AtCYP6K1 regulates ovarian development and reproduction by catalyzing JH biosynthesis in the small hive beetle, Aethina tumida.

Insect cytochrome P450 monooxygenases (CYPs) exert essential functions in xenobiotic detoxification and growth and development. Nevertheless, the physiological roles of the majority of CYP genes remain poorly characterized in the small hive beetle (SHB), Aethina tumida. Screening of CYP genes from the ovarian transcriptome of SHBs identified AtCYP6K1 as an ovary-enriched gene. RT-qPCR analysis revealed that ovarian AtCYP6K1 expression increased with female adult age, reaching the highest level at day 7 post-eclosion. RNA interference (RNAi)-mediated silencing of AtCYP6K1 caused defective ovarian development and markedly reduced female fecundity, egg hatchability, and juvenile hormone III (JHIII) titers. Furthermore, knockdown of AtCYP6K1 significantly downregulated the expression of key genes associated with vitellogenin synthesis (Vg1, Vg2, VgR) and the juvenile hormone signaling pathway (Met, Kr-h1). Notably, in vitro overexpression assays confirmed that AtCYP6K1 catalyzes JH III synthesis from methyl farnesoate (MF) with activity similar to CYP15A1. These findings establish that AtCYP6K1 plays an essential role in female reproduction by directly participating in JH III biosynthesis, which in turn regulates vitellogenin synthesis and ovarian maturation. This study advances our understanding of endogenous physiological functions of insect CYP6 proteins and the reproductive regulatory mechanism of SHB, and offers insights for developing future population management strategies against this pest.