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.

昆虫成虫的变态通常以未检测到的幼体激素（JH）水平进行。在红色面粉甲虫Tribolium castaneum的成年发育中，成年角质层的生物合成继而发生色素沉着和硬化，并且在食堂成虫中可见角质层的暗色。在这里，我们更详细地研究了成人表皮形成的分子机制。我们注意到，第0天an的外源性JH模拟（JHM）处理不会抑制色素沉着或硬化，而是提前两天诱导成年表皮的早熟色素沉着。定量RT-PCR分析显示，蜕皮激素诱导的蛋白75B（E75）在JHM处理的p中被下调。同时，酪氨酸羟化酶（Th）是一种与表皮色素沉着和硬化有关的酶，可通过外源性JHM处理早熟地诱导，而结构性表皮蛋白CPR27被下调。RNA干扰介导的E75的敲低导致早熟的成人表皮色素沉着，类似于JHM治疗引起的表型。值得注意的是，用E75敲低观察到Th的上调以及CPR27的抑制。同时，JHM处理抑制了涉及黑色素合成的基因的表达，例如Yellow-y和Laccase 2，但E75组合式并没有导致其表达明显降低。综上所述，这些结果提供了对成人表皮形成调节机制的见解。成年表皮形成相关基因的转录在适当的时机进行，无法检测到JH，外源性JHM处理会干扰它们的转录。对于其中一些基因，如Th和CPR27，E75参与转录调控。这项研究揭示了JHM作为杀虫剂的分子作用方式。外源性JHM处理扰乱了成年表皮形成相关基因的表达，导致成年的成年杀。