Elasticity of trachea in the silkworm: A role of gene <i>BmMuc91C</i>

Respiration is a vital process essential for organism survival, with most terrestrial insects relying on a sophisticated tubular tracheal network. In the current study, a gene with repetitive sequence was identified within the silkworm genome. Designated as BmMuc91C, it contains a dozen repeated motifs "PSSSYGAPX" and "GGYSSGGX" in its sequence. BmMuc91C exhibits specific expression in the tracheal system of silkworm larvae, with significantly higher expression levels during the molting stage. Overexpression of BmMuc91C in individual silkworms resulted in a marked increase in tracheal diameter, particularly during the molting stage. Immunofluorescence staining using a BmMuc91C antibody revealed a noticeable thickening of the apical extracellular matrix in the trachea. Tensile testing confirmed a considerable enhancement in tracheal elasticity. Additionally, a BmMuc91C mutation strain of silkworms was generated using the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 system. Although no significant differences were observed in the growth, development, and molting of BmMuc91C mutant silkworms, mechanical tests demonstrated a decrease in tracheal elasticity. Transcriptomic techniques revealed that a significant number of cuticular and chitin-binding proteins were among the differentially expressed genes between mutant and wild-type silkworms. Furthermore, the recombined BmMuc91C protein was successfully expressed using the Escherichia coli system. Cross-linking experiments with horseradish peroxidase demonstrated the formation of macromolecular complexes of BmMuc91C, which exhibited spontaneous luminescent properties under ultraviolet light. This research sheds light on the role of elastic proteins in insect tracheae and provides valuable insights for the development of elastic biomaterials.