Limited association of m⁶A RNA modification with post-transcriptional regulation during honeybee caste...

Honeybee caste differentiation is a prime example of phenotypic plasticity in eusocial insects and serves as a valuable model for epigenetic studies. Various epigenetic modifications such as non-coding RNAs and histone modifications, have been implicated in caste differentiation. However, the role of some epigenetic modifications such as DNA methylation still remains controversial. Similarly, mRNA m⁶A modification has been suggested to participate in honeybee caste differentiation. Here we used Oxford Nanopore technology to compare m⁶A modifications in 2d, 4d, and 6d queen and worker larvae. Our data indicated that m⁶A modification is unlikely to exert a direct role in the caste differentiation at the developmental stages examed in this study. We identified 363, 1074, and 1000 differentially expressed transcripts (DETs) in 2d, 4d, and 6d queen-worker larval comparisons, respectively, 74 of which were mapped in seven key KEGG pathways (e.g., mTOR, Notch, FoxO, MAPK, Wnt, Hippo, Hedgehog) associated with honeybee caste differentiation. In contrast, only 16, 39, and 11 differentially m⁶A-modified transcripts (m⁶A-DMTs) were found in the 2d, 4d and 6d comparisons, with very few m⁶A-DMTs involved in key pathways for caste differentiation. Notably, no correlation was observed between m⁶A modification levels and transcript expression, including DETs and m⁶A-DMTs. The motifs of m⁶A modifications remained unchanged across all three developmental time points. When the original samples were randomly regrouped, the number of m⁶A-DMTs did not change significantly, but the DETs related to caste differentiation were dramatically altered. Therefore, the functional relevance of m⁶A in honeybee caste differentiation is still under debate and requires more comprehensive studies.