Abstract
5-methylcytosine (5mC) is an important epigenetic mark in eukaryotes.
Little information about its role exists for invertebrates. How 5mC
contributes to phenotypic variation in invertebrates can be investigated
by experimental alteration of methylation patterns. Here, we apply new
non-nucleoside DNA methyltransferase inhibitors (DNMTi) to introduce
global changes into the methylome of mollusk species. Flavanone
inhibitor Flv1 was highly efficient in reducing 5mC in the freshwater
snails Biomphalaria glabrata and Physa acuta, and to a
lesser degree, probably due to lower stability in sea water, in the
oyster Crassostrea gigas. Flv1 has no toxic effects and
significantly decreased the 5mC level in the treated B. glabrata
generation and in its untreated offspring. Drug treatment triggers
significant variation in the morphometric traits in both generations. An
epigenotyping by sequencing method corroborates hypomethylation effect
of Flv1 in both B. glabrata generations and identifies one
Differential Methylated Region (DMR) out of 8, found both in
Flv1-exposed snails and its progeny, demonstrating a multigenerational
effect of an induced epimutation. By targeted bisulfite sequencing, we
confirmed hypomethylation in a locus associated with reduced gene
expression.