Abstract: Insect cuticular proteins, as the major extracellular matrix components of the insect cuticle, are highly diverse in type and sequence characteristics. They play crucial roles in the construction and functional regulation of the cuticle structure, contributing significantly to the integrity and diversity of the insect exoskeleton. The Asian corn borer Ostrinia furnacalis is one of the main stem-boring pests of maize and other crops in China, posing a serious threat to food security. Previous studies have identified a highly abundant CPH family cuticular protein, OfCPH-1, in the head capsule of O. furnacalis larvae. OfCPH-1 can bind to chitosan and form liquid-liquid phase separation(LLPS) condensates. This study focuses on the chitin-binding ability and LLPS behavior of OfCPH-1 to explore its potential role in cuticle assembly. Chitin-binding assays demonstrated that OfCPH-1 has strong binding affinity to α-chitin, despite lacking any previously reported canonical chitin-binding domains in its sequence. LLPS assays showed that full-length OfCPH-1 undergoes LLPS at room temperature in response to changes in ionic strength, forming condensates with an average diameter of(571.99±8.67) nm, whereas truncated peptides containing the 18 aa motif or AH-rich motif failed to undergo phase separation. The LLPS capability of OfCPH-1 is likely due to the multiple intrinsically disordered regions(IDRs) in its sequence. Fourier-transform infrared spectroscopy(FTIR) analysis further revealed that, upon phase separation, OfCPH-1 undergoes a conformation shift from random coils to β-sheet structures. This study provides new perspective for understanding the biological function of CPH family cuticular proteins and supports the potential development of novel green pesticides by targeting the LLPS behavior of insect cuticular proteins.

Key words: insect cuticular protein, Ostrinia furnacalis, liquid-liquid phase separation