CYP6A14 is the major P450 epoxidase responsible for juvenile hormone Ⅲ skipped bisepoxide biosynthesis in Aphis craccivora

Juvenile hormones (JHs) play a crucial role in regulating development and reproduction in insects. In the cowpea aphid ( Aphis craccivora ), JH Ⅲ acid (JH ⅢA) can be converted into JH Ⅲ skipped bisepoxide (JHSB 3 ); however, the enzymes and pathways involved in this conversion remain elusive. In this study, we identified Ac -CYP6A14 as a key JH epoxidase and characterized its role in the JHSB 3 biosynthetic pathway in A. craccivora . In addition, we found that JHSB 3 titers were significantly higher in embryonic tissues than in maternal tissues. Transcriptomic analysis revealed six upregulated genes in the embryo. Temporal expression pattern analysis revealed that only Ac - CYP6A14 showed concordance with JHSB 3 titers. Silencing Ac - CYP6A14 decreased JHSB 3 titers. In vitro catalytic experiments demonstrated that Ac -CYP6A14 catalyzes the conversion of JH Ⅲ into JHSB 3 . Furthermore, we found that the “epoxidation then esterification” sequence is not feasible. Collectively, our findings elucidated the JHSB 3 biosynthetic pathway, in which JH ⅢA is first esterified to form JH Ⅲ, followed by epoxidation to JHSB 3 by Ac -CYP6A14.