Unique DNA Methylation Profiles Are Associated with cis-Variation in Honey Bees

Abstract

DNA methylation is an important epigenetic modification that mediates diverse processes such as cellular differentiation, phenotypic plasticity, and genomic imprinting. Mounting evidence suggests that local DNA sequence variation can be associated with particular DNA methylation states, indicating that the interplay between genetic and epigenetic factors may contribute synergistically to the phenotypic complexity of organisms. Social insects such as ants, bees, and wasps have extensive phenotypic plasticity manifested in their different castes, and this plasticity has been associated with variation in DNA methylation. Yet, the influence of genetic variation on DNA methylation state remains mostly unknown. Here we examine the importance of sequence-specific methylation at the genome-wide level, using whole-genome bisulfite sequencing of the semen of individual honey bee males. We find that individual males harbor unique DNA methylation patterns in their semen, and that genes that are more variable at the epigenetic level are also more likely to be variable at the genetic level. DNA sequence variation can affect DNA methylation by modifying CG sites directly, but can also be associated with local variation in cis that is not CG-site specific. We show that covariation in sequence polymorphism and DNA methylation state contributes to the individual-specificity of epigenetic marks in social insects, which likely promotes their retention across generations, and their capacity to influence evolutionary adaptation.

Keywords: Apis mellifera; DNA methylation; allele-specific methylation; epigenetics; phenotypic plasticity.

Fig. 1.

—Individual variability and consistency in DNA methylation in honey bee semen. (A) Overlap of mCGs across the four individual HC semen samples. (B) Methylation level (%) of mCGs in each individual semen sample. (C) Proportion of mCGs showing methylation levels of respectively 100%, [95–100%], [90–95%], [50–90%], and <50% in each individual semen sample. Violin plots represent median, interquartile range, 95% confidence interval and kernel density plot.

Fig. 2.

—Differential methylation in male honey bees’ semen. Volcano plots of significantly differentially methylated CGs in pairwise comparisons (Fisher’s exact tests, q-value ≤0.01 and methylation difference >25%) between the four individual HC semen samples. (A) Sperm i47 (blue) versus Sperm i49 (purple). (B) Sperm i47 (blue) versus Sperm i51 (brown). (C) Sperm i47 (blue) versus Sperm i53 (orange). (D) Sperm i49 (purple) versus Sperm i51 (brown). (E) Sperm i49 (purple) versus Sperm i53 (orange). (F) Sperm i51 (brown) versus Sperm i53 (orange). Nonsignificant sites are represented in gray.

Fig. 3.

—Differentially methylated genes (DMGs) have a higher tendency to exhibit sequence polymorphisms compared with consistently methylated genes (CMGs). (A, E) Sperm i47. (B, F) Sperm i49. (C, G) Sperm i51. (D, H) Sperm i53. Bar plots in (E–H) represent mean ± SE. (A–D): χ² tests: Sperm i47, χ² = 136.9, df (degrees of freedom) = 1, P < 0.00001; Sperm i49, χ² = 135.6, df = 1, P < 0.00001; Sperm i51, χ² = 111.0, df = 1, P < 0.00001; Sperm i53, χ² = 172.4, df = 1, P < 0.00001. (E–H): Wilcoxon rank sum tests: Sperm i47, W = 81500, P < 0.00001; Sperm i49, W = 91156, P < 0.00001; Sperm i51, W = 90308, P < 0.00001; Sperm i53, W = 82917, P < 0.00001. ***P < 0.00001.

Fig. 4.

—Direct bisulfite PCR sequencing of (A) oxysterol-binding protein-related protein 2 (GB52517), (B) protocadherin-like wing polarity protein stan (GB51276), and (C) atrial natriuretic peptide-converting enzyme (GB54775). Exons are represented in gray. CG sites of the reference genome are represented by open ovals. WGBS LC and HC individual samples are represented. Lines labeled 1–8 are direct bisulfite PCR sequencing individual samples. mCGs are represented as red ovals. Orange ovals indicate where C/T peaks were present indicating sites with intermediate (<100%) methylation. SNPs are represented as blue rectangles. Insertions are represented as green polygons. Deletions are represented as purple dashed lines. Stars indicate SNPs adding CG sites absent from the reference genome. Arrows indicate SNPs removing CG sites present in the reference genome.