Abstract: 【Aim】This study aims to explore the potential regulatory roles of the demethylase AmALKBH genes in Apis mellifera ligustica in the growth and developmental process and mRNA methylation, so as to provide the new theoretical basis and references for the further functional study of AmALKBHs. 【Methods】 Multiple software was used to bioinformatically analyze the five AmALKBH genes (AmALKBH1, AmALKBH4, AmALKBH6, AmALKBH7 and AmALKBH8), homologous sequence alignment was performed and phylogenetic tree of ALKBHs from different species was constructed. qRT-PCR was employed to examine the expression levels of these five AmALKBH genes in different developmental stages (egg, larva and pupa), brains of the 1-, 7- and 18-day-old adult workers, and various tissues (antennae, brain, hypopharyngeal gland, crop, midgut, ileum, rectum, fat body and venom gland) of the 1-day-old adult workers of A. m. ligustica. Additionally, high performance liquid chromatography tandem triple quadrupole mass spectrometry (HPLC-QqQ-MS/MS) was used to quantify the levels of mRNA m⁶A methylation in samples throughout the above different developmental stages and brains of the 1-, 7- and 18-day-old adult workers of A. m. ligustica. 【Results】The five AmALKBH genes (AmALKBH1, AmALKBH4, AmALKBH6, AmALKBH7 and AmALKBH8) in A. m. ligustica encode proteins of 311, 297, 229, 228 and 585 amino acids, respectively, with conserved motifs and structural domains, being hydrophilic spherical proteins. These five proteins are different in their physicochemical properties. The five AmALKBHs of A. m. ligustica exhibited 12.92%-46.46% amino acid sequence identities with ALKBHs of Drosophila melanogaster, Homo sapiens, Mus musculus, Arabidopsis thaliana and Escherichia coli, and showed the closest genetic relationships with ALKBHs from A. cerana, Vespa velutina, and  Bombus terrestris.Along with the embryonic development, there was a progressive reduction in the expression levels of AmALKBH4 and AmALKBH7, concomitant with an elevation in the expression level of AmALKBH6. The expression level of AmALKBH8 reached its peak in the middle stage of egg development (36 h post oviposition), and then declined thereafter. The expression of AmALKBH1 was undetectable. During the larval and pupal stages, the expression levels of all the five AmALKBH genes gradually increased with the developmental progress, reaching the peak in the brown-eyed white pupae and brown-eyed light-colored pupae before decreasing slightly in the newly emerged adults. These five AmALKBH genes were highly expressed in the antennae and brain of the 1-day-old adult workers of A. m. ligustica. The expression levels of AmALKBH6, AmALKBH7 and AmALKBH8 in the brains of the 1-day-old adult workers were significantly lower than those in the brains of the 7- and 18-day-old adult workers. An obvious decrease in the level of mRNA m⁶A methylation was observed from the early stage to middle stage of egg development, with a notable increase in the late stage of egg development (60 h post oviposition). The level of mRNA m⁶A methylation exhibited a similar trend as the gene expression level, peaking earlier in the pink-eyed white pupae. While the mRNA m⁶A methylation level was the highest in the brains of the 1-day-old adult workers but reduced in the brains of the 7- and 18-day-old adult workers. 【Conclusion】 This study result demonstrated the high conservation of the ALKBH gene family among species. Differences in the sequence characteristics, protein structure and expression patterns were observed among the five AmALKBH genes in A. m. ligustica, indicating their distinct functional features. The expression levels of AmALKBH genes and the levels of mRNA m⁶A methylation dynamically changed throughout the developmental stages of A. m. ligustica and were found to be correlated, suggesting a potential regulatory role of AmALKBH genes in the growth and development of A. m. ligustica. These findings lay a solid foundation for uncovering the molecular mechanisms, by which AmALKBH genes regulate growth and development, providing a new theoretical basis for further enhancing the value of honeybees in basic research and pollination applications.

Key words: Apis mellifera ligustica, ALKBH gene family, demethylase, mRNA m⁶A methylation, developmental stages