<i>TcALS</i> knockdown accelerates metamorphosis via insulin signaling in <i>Tribolium castaneum</i>

Insect metamorphosis is an evolutionary adaptation precisely regulated by nutritional and hormonal cues. The red flour beetle Tribolium castaneum, a major stored-product pest, serves as an excellent model for studying this process. The acid-labile subunit (ALS) of insulin-like growth factor forms a trimer with insulin-like peptides and their binding proteins, prolonging insulin half-life. However, its role in T. castaneum metamorphosis remains poorly understood. Here, we found that TcALS protein is structurally conserved with a characteristic horseshoe shape. Spatiotemporal analysis showed lowest TcALS expression during the pupal stage. TcALS knockdown advanced pupation by approximately three days without altering the final emergence rate. Transcriptome and targeted qRT-PCR analyses uncovered a strict temporal hierarchy: glycosylation, autophagy, carbohydrate metabolism genes were upregulated within 24 h, whereas insulin, juvenile hormone, and 20-hydroxyecdysone signaling genes remain unchanged until 48 h. Mechanistically, TcALS knockdown diverted glucose into the hexosamine pathway and activated glycosylation; the resulting advanced glycation end-products triggered endoplasmic reticulum and oxidative stress, thereby initiating autophagy. Subsequently, insulin-juvenile hormone signaling was suppressed while 20-hydroxyecdysone levels increased, collectively driving larvae to pupate precociously. Thus, our findings establish TcALS as a nutrient-sensitive checkpoint linking early glycosylation-autophagy responses to late insulin-hormonal signaling, providing novel insights into the mechanisms of pest adaptation to nutrition.