Zona pellucida proteins Piopio and Dumpy are essential for the apical extracellular matrix assembly and survival of <scp> <i>Locusta migratoria</i> </scp>

BACKGROUND: The apical extracellular matrix (aECM) forms a critical interface between epithelial cells and the external environment. Its integrity, stability, and flexibility are crucial for the survival of organisms. However, the molecular mechanisms governing the correct assembly and maturation of the insect aECM remain poorly understood, impeding the development of pest control strategies. RESULTS: The conserved zona pellucida (ZP) protein Piopio in Locusta migratoria (LmPio) was identified and characterized. LmPio is predominantly expressed in ectoderm-derived tissues, and the protein localizes within the cuticle assembly zone in the apical region of epidermal cells. RNA interference (RNAi)-mediated knockdown of LmPio resulted in 71.89% mortality, associated with cuticle thinning, reduced lamellae number, and impaired adhesion between the epidermis and cuticle. Furthermore, the transport of cuticular lipids and proteins to the cuticle was severely disrupted, although chitin content remained unchanged. In immunoprecipitation experiments we identified Dumpy (LmDpy), another ZP protein, as a potential partner of LmPio. Knockdown of LmDpy resulted in 100% mortality of locusts, with a phenotype highly similar to that induced by LmPio knockdown. Co-immunoprecipitation and pull-down assays confirmed that LmPio directly interacted with LmDpy to jointly regulate aECM assembly and maturation. CONCLUSION: Our results demonstrate that LmPio and LmDpy are essential for the formation of the assembly zone and the maintenance of cuticle integrity. These findings reveal the critical role of ZP proteins in aECM assembly and cuticle morphogenesis. Knockdown of LmPio or LmDpy resulted in high mortality, validating these genes as promising targets for the RNAi-based control of this pest. © 2026 Society of Chemical Industry.