Abstract
Holometabolous insects stop feeding at the final larval instar stage and then undergo metamorphosis; however, the mechanism is unclear. In the present study, using the serious lepidopteran agricultural pest Helicoverpa armigera as a model, we revealed that 20-hydroxyecdysone (20E) binds to the dopamine receptor (DopEcR), a G protein-coupled receptor, to stop larval feeding and promote pupation. DopEcR was expressed in various tissues and its level increased during metamorphic molting under 20E regulation. The 20E titer was low during larval feeding stages and high during wandering stages. By contrast, the dopamine (DA) titer was high during larval feeding stages and low during the wandering stages. Injection of 20E or blocking dopamine receptors using the inhibitor flupentixol decreased larval food consumption and body weight. Knockdown of DopEcR repressed larval feeding, growth, and pupation. 20E, via DopEcR, promoted apoptosis; and DA, via DopEcR, induced cell proliferation. 20E opposed DA function by repressing DA-induced cell proliferation and AKT phosphorylation. 20E, via DopEcR, induced gene expression and a rapid increase in intracellular calcium ions and cAMP. 20E induced the interaction of DopEcR with G proteins αs and αq. 20E, via DopEcR, induced protein phosphorylation and binding of the EcRB1-USP1 transcription complex to the ecdysone response element. DopEcR could bind 20E inside the cell membrane or after being isolated from the cell membrane. Mutation of DopEcR decreased 20E binding levels and related cellular responses. 20E competed with DA to bind to DopEcR. The results of the present study suggested that 20E, via binding to DopEcR, arrests larval feeding and promotes pupation.
Highlights
The post-embryo development of holometabolous insects involves larval, pupal, and adult stages
This study presents evidence that G protein-coupled receptors (GPCRs) function as cell membrane receptors of steroid hormones, and the interaction between the endocrine system and the nervous system
The mRNA levels of dopamine receptor (DopEcR) in the head were upregulated by 20E induction in a time and dose-dependent manner, as assessed using quantitative real-time reverse transcription PCR analysis (Fig 1D)
Summary
The post-embryo development of holometabolous insects involves larval, pupal, and adult stages. The insect molting hormone 20-hydroxyecdysone (20E) promotes metamorphosis by upregulating 20E-pathway gene expression [1] and by counteraction with the juvenile hormone [2] and insulin [3]. 20E initiates gene expression by binding to its nuclear receptor ecdysteroid hormone receptor B1 (EcRB1), which forms a transcription complex with ultraspiracle protein (USP1) and binds to the ecdysone response element (EcRE) [4]. 20E, via GPCR, Phospholipase C (PLC), and calcium-signaling pathways, regulates protein phosphorylation, including that of USP1 [10], cyclin dependent kinase 10 (CDK10) [11], and catalytic domain of protein kinase A (PKAC1) [12] to form the 20E transcription complex EcRB1/USP1 and promote gene expression during insect metamorphosis [10, 13] In Helicoverpa armigera, ErGPCR-1 [8] and ErGPCR-2 [9] transmit 20E signals in the cell membrane. 20E, via GPCR, Phospholipase C (PLC), and calcium-signaling pathways, regulates protein phosphorylation, including that of USP1 [10], cyclin dependent kinase 10 (CDK10) [11], and catalytic domain of protein kinase A (PKAC1) [12] to form the 20E transcription complex EcRB1/USP1 and promote gene expression during insect metamorphosis [10, 13]
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