Intergenerational transfer of DNA methylation marks in the honey bee

Abstract

The evolutionary significance of epigenetic inheritance is controversial. While epigenetic marks such as DNA methylation can affect gene function and change in response to environmental conditions, their role as carriers of heritable information is often considered anecdotal. Indeed, near-complete DNA methylation reprogramming, as occurs during mammalian embryogenesis, is a major hindrance for the transmission of nongenetic information between generations. Yet it remains unclear how general DNA methylation reprogramming is across the tree of life. Here we investigate the existence of epigenetic inheritance in the honey bee. We studied whether fathers can transfer epigenetic information to their daughters through DNA methylation. We performed instrumental inseminations of queens, each with four different males, retaining half of each male's semen for whole genome bisulfite sequencing. We then compared the methylation profile of each father's somatic tissue and semen with the methylation profile of his daughters. We found that DNA methylation patterns were highly conserved between tissues and generations. There was a much greater similarity of methylomes within patrilines (i.e., father-daughter subfamilies) than between patrilines in each colony. Indeed, the samples' methylomes consistently clustered by patriline within colony. Samples from the same patriline had twice as many shared methylated sites and four times fewer differentially methylated regions compared to samples from different patrilines. Our findings indicate that there is no DNA methylation reprogramming in bees and, consequently, that DNA methylation marks are stably transferred between generations. This points to a greater evolutionary potential of the epigenome in invertebrates than there is in mammals.

Keywords: Apis mellifera; DNA methylation; epigenetic inheritance; epigenetic remodeling.

Fig. 1.

Experimental design. For each replicate (×3), we collected the semen from four drones and split each semen into two equal parts. We performed instrumental insemination of virgin queens each with half the semen of four drones, before allowing each queen to lay and produce worker offspring. We collected the hind legs of each drone and worker and performed microsatellite genotyping to assign them to their respective patriline. We performed WGBS of each drone’s half semen and thorax, as well as a pool of 20 worker thoraxes from each patriline to analyze their DNA methylation profiles.

Fig. 2.

Patterns of DNA methylation in drones, semen, and workers. (A–C) DNA methylation density (% of mCpGs out of all CpGs) across all patrilines for drone, semen, and worker samples in colony B1 (A), B2 (B), and B4 (C). Box plots represent median, interquartile range (IQR), and 1.5 × IQR. (D–F) DNA methylation level [% of C out of (C + T) at each CpG site] of mCpGs across all patrilines for drone, semen, and worker samples in colony B1 (D), B2 (E), and B4 (F). Violin plots represent median, interquartile range (IQR), 1.5 × IQR, and kernel density plot. Different lowercase letters represent significant differences within each colony (GLMMs, all P < 0.0003).

Fig. 3.

Clustering of samples according to their methylation status in each colony. (A–C) Hierarchical clustering (average agglomerative method on correlation distances) of methylation status of all shared mCpGs across all patrilines for drone (D), semen (S), and worker (W) samples in colony B1 (A), B2 (B), and B4 (C). Bootstrap support values (%) are depicted above each node. Heatmaps show the level of DNA methylation for each site (ranging from 0%, yellow to 100%, red). (D–F) PCA of DNA methylation level of all shared mCpGs across all patrilines for drone (D), semen (S), and worker (W) samples in colony B1 (D), B2 (E), and B4 (F). Percentage of variance explained is depicted on each axis. (G–I) Exemplar genome browser snapshots showing gene body methylation patterns in each sample in colony B1 (G), B2 (H), and B4 (I). Vertical lines show the level of DNA methylation for each CpG. Different colors represent different patrilines.

Fig. 4.

Overlap of mCpGs within and between patrilines in colonies B1, B2, and B4. (A–C) Semen vs. worker comparison showing the proportion of (A) shared mCpGs, (B) semen-specific mCpGs, and (C) worker-specific mCpGs. (D–F) Drone vs. worker comparison showing the proportion of (D) shared mCpGs, (E) drone-specific mCpGs, and (F) worker-specific mCpGs. (G–I) Semen vs. drone comparison showing the proportion of (G) shared mCpGs, (H) semen-specific mCpGs, and (I) drone-specific mCpGs. Box plots represent median, IQR, and 1.5 × IQR. GLMMs: ****P < 0.0001.

Fig. 5.

DMRs within and between patrilines. (A–C) Number of DMRs between semen and workers from different patrilines and from the same patriline in colony B1 (A), B2 (B), and B4 (C). (D–F) Number of DMRs between drones and workers from different patrilines and from the same patriline in colony B1 (D), B2 (E), and B4 (F). (G–I) Number of DMRs between semen and drones from different patrilines and from the same patriline in colony B1 (G), B2 (H), and B4 (I). Box plots represent median, IQR, and 1.5 × IQR. ***P < 0.001; ****P < 0.0001.