METTL3/METTL14-mediated RNA m6A modification is involved in male reproductive development in Bactrocera dorsalis.

RNA N⁶-methyladenine (m⁶A) modification represents a pivotal epigenetic modification that facilitates the remodeling of gene expression and regulates a variety of biological processes via certain post-transcriptional mechanisms. However, the specific function of RNA m⁶A modification in insect male reproduction remains unclear. In this study, we explored the molecular mechanism by which METTL3/METTL14-mediated RNA m⁶A modification regulates male reproduction in the invasive pest Bactrocera dorsalis. The results showed that BdMettl3 and BdMettl14 were highly expressed in fat body (FB) and male accessory glands (MAGs). Knockout of BdMettl3 or BdMettl14 decreased the expression level of m⁶A in B. dorsalis, resulting in testicular deformities and a significant reduction of viable sperm number. Specifically, BdMettl3 or BdMettl14 knockout reduced the titer of 20-hydroxyecdysone (20E, the active form of ecdysone) in males. The messenger RNA (mRNA) of Disembodied, one of the 20E synthesis genes, was modified by m⁶A, and its expression increased the titer of 20E. The mRNA m⁶A level of Disembodied obviously decreased after the knockout of BdMettl3 or BdMettl14, suggesting that RNA m⁶A modification regulates testis development and fecundity by modulating 20E synthesis. Taken together, this study indicates that METTL3/METTL14-mediated RNA m⁶A modification presents a new regulatory mechanism for male reproduction in B. dorsalis, serving as a potential target for the control of B. dorsalis.