Knockout of Resilin-like genes results in diminished wing size in silkworm moths

The insect cuticle, an important protective barrier covering the body surface, is mainly composed of cuticular proteins and chitin fibers, which together form the insect exoskeleton system. Resilin, an arthropod elastic protein, has attracted attention due to its unique mechanical properties. In this study, we identified BmCPR151, a short resilin-like cuticular protein from Bombyx mori (B. mori). The gene contains a 699-bp CDS encoding a 233-amino-acid full-length protein with shortened repetitive domains, distinguishing it from Drosophila resilin and the previously reported silkworm BmCPR140. Using the CRISPR/Cas9 gene-editing tool, we successfully constructed a systemic BmCPR151 knockout homozygous mutant, and the moths of this mutant exhibited smaller wing area, a significantly thinned wing membrane, and thinner wing veins. Furthermore, based on the previously obtained BmCPR140 knockout mutant, a Double-KO (BmCPR151-KO & BmCPR140-KO) homozygous mutant was generated. Comparative analysis of the three mutants (BmCPR140-KO, BmCPR151-KO, and Double-KO) and the wild type showed that all mutants had decreased wing area and wing vein width. The wing rigidity of female moths decreased by 36.91%, 43.65%, and 45.78%, while that of male moths decreased by 26.54%, 27.08%, and 32.25%, respectively. Moreover, wing patterns were markedly faded in the Double-KO mutant. Transcriptomic analysis revealed that a large number of cuticular proteins were significantly differentially expressed in the wings of Double-KO silkworms, among which 98 cuticular proteins were down-regulated. Collectively, these results indicate that insect cuticular proteins have a relative expression balance, and the loss of resilin-like proteins disrupts the balance, thereby affecting wing development. This study provides a reference for further exploring the functions of insect resilin-like proteins.