Context-dependent gene expression in eukaryotes is controlled by several mechanisms including cytosine methylation that primarily occurs in the CG dinucleotides (CpGs). However, less frequent non-CpG asymmetric methylation has been found in various cell types, such as mammalian neurons, and recent results suggest that these sites can repress transcription independently of CpG contexts. In addition, an emerging view is that CpG hemimethylation may arise not only from deregulation of cellular processes but also be a standard feature of the methylome. Here, we have applied a novel approach to examine whether asymmetric CpG methylation is present in a sparsely methylated genome of the honeybee, a social insect with a high level of epigenetically driven phenotypic plasticity. By combining strand-specific ultra-deep amplicon sequencing of illustrator genes with whole-genome methylomics and bioinformatics, we show that rare asymmetrically methylated CpGs can be unambiguously detected in the honeybee genome. Additionally, we confirm differential methylation between two phenotypically and reproductively distinct castes, queens and workers, and offer new insight into the heterogeneity of brain methylation patterns. In particular, we challenge the assumption that symmetrical methylation levels reflect symmetry in the underlying methylation patterns and conclude that hemimethylation may occur more frequently than indicated by methylation levels. Finally, we question the validity of a prior study in which most of cytosine methylation in this species was reported to be asymmetric.

Apis_mellifera_amplicon_bisulfite_sequencing_queen_2Amplicon bisulfite sequencing reads from Apis mellifera newly-emerged queen brains (5 pooled brains). Archive contains four Illumina Miseq libraries (paired end).Apis_mellifera_amplicon_bisulfite_sequencing_queen_3Amplicon bisulfite sequencing reads from Apis mellifera newly-emerged queen brains (5 pooled brains). Archive contains four Illumina Miseq libraries (paired end).queen3.tarApis_mellifera_amplicon_bisulfite_sequencing_worker_1Amplicon bisulfite sequencing reads from Apis mellifera newly-emerged worker brains (5 pooled brains). Archive contains four Illumina Miseq libraries (paired end).worker1.tarApis_mellifera_amplicon_bisulfite_sequencing_worker_2Amplicon bisulfite sequencing reads from Apis mellifera newly-emerged worker brains (5 pooled brains). Archive contains four Illumina Miseq libraries (paired end).worker2.tarApis_mellifera_amplicon_bisulfite_sequencing_worker_3Amplicon bisulfite sequencing reads from Apis mellifera newly-emerged worker brains (5 pooled brains). Archive contains four Illumina Miseq libraries (paired end).worker3.tarApis_mellifera_amplicon_bisulfite_sequencing_queen_1Amplicon bisulfite sequencing reads from Apis mellifera newly-emerged queen brains (5 pooled brains). Archive contains four Illumina Miseq libraries (paired end).