85,5%. A B. straminea foi suscetível às cepas LE, SJ e WVS, com taxas de infecção de 11,0 a 24,6%. A B. tenagophila foi suscetível a LE e Sj com taxas de infecção de 2,5 a 6,5%. As médias de cercárias da cepa WVS, eliminadas por dia por B. straminea e B. glabrata variaram de 93 ± 59 e 782 ± 1.120, respectivamente.

The levels of infectivity of Schistosoma mansoni for the three species of Biomphalaria, intermediate hosts of parasite in Brazil were studied after exposing of molluscs to miracidia in the laboratory and in the field. The LE and SJ strains of S. mansoni, maintained in laboratory were used in these experiments as well as the WVS and RFS strains ohtained from faeces of schoolchildren from Belo Horizonte, MG. The results show the high level of infectivity of S. mansoni for B. glabrata with infection rates varying from 4.7 to 85-5%. The snail B. straminea was susceptible to LE, SJ and WVS strains, with infection rates of 11.0 to 24.6%, B. tenagophila was susceptible only to LE and SJ strains with infection rates of 2.5 to 6.5%. The mean number of cercariae of the WVS strain shed per day, by B. straminea and B. glabrata were 93 ± 59 and 782 ± 1,120, respectively.

Nestes experimentos a relação de larvas de Schistosoma mansoni, macho e fêmea, em Biomphalaria glabrata de Belo Horizonte e de Ribeirão das Neves, MG, Brasil, criada no laboratório ou capturada no campo variou de 1:1 a 1: 1 ,3 ou 1,4:1. Cercánas de 39 moluscos mantidos a 25 ± 0,5°C, inoculadas em camundongos originaram 76,6% de vermes machos e 23,4% de fêmeas; cercárias de 32 exemplares mantidos a 27 ± 0,5°C, originaram 43,4% de vermes machos e 56,6% de fêmeas (p<0,05). Cercárias de moluscos capturados no Barreiro e Ressaca, Belo Horizonte, em meses quentes, originaram 45,7 a 47,7% de vermes machos e 52,3 a 54,3% de fêmeas. Moluscos capturados em meses frios (20 + 3,0°C) no Gorduras, Belo Horizonte, produziram número menor de cercárias, que originaram 51,6% de machos e 48,4% de fêmeas. Portanto, nessa região, as infecções dos hospedeiros vertebrados com S. mansoni, poderão ser mais graves no verão devido a carga parasitária e número maior de ovos.

In these experiments the ratio of male to female S. mansoni larvae in D. glabrata from Belo Horizonte and Ribeirão das Neves Minas Gerais, Brazil, either reared in laboratoty or collected in the field, varied from 1:1 to 1:1.3 or 1.4:1. Cercariae of LE strain of Schistosoma mansoni, shed by 39 snails maintained at 25±0.5ºC were used to infect mice on a weekly basis. Subsequent perfusion resulted in 76.6% male and 23.4% female worms. The cercariac produced by 32 infected snails maintained at 27+0.5°C were inoculated into mice and produced 43.4% male and 56.6% female worms (p<0.05). Cercariae eliminated by snails collected in Barreiro and Ressaca, Belo Horizonte, during hot months, produced 45.7 to 47.7% male and 52.3 to 54.3% female worms. A lower number of cercariae shed by snails collected in Gorduras, Belo Horizonte, at 20+3.0°C, produced 51.6% male and 48.4% female worms. Thus, in this region the infection of vertebrate hosts with S. mansoni cercariae would be more severe in the summer due to the higher level of parasites and the number of eggs.

The compatibility of Biomphalaria tenagophila, B. straminea and B. glabrata from Minas Gerais with different strains of Schistosoma mansoni was evaluated using the method of Frandsen (1979b) in standardized experiments. One hundred and fifty of each species of snail were individually exposed in the laboratory to 50 miracidia of S. mansoni lines LE, SJ and AL. The cercariae from the infected snails were counted and used to calculate TCP/100 indices, which were compared with those of Frandsen (1979b). For B. tenagophila the TCP/100 indices varied from 37,996 to 74,266 (class II and III). The snail was poorly compatible with LE (class II) and compatible with SJ and AL (class III). For B. straminea the indices varied from 9,484 to 20,508. The snail was not very compatible with SJ (class I) and poorly compatible with LE and AL (class II). For B. glabrata the indices varied from 588,828 to 1,039,065. The snails was extremely compatible (class VI) with the three lines of S. mansoni. These results confirm the epidemiological importance of B. glabrata in Brazil followed by B. tenagophila and B. straminea.

Espécimes de Sarasinula marginata foram coletadas em hortas e jardins residenciais de Belo Horizonte, Minas Gerais. A suscetibilidade desta espécie de lesma ao Angiostrongylus costaricensis foi verificada em laboratório, utilizando-se 15 exemplares da geração F1. Foi demonstrada uma positividade de 80,0%

Specimens of Sarasinula marginata were collected in kitchen and house gardens of Belo Horizonte, Minas Gerais. The suscetibility of these molluscs for Angiostrongylus costaricensis was tested by infecting 15 laboratory - reared slugs (F1). The positivity demonstrated was of 80.0%.