Genetic markers from Biomphalaria tenagophila (Gastropoda: Pulmonata: Planorbidae) obtained by the double stringency polymerase chain reaction technique

Abstract

Biomphalaria tenagophila, one of the intermediate hosts of the trematoda Schistosoma mansoni, is a simultaneous hermafrodite snail species. In order to analyse the genetic structure of these populations, we performed a double-stringency PCR technique to obtain genetic markers with microsatellites and arbitrary primers in a single reaction.Key words: Biomphalaria -DNA polymorphism -PCR Biomphalaria are simultaneous hermaphrodite freshwater snails, which can breed mainly by cross (Paraense 1955) but also by self-fertilization (Tuan & Simões 1998). Analysis of the effects of reproduction in freshwater snails using genetic markers such as isozymes (Bandoni et al. 1995) shows heterozygote deficiencies indicating that selfing could be a regular mating strategy creating complex patterns of population structure exhibiting genetic differences that can cause differences in susceptibility to Schistosoma mansoni (Hofman et al. 1998).Molecular markers are in large use for freshwater snails diagnosis, mainly PCR based protocols which show a great power for quick and simple characterization of genetic variation within and among populations (Avise 1994). DS-PCR described here combines the specificity of microsatellites and the simplicity of amplification of RAPDS markers in a double stringency condition leading to the amplification of selective population of DNA. The technique produced genetically informative co-dominant markers in Drosophila mercatorum (Matioli & Brito 1995).We applied DS-PCR to laboratory strains B. tenagophila from Tremembé, São José dos Campos and Bananal, all from endemic areas in São Paulo, SP; and a strain from Taim, RS, a non-endemic area. In order to observe intra-population variation we used 12 B. tenagophila snails collected from rice paddies at Tremembé, SP, a site with a long history of S. mansoni transmission (Silva 1992). The hepatopancreas-ovotestis complex were extracted from snails previously identified by morphological reproductive traits (according to Paraense 1975), and free from S. mansoni infection. We used a standard phenol-chloroform protocol for DNA extraction (Sambrock et al. 1989) which produced a clear pattern of DNA (Fig. 1). The temperature profile was the following: a first denaturation step at 94 o C for 2 min+15 cycles at 94 o C for 30s-52 o C for 45s and 72 o C for 60s + 25 cycles at 94 o C for 30s-35 o C for 45s-a ramp of 60s to 72 o C for 60s. Finally extension at 70 o C for 10 min. The products were analyzed by agarose gel electrophoresis (1.4% in TBE buffer) and stained with ethidium bromide (EtBr).The interpopulational profiles of the six primer combinations show a significant degree of genetic variation, suggesting heterogeneous genetic population patterns (Fig. 2).At the intrapopulational level DS-PCR generates polymorphisms that can contribute with the comparison of populations derived from different environmental conditions (Fig. 3).We concluded that DS-PCR is an excellent tool that can be applied to identify genetic polymorphisms within and among hermaphrodite snail...

Schistosome/mollusk: genetic compatibility

“…The understanding of the genetic basis for schistosome resistance/or susceptibility in these snail species is especially important, because schistosome eradication programs may benefit from the knowledge of the genetic polymorphism of these snails since association between hosts and parasite tend to generate and maintain genetic polymorphism (May, 1985), as has been predicted in models of schistosome-snail interactions (Morand et al, 1996). Several molecular biology techniques for studies of genetic variability in schistosomiasis vectors have been used Spatz et al, 1999;Knight et al, 1991Knight et al, , 2000Jannotti-Passos and Souza, 2000;Vidigal et al, 2000Vidigal et al, , 2001Caldeira et al, 2000Caldeira et al, , 2001Souza and Passos, 2001;Tuan and Bortolato, 2001). …”

Genetic markers between Biomphalaria glabrata snails susceptible and resistant to Schistosoma mansoni infection

“…Several techniques of molecular biology for studies of genetic variability in schistosomiasis vectors have been used (Knight et al 1991, Milller et al 1996, Spatz et al 1999, JannottiPassos & Souza 2000, Knight et al 2000, Souza & JannottiPassos 2001, Tuan & Bortolato 2001.…”

Polimorfismo dos genes mitocondrial 16S rRNA e nuclear ITS-2 em populações de Biomphalaria tenagophilada bacia litorânea do estado de São Paulo e estudo da suscetibilidade dos caramujos ao Schistosoma mansoni

“…Os resultados moleculares em Biomphalaria da região neotropical, obtidos após introdução da reação em cadeia da polimerase (PCR), foram obtidos com marcadores moleculares arbitrários (Vidigal et al, 1998;Tuan & Bortolato, 2001). Os resultados já indicavam a alta diversidade genética populacional principalmente das espécies hospedeiras intermediárias do S. mansoni.…”

“…Populações de caramujos do gênero Biomphalaria estão sujeitas a influencia de gargalos populacionais locais causados por aplicação de moluscicidas e alta suscetibilidade aos parasitas, que podem levar a redução da variabilidade genética. São dados que explicam padrões variáveis de polimorfismo genéticos encontrados ao mesmo tempo em populações da mesma espécie de Biomphalaria numa mesma área geográfica ou em áreas adjacentes (Hoffman et al, 1998;Tuan & Bortolato, 2001;Tuan & Santos, 2007).…”