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

Research on the effects of ozone (O 3 ) on reproductive development and population stability remains limited. This study investigates the impact of O 3 on Drosophila , a classic model organism for sex determination. Flies were exposed to 1.0 ppm O 3 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 O 3 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. O 3 exposure also resulted in a decrease in global m 6 A levels, with m 6 A-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 m 6 A level on Sxl mRNA was reduced. Importantly, YT521-B overexpression restored the dysregulation of O 3 -induced sex regulatory pathways at the molecular level. Our results indicate that O 3 disrupts m 6 A 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.