25DNA methylation of cytosine residues across the genome influences how many genes and phenotypes 26 are regulated. As such, understanding the role of DNA methylation and other epigenetic mechanisms 27 has become very much a part of mapping genotype to phenotype, a major question in evolutionary 28 biology. Ideally, we would like to manipulate DNA methylation patterns on a genome-wide scale, to 29 elucidate the role of epigenetic modifications in phenotypic expression. Recently, the demethylating 30 agent 5-aza-2'-deoxycytidine (5-aza-dC; commonly used in the epigenetic treatment of certain 31 cancers), has been deployed to explore the epigenetic regulation of a number of traits of interest to 32 evolutionary ecologists. Recently, we showed that treatment with 5-aza-dC shifted patterns of sex 33 allocation as predicted by genomic conflict theory in the parasitoid wasp Nasonia vitripennis. This was 34 the first (albeit indirect) experimental evidence for genomic conflict over sex allocation facilitated by 35 DNA methylation. However, this work lacked confirmation of the effects of 5-aza-dC on DNA 36 methylation, drawing commentary on the efficacy of 5-aza-dC in a novel system. Here, using whole-37 genome bisulphite sequencing, we demonstrate unequivocally that 5-aza-dC disrupts methylation 38 across the Nasonia vitripennis genome. We show that disruption leads to both hypo-and hyper-39 methylation, may vary across tissues and time of sampling, and that the effects of 5-aza-dC are 40 context-and sequence specific. We conclude that 5-aza-dC has the potential to be repurposed as a 41 tool in evolutionary ecology for studying the role of DNA methylation. 42 43 44 45 46 47 48 49 50 51