Juvenile hormone biosynthetic genes are critical for regulating reproductive diapause in the cabbage beetle.

In insects, the juvenile hormone (JH) biosynthetic pathway regulates the in vivo JH titer. Thus, its downregulation potentially contributes to the lowering of JH titers typically observed in insects undergoing reproductive diapause, a developmental arrest at the adult stage. However, no systematic evidence has yet been presented to demonstrate the physiological and genetic roles of JH biosynthetic genes in reproductive diapause. In this work, we performed RNA interference (RNAi)-based reverse genetic analyses by targeting JH biosynthetic genes, followed by analysis of the reproductive diapause traits in Colaphellus bowringi, an economically important cabbage beetle. We identified a total of 22 genes encoding homologues of enzymes involved in the mevalonate pathway and the JH branch of JH biosynthesis in C. bowringi. Among these, 18 genes showed significant downregulation of their expression in the long day-induced diapausing females, compared to the short day-induced reproductive females. RNAi knockdown of almost any one of the 18 genes in reproductive females reduced the expression of the JH-responsive gene, Krüppel homolog1 (Kr-h1), indicating a lowered circulating JH. Most importantly, depleting transcripts of 3-hydroxy-3-methylglutaryl-CoA reductase 2 (HMGR2), farnesyl-pyrophosphate synthase 1 (FPPS1) and juvenile hormone acid methyltransferase 1 (JHAMT1) induced diapause-associated traits, including immature and inactive ovaries, large accumulations of lipids and adult burrowing behavior. Meanwhile, genes related to ovarian development, lipid accumulation and stress response showed expression patterns like those of diapausing females. RNAi-mediated diapause phenotypes could be reversed to reproductive phenotypes by application of methoprene, a JH receptor agonist. These results suggest that photoperiodic reproductive diapause in C. bowringi is triggered by transcriptional suppression of JH biosynthetic genes, with HMGR2, FPPS1 and JHAMT1 playing a critical role in this process. This work provides sufficient evidence to reveal the physiological roles of JH biosynthetic genes in reproductive diapause.