Involvement of the transcription factor E75 in adult cuticular formation in the red flour beetle Tribolium castaneum

Insect adult metamorphosis generally proceeds with undetectable levels of juvenile hormone (JH). In adult development of the red flour beetle Tribolium castaneum, biosynthesis of adult cuticle followed by its pigmentation and sclerotization occurs, and dark coloration of the cuticle becomes visible in pharate adults. Here, we examined the molecular mechanism of adult cuticular formation in more detail. We noticed that an exogenous JH mimic (JHM) treatment of Day 0 pupae did not inhibit pigmentation or sclerotization, but instead, induced precocious pigmentation of adult cuticle two days in advance. Quantitative RT-PCR analyses revealed that ecdysone-induced protein 75B (E75) is downregulated in JHM-treated pupae. Meanwhile, tyrosine hydroxylase (Th), an enzyme involved in cuticular pigmentation and sclerotization, was precociously induced, whereas a structural cuticular protein CPR27 was downregulated, by exogenous JHM treatment. RNA interference-mediated knockdown of E75 resulted in precocious adult cuticular pigmentation, which resembled the phenotype caused by JHM treatment. Notably, upregulation of Th as well as suppression of CPR27 were observed with E75 knockdown. Meanwhile, JHM treatment suppressed the expression of genes involved in melanin synthesis, such as Yellow-y and Laccase 2, but E75 knockdown did not result in marked reduction in their expression. Taken together, these results provided insights into the regulatory mechanisms of adult cuticular formation; the transcription of genes involved in adult cuticular formation proceeds in a proper timing with undetectable JH, and exogenous JHM treatment disturbs their transcription. For some of these genes such as Th and CPR27, E75 is involved in transcriptional regulation. This study shed light on the molecular mode of action of JHM as insecticides; exogenous JHM treatment disturbed the expression of genes involved in the adult cuticular formation, which resulted in lethality as pharate adults.