Proteomic Analysis of the Midgut Contents of Silkworm in the Pupal Stage

Simple Summary During the pupal stage, the midgut of Bombyx mori undergoes remarkable changes, condensing into the midgut lumen and, subsequently, undergoing decomposition. To comprehensively analyze this process of change, we utilized mass spectrometry to examine the midgut contents in the early and later stages of pupation. We detected a large number of enzymes involved in metabolism, proteases, inhibitors, chitin-binding proteins, chitinases, and storage proteins in the midgut contents during the pupal stage, indicating significant alterations within the pupal midgut lumen. Additionally, we have identified six proteases with potential gelatinolytic activity, which may be involved in the degradation of the midgut contents during the late stages of pupation. This research provides valuable insights into the mechanisms of tissue digestion and midgut remodeling in Bombyx mori pupae. The results serve as an important reference for comprehending the intricate processes taking place during this developmental stage in lepidopteran insects. Abstract The silkworm Bombyx mori, a lepidopteran insect, possesses an 8–10-day pupal stage, during which significant changes occur in the midgut, where it first condenses into the yellow body, and then undergoes decomposition. To gain insights into this transformation process, proteomics was performed on Bombyx mori midgut contents on day 2 and day 7 after pupation. The results revealed the identification of 771 proteins with more than one unique peptide. An analysis using AgriGO demonstrated that these proteins were predominantly associated with catalytic activity. Among the identified proteins, a considerable number were found to be involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, nucleic acid degradation, and energy support. Additionally, variations in the levels of certain proteases were observed between the midgut contents on day 2 and day 7 after pupation. An in-depth analysis of the two-dimensional electrophoresis of the midgut contents on day 7 after pupation led to the identification of twelve protein spots with potential gelatinolytic activity. Among these, six proteases were identified through mass spectrometry, including the p37k protease, vitellin-degrading protease, chymotrypsin-2, etc. These proteases may be responsible for the digestion of the yellow body during the later stages of pupal development.