Evolution of m6A-related genes in insects and the function of METTL3 in silkworm embryonic development.

N6-methyladenosine (m6A) plays a key role in many biological processes. However, the function and evolutionary relationship of m6A-related genes in insects remain largely unknown. Here we analysed the phylogeny of m6A-related genes among 207 insect species and found that m6A-related genes are evolutionarily conserved in insects. Subcellular localization experiments of m6A-related proteins in BmN cells confirmed that BmYTHDF3 was localized in the cytoplasm, BmMETTL3, BmMETTL14, and BmYTHDC were localized in the nucleus, and FL2D was localized to both the nucleus and cytoplasm. We examined the expression patterns of m6A-related genes during the embryonic development of Bombyx mori. To elucidate the function of BmMETTL3 during the embryonic stage, RNA sequencing was performed to measure changes in gene expression in silkworm eggs after BmMETTL3 knockdown, as well as in BmN cells overexpressing BmMETTL3. The global transcriptional pattern showed that knockdown of BmMETTL3 affected multiple cellular processes, including oxidoreductase activity, transcription regulator activity, and the cation binding. In addition, transcriptomic data revealed that many observed DEGs were associated with fundamental metabolic processes, including carbon metabolism, purine metabolism, amino acid biosynthesis, and the citrate cycle. Interestingly, we found that knockdown of BmMETTL3 significantly affected Wnt and Toll/Imd pathways in embryos. Taken together, these results suggest that BmMETTL3 plays an essential role in the embryonic development of B. mori, and deepen our understanding of the function of m6A-related genes in insects.

Imaginal disc growth factor maintains cuticle structure and controls melanization in the spot pattern formation of Bombyx mori.

The complex stripes and patterns of insects play key roles in behavior and ecology. However, the fine-scale regulation mechanisms underlying pigment formation and morphological divergence remain largely unelucidated. Here we demonstrated that imaginal disc growth factor (IDGF) maintains cuticle structure and controls melanization in spot pattern formation of Bombyx mori. Moreover, our knockout experiments showed that IDGF is suggested to impact the expression levels of the ecdysone inducible transcription factor E75A and pleiotropic factors apt-like and Toll8/spz3, to further control the melanin metabolism. Furthermore, the untargeted metabolomics analyses revealed that BmIDGF significantly affected critical metabolites involved in phenylalanine, beta-alanine, purine, and tyrosine metabolism pathways. Our findings highlighted not only the universal function of IDGF to the maintenance of normal cuticle structure but also an underexplored space in the gene function affecting melanin formation. Therefore, this study furthers our understanding of insect pigment metabolism and melanin pattern polymorphisms.