Asynchronous programmed cell death occurrence in the silk glands at pupation (<i>Bombyx mori</i>)

Silk glands are crucial for silk protein synthesis and secretion. However, the degeneration process takes place at pupation, as mediated by programmed cell death (PCD). The differences of PCD in the anterior silk gland (ASG) and middle silk gland (MSG) with different physiological functions remain unexplored. In this study, we investigated the occurrence of autophagy and apoptosis in the MSG and the ASG of silkworms from the mature silkworm to the 1st d of pupation (P1) based on Ca²⁺ homeostasis. The results showed high autophagy but low apoptosis at the early stage. After Ca²⁺ levels peak, calpain-mediated autophagy-related protein 5 (ATG5) cleavage drove the autophagy-to-apoptosis conversion. The ASG exhibited significant apoptosis at the P1 stage (P < 0.001), showing a 24 h delay compared to MSG. The peak of Ca²⁺ levels coincided with the onset of significant apoptosis. Further investigation revealed that delayed inositol-1,4,5-trisphosphate receptor (IP3R) protein expression in ASG, resulting in delayed Ca²⁺ release from the endoplasmic reticulum and lagging ATG5 cleavage, is responsible for the delayed onset of apoptosis in the ASG. The rescue of 20-hydroxyecdysone-induced apoptosis by IP3R inhibitors further confirmed this mechanism. The results indicate that delayed expression of IP3R in the ASG regulates Ca²⁺ release from the endoplasmic reticulum and calpain-mediated ATG5 cleavage, leading to asynchronous PCD in different parts of the silk gland. This finding not only reveals the differences in the regulation of PCD between the secretory region (MSG) and non-secretory region (ASG) but also provides new insights into the tissue-specific degradation mechanisms in insect metamorphosis.