<i>fmd</i>/<i>fmd2/DHX16</i>‐<i>dsx</i> cascade in sex determination of one Hemiptera species, <i>Laodelphax striatellus</i>

Although recent investigations have identified a novel regulatory cascade involving female determiner (fmd), female determiner 2 (fmd2), and doublesex (dsx) in Hemiptera species, the evolutionary conservation and molecular mechanisms governing the fmd/fmd2-dsx pathway remain poorly characterized. The small brown planthopper, Laodelphax striatellus (Hemiptera: Delphacidae), represents an ideal model system for elucidating the evolutionary dynamics of sex determination pathways, as its underlying mechanism remains undefined. Here, we demonstrate that the fmd2 homolog (Lsfmd2) in L. striatellus is functionally involved in sex determination through a specific splicing isoform, LsFMD2³¹⁵, the protein of which contains a specific carboxyl-terminal sequence - GTGGGYGGGGKQRGGGRGQRHTPY. Functional characterization in 293T cells revealed that LsFMD2³¹⁵ interacts with LsFMD-F to regulate female-specific splicing of Lsdsx. Importantly, we identified DHX16, an ATP-dependent RNA helicase and pre-messenger RNA splicing factor, as a novel binding partner of LsFMD2³¹⁵. RNA interference-mediated depletion of DHX16 resulted in the ectopic expression of male-specific Lsdsx isoforms in female individuals and induced significant ovipositor malformations. Intriguingly, while LsFMD2³¹⁵ exhibited simultaneous binding capacity with both LsFMD-F and LsDHX16, no direct interaction was observed between LsDHX16 and LsFMD-F, indicating that LsFMD2³¹⁵ serves as a molecular scaffold in the fmd/fmd2/DHX16-dsx regulatory cascade. These findings provide robust evidence for the evolutionary conservation of the fmd/fmd2-dsx regulatory cascade across Hemiptera species. Moreover, the newly identified component DHX16 significantly enhances our mechanistic understanding of this non-canonical sex determination pathway.