Evolutionary recruitment of a de novo gene by paired box 6 (Pax-6) regulates mating in fruit flies

The post-mating decline in female sexual receptivity is a key reproductive adaptation, yet its genetic basis, evolutionary origins, and transcriptional regulation remain poorly understood across divergent insect lineages. Although Pax factors are well-known as conserved developmental regulators, their proposed repurposing in mating contexts requires urgent investigation. Here, we identify a Pax-6-regulated seminal protein, Seminal C-type lectin (SCF), that controls post-mating behavior in females of the invasive genus Bactrocera . SCF is originated de novo from non-coding sequence and is directly activated by Pax-6 via binding to its promoter region. SCF encodes a secreted C-type lectin whose function depends on a canonical calcium-dependent ligand-binding surface. Transferred to females during mating, SCF significantly suppresses female remating receptivity. Loss-of-function experiments show that knockdown of either Pax-6 or SCF abolishes the mating-inhibitory effect of seminal fluid proteins and leads to a marked increase in female remating rates. Moreover, SCF exhibits conserved expression and function across Bactrocera species, indicating it has acquired a stable reproductive regulatory role within the genus. Our findings reveal how a Pax family gene governs female mating behavior by regulating a recently evolved effector, providing insights into the genetic and evolutionary mechanisms underlying female reproductive strategies and uncovering a novel function for the highly conserved Pax family in behavioral regulation.