Chitin and cuticle proteins form the cuticular layer in the spinning duct of silkworm.

Chitin is found in the exoskeleton and peritrophic matrix of arthropods, but recent studies have also identified chitin in the spinning duct of silk-spinning arthropods. Here, we report the presence and function of chitin and cuticle proteins ASSCP1 and ASSCP2 in the spinning duct of silkworm. We show that chitin and these proteins are co-located in the cuticular layer of the spinning duct. Ultrastructural analysis indicates that the cuticular layer has a multilayer structure by layered stacking of the chitin laminae. After knocking down ASSCP1 and ASSCP2, the fine structure of this layer was disrupted, which had negative impacts on the mechanical properties of silk. This work clarifies the function of chitin in the spinning duct of silkworm. Chitin and cuticle proteins are the main components of the cuticular layer, providing the shearing stress during silk fibrillogenesis and regulating the final mechanical properties of silk. STATEMENT OF SIGNIFICANCE: Recent studies have identified chitin in the spinning duct of silk-spinning arthropods. However, the role of chitin in this specific organ remains unclear. This study reports that chitin and cuticle proteins form the cuticular layer, a unique structure of the spinning duct of silkworm. This layer with a precise laminate structure gives the spinning duct flexible properties, provides shearing forces for silk fibrillogenesis, and contributes to silk final mechanical properties. Our work clarifies the component, ultrastructure, and biological significance of the silkworm cuticular layer, describes the specific process of silk fiber formation, and proposes new molecular targets (chitin and cuticle proteins) for the improvement of animal silks.