Abstract
The understanding of the molecular mechanisms that allows the rapid adaptation of mollusks that are vector of parasites, to new environments is important for disease control. Rapid adaptation is difficult to explain by traditional Mendelian genetics and there is strong evidence supporting that epigenetic mechanisms, are behind rapid adaptations in other species. I studied one epigenetic mark called DNA methylation that has demonstrated to be environmentally modulated and to play a role in phenotypic plasticity in many species, principally plants and vertebrates. Nevertheless, the role of DNA methylation in generating phenotypic variation in invertebrates has been poorly studied. I addressed the question of the role of DNA methylation in the generation of phenotypic plasticity and its heritability in the snail Biomphalaria glabrata, the intermediate host of the parasite Schistosoma mansoni, the causal agent of schistosomiasis, a neglected tropical disease. DNA methylation in B. glabrata has been found to be modulated by the infection of the parasite S. mansoni and by environmental stress, furthermore, it was demonstrated that DNA methylation affects its gene expression, suggesting that DNA methylation can affect phenotypic variation and therefore the adaptation of the snail to new environments. To study the role of DNA methylation in the generation of phenotypic variation, experimental manipulation of the DNA methylation in the snail was necessary. Therefore, two approaches were proposed in this thesis to introduce epimutations in the snail B. glabrata: 1) Random epi-mutagenesis using chemical DNA methyltransferase (DNMT) inhibitors and by consequent segregation of epimutations in self-fertilization lines and 2) Methylate the cytosines of a targeted locus with a targeted epigenome editing tool consisting in the use of the DNA methyltransferase (DNMT3) construct fused to the nuclease-inactivated dCas9. For the random epi-mutagenesis approach, a novel DNMT inhibitor has shown methylation inhibiting effects in two subsequent generations, showing a2multigenerational epigenetic effect and without showing toxic effects in either survival nor fecundity of the snail B. glabrata. In addition, the inhibitor Flv1 has been shown to be effective in other two mollusk species, the freshwater snail Physa acuta and the pacific oyster Crassostrea gigas, which suggests that this inhibitor represents a molecular tool to modulate the methylation of DNA in other mollusks. In the case of the targeted epimutagenesis approach, I used a transfection method that allows introducing two plasmid vectors with an SV40 viral promoter in vivo in embryos of the snail B. glabrata. The transfection was performed at the gastrula stage, which resulted in mosaic incorporation of the vector into the transfected cells. However, the method was able to methylate some CpG sites of the targeted gene.