Analysis of histomorphometric and proteome dynamics inside the silk gland lumen of Bombyx mori revealed the dynamic change of silk protein during the molt stage.

Silkworms spin different silks at different growth stages for specific purposes. The silk spun before the end of each instar is stronger than that at the beginning of each instar and cocoon silk. However, the compositional changes in silk proteins during this process are unknown. Consequently, we performed histomorphological and proteomic analyses of the silk gland to characterize changes from the instar end to the next instar beginning. The silk glands were collected on day 3 of third- and fourth-instar larvae (III-3 and IV-3) and the beginning of fourth-instar larvae (IV-0). Proteomic analysis identified 2961 proteins from all silk glands. Silk proteins P25 and Ser5 were significantly more abundant in III-3 and IV-3 than in IV-0, and many cuticular proteins and protease inhibitors increased significantly in IV-0 compared with III-3 and IV-3. This shift may cause mechanical property differences between the instar end and beginning silk. Using section staining, qPCR, and western blotting, we found for the first time that silk proteins were degraded first and then resynthesized during the molting stage. Furthermore, we revealed that fibroinase mediated the changes of silk proteins during molting. Our results provide insights into the molecular mechanisms of silk proteins dynamic regulation during molting.