Stage-specific functional divergence of yolk protein genes in Spodoptera frugiperda

Yolk protein genes (YPs) are central to insect reproduction, yet the extent of functional divergence among members of the same gene family remains poorly understood in many pest species. Here, we identified five YP genes in Spodoptera frugiperda. Gene structure analysis showed that four YP genes are intronless, whereas YP3 possesses a two-exon structure. Conserved domain analysis revealed that all five YP proteins contain a PFM_spherulin-2a-like domain. We then examined their developmental expression profiles to assess potential stage-specific specialization. YP1 and YP2 were highly expressed in eggs, whereas YP4 and YP5 were more strongly expressed during the pupal and adult stages, with YP3 showing no marked enrichment at either targeted stage. Based on these expression patterns, four genes were selected for RNA interference assays at two developmental stages to evaluate their functions during embryogenesis and reproductive development. Knockdown of YP1 and YP2 in eggs significantly reduced hatchability, whereas pupal-stage knockdown of these genes did not affect fecundity or offspring hatchability. In contrast, knockdown of YP4 and YP5 in eggs caused little or no effect on hatchability, whereas pupal-stage knockdown reduced fecundity and offspring hatchability and impaired ovarian development. In addition, YP4 knockdown disrupted adult emergence, leading to incomplete eclosion and malformed adults. These results show that YP genes in S. frugiperda are not functionally redundant, but instead exhibit clear stage-specific specialization. YP1 and YP2 are primarily associated with embryonic development and egg viability, whereas YP4 and YP5 contribute more to later reproductive performance, with YP4 additionally affecting successful adult emergence. This study provides new evidence for stage-specific functional divergence within the YP gene family in a major agricultural pest, which may pave the ways for developing RNAi-based strategies for green pest control in S. frugiperda.