DNA methylation dynamics, metabolic fluxes, gene splicing, and alternative phenotypes in honey bees

Abstract

In honey bees (Apis mellifera), the development of a larva into either a queen or worker depends on differential feeding with royal jelly and involves epigenomic modifications by DNA methyltransferases. To understand the role of DNA methylation in this process we sequenced the larval methylomes in both queens and workers. We show that the number of differentially methylated genes (DMGs) in larval head is significantly increased relative to adult brain (2,399 vs. 560) with more than 80% of DMGs up-methylated in worker larvae. Several highly conserved metabolic and signaling pathways are enriched in methylated genes, underscoring the connection between dietary intake and metabolic flux. This includes genes related to juvenile hormone and insulin, two hormones shown previously to regulate caste determination. We also tie methylation data to expressional profiling and describe a distinct role for one of the DMGs encoding anaplastic lymphoma kinase (ALK), an important regulator of metabolism. We show that alk is not only differentially methylated and alternatively spliced in Apis, but also seems to be regulated by a cis-acting, anti-sense non-protein-coding transcript. The unusually complex regulation of ALK in Apis suggests that this protein could represent a previously unknown node in a process that activates downstream signaling according to a nutritional context. The correlation between methylation and alternative splicing of alk is consistent with the recently described mechanism involving RNA polymerase II pausing. Our study offers insights into diet-controlled development in Apis.

Fig. 1.

Annotation of the insulin/TOR network in Apis showing methylated and differentially methylated genes. In those cases in which more than one paralog has been found only one is referred to by the consensus protein name, with the others designated by the GB numbers from the official honey bee gene list (www.beebase.org). The nutrient-independent activation of the PI3K pathway by ALK is highlighted in pink. Gene annotation is shown in Table S3.

Fig. 2.

Simplified gene model of Amalk and its pattern of methylation in different phenotypes. (A) alk gene model and the positions of methylated CpGs. (B) CpG percentage methylation levels in alk intron 26 (highlighted in blue) in the adult brain and larval heads of both queens and workers.

Fig. 3.

Relative expression of alk and anti-alk in selected tissues. (A) Section of the alk gene model showing the transcript variants produced by alternative splicing of alk and anti-alk and the positions of PCR primers used for variant-specific amplifications. (B) Expression of alternative transcripts alk-A, alk-B, and anti-alk-A, anti-alk-B in adult brain relative to larval head. Quantitative data for alk-C representing total expression of the alk gene were used for normalization. alk-A represents the short transcript with no exon 25, whereas alk-B contains exon 25. anti-alk-B contains an alternatively spliced exon 3 of anti-alk, which is missing in anti-alk-A. Blue bars, workers; red bars, queens. (C) Expression of the same transcripts in the adult queen brain relative to ovary. The reference gene was ugt. (experimental details in SI Materials and Methods). Two biological replicates were used in each experiment. Error bars represent SD.