YT521-B-mediated RNA m6A modification was involved in O3-induced male-biased sex ratio in Drosophila melanogaster.

Research on the effects of ozone (O3) on reproductive development and population stability remains limited. This study investigates the impact of O3 on Drosophila, a classic model organism for sex determination. Flies were exposed to 1.0 ppm O3 for 8 h per day over 7 days. We observed a significant decrease in the female proportion of fruit fly offspring, with F2 females exhibiting partial masculinization traits. Experiments have demonstrated that O3 exposure reduced the expression of female-specific Sxl-F transcripts while increasing the expression of male-specific Sxl-M transcripts and male splicing subtypes in the female heads. Additionally, female-related genes (da, snf, tra, tra2, ix) were downregulated, whereas male-related genes (mle, msl-1, msl-2, msl-3) were upregulated. O3 exposure also resulted in a decrease in global m6A levels, with m6A-related genes (METTL14, METTL3, Nito, Xio, Vir, fl(2)d, YT521-B, YTHDF, and ALKB) being upregulated in the F0 generation but downregulated in the F1 and F2 generations. Notably, the m6A level on Sxl mRNA was reduced. Importantly, YT521-B overexpression restored the dysregulation of O3-induced sex regulatory pathways at the molecular level. Our results indicate that O3 disrupts m6A modification on Sxl mRNA, which impairs female-specific splicing and disrupts the balance of the sex determination network, ultimately leading to a distorted offspring sex ratio and potential ecological imbalance.