RNA m⁶A Modification Functions in Larval Development and Caste Differentiation in Honeybee (Apis mellifera)

Genetically identical female honeybee larvae with different diets develop into sterile workers or fertile queens. It remains unknown whether the reversible RNA N⁶-methyladenosine (m⁶A) mark functionally impact this "caste differentiation." Here, we profile the transcriptome-wide m⁶A methylome of honeybee queen and worker larvae at three instar stages and discover that m⁶A methylation dynamics are altered by differential feeding. Multiple methylome comparisons show an obvious increase in m⁶A marks during larval development and reveal a negative correlation between gene expression and m⁶A methylation. Notably, we find that worker larvae contain more hypermethylated m⁶A peaks than do queen larvae, and many caste-differentiation-related transcripts are differentially methylated. Chemical suppression of m⁶A methylation in worker larvae by 3-deazaadenosine (DAA) reduces overall m⁶A methylation levels and triggers worker larvae to develop queen caste features. Thus, our study demonstrates that m⁶A functionally impacts caste differentiation and larval development, yet it does not exclude potential contributions from other factors.