Disruption of Yellow-E3 Impairs Both Adult Molting and Cuticular Melanization in the Honeybee (apis Mellifera)

BACKGROUND:The insect yellow gene family plays crucial roles in cuticle pigmentation, waterproofing, courtship, molting, and eggshell development. However, the specific physiological function of yellow-e3 remains unclear. RESULTS:In this study, we first generated yellow-e3 mutant drones using CRISPR/Cas9, achieving mutation rate of 87.4%, primarily (61.6%) comprising 5-bp deletions. But no discernible phenotypic differences were observed in these mutants. We therefore performed RNAi knockdown by injecting yellow-e3 siRNA into 2-day-old pupae, which significantly reduced eclosion rate (36.14 vs. 72.53% in controls). Individuals that failed to eclose were arrested during molting, unable to shed the pupal cuticle, and exhibited overall yellow cuticles. Eclosed adults, by contrast, showed normal cuticle coloration. Further analysis revealed that non-eclosed individuals had significantly upregulated expression of ecdysone signaling genes USP and E75, but downregulated E74, accompanied by markedly reduced expression of melanin synthesis genes (TH, yellow-y, and tan). These changes ultimately disrupted melanin deposition, leading to cuticular yellowing. CONCLUSIONS:We propose that genetic compensation may conceal phenotypic defects in CRISPR-generated mutants. This study provides the first evidence that silencing yellow-e3 in A. mellifera disrupts molting, reduces the eclosion rate, and inhibits cuticular melanization.