Validating the Demethylating Effects of 5-aza-2′-deoxycytidine in Insects Requires a Whole-Genome Approach

Abstract

We previously demonstrated that treatment with the demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) alters the offspring sex ratios produced by females of the parasitoid wasp Nasonia vitripennis . Females allocate offspring sex ratio in line with Local Mate Competition theory, producing more or less female-biased sex ratios as the number of other females laying eggs on a patch varies, thereby reducing competition amongst their sons for mates. Interestingly, treatment with 5-aza-dC did not ablate the facultative sex allocation response. Instead, sex ratios became less female-biased, a shift in the direction of the optimum sex ratio for paternally-inherited alleles according to genomic conflict theory. This was the first (albeit indirect) experimental evidence for genomic conflict over sex allocation. Ellers et al. (2019) have since assayed the effects of 5-aza-dC on DNA methylation in ten Nasonia genes, finding no evidence of demethylation in these 10 genes, from which they conclude that 5-aza-dC has no demethylating capability in Nasonia vitripennis. Quantifying the efficacy of 5-aza-dC in terms of demethylation is indeed crucial to in-depth interpretation of studies using 5-aza-dC to link phenotypes to epigenetic regulation. Here, we outline the mode of action of 5-aza-dC and demonstrate that determining the efficacy of 5-aza-dC in insect systems requires a whole-genome approach.

Phenotypic Plasticity: What Has DNA Methylation Got to Do with It?

“…They all need to be present in cells at the S phase of the cell cycle to have an effect, which means that tissues undergoing rapid cell division may show preferential effects of these inhibitors. Treatment with these inhibitors has also been reported to cause hypermethylation of some loci [113], making interpretation of phenotypes and effects on gene expression difficult.…”

Genomics of sex allocation in the parasitoid wasp Nasonia vitripennis

“…In terms of our own work on sex allocation, we have shown for example that facultative sex allocation under local mate competition is not associated with changes in gene expression [ 17 , 18 ], even though female oviposition of eggs is associated with major changes to the transcriptome (in particular, a down-regulation of metabolic processes: [ 17 , 54 ]). We have also shown though that disrupting patterns of DNA methylation changes the pattern of facultative sex allocation [ 85 , 86 ], suggesting that the regulation of gene expression via DNA methylation is important for facultative sex allocation.…”

Chromatin Structure and Function in Mosquitoes

“…Second, the systemic cytotoxic effects of these treatments are known from studies in other organisms, including Drosophila (Katz, 1985;Cunha et al, 2002). In these studies, the effects and toxicity of the drugs have been shown to be variable across developmental stages, tissues, and cell types (Laurent et al, 2010;Foret et al, 2012;Rasmussen et al, 2016;Cook et al, 2019) and also depend on the drug dosage (Yang et al, 2006;Cook et al, 2019). The study by Claudio-Piedras et al (2019) did not report toxicity in the mosquito An.…”

“…The mutagenic effects of this drug on DNA, which is the result of the formation of the Dnmt2nucleoside adduct and the subsequent repair, is expected to be proportional to the number of cytosines in the DNA that are targeted by Dnmt2. Therefore, such a global toxicity does not seem to agree with the Diptera's low Dnmt2 activity, and instead a marginal effect would be expected (Stresemann and Lyko, 2008;Cook et al, 2019). Beyond these initial observations, to validate the function of DNA methylation in mosquitoes, it will be necessary to silence the Dnmt2 enzyme, with iRNA or CRISPR/cas9, and to study the genome-wide effects at the level of DNA methylation, using bisulfite sequencing, and at the level of gene expression by RNA-seq.…”