Adenosine Monophosphate-Activated Protein Kinase (AMPK) Phosphorylation Is Required for 20-Hydroxyecdysone Regulates Ecdysis in Apolygus lucorum.

Abstract

The plant mirid bug Apolygus lucorum is an omnivorous pest that can cause considerable economic damage. The steroid hormone 20-hydroxyecdysone (20E) is mainly responsible for molting and metamorphosis. The adenosine monophosphate-activated protein kinase (AMPK) is an intracellular energy sensor regulated by 20E, and its activity is regulated allosterically through phosphorylation. It is unknown whether the 20E-regulated insect's molting and gene expression depends on the AMPK phosphorylation. Herein, we cloned the full-length cDNA of the AlAMPK gene in A. lucorum. AlAMPK mRNA was detected at all developmental stages, whereas the dominant expression was in the midgut and, to a lesser extent, in the epidermis and fat body. Treatment with 20E and AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AlCAR) or only AlCAR resulted in activation of AlAMPK phosphorylation levels in the fat body, probed with an antibody directed against AMPK phosphorylated at Thr172, enhancing AlAMPK expression, whereas no phosphorylation occurred with compound C. Compared to compound C, 20E and/or AlCAR increased the molting rate, the fifth instar nymphal weight and shortened the development time of A. lucorum in vitro by inducing the expression of EcR-A, EcR-B, USP, and E75-A. Similarly, the knockdown of AlAMPK by RNAi reduced the molting rate of nymphs, the weight of fifth-instar nymphs and blocked the developmental time and the expression of 20E-related genes. Moreover, as observed by TEM, the thickness of the epidermis of the mirid was significantly increased in 20E and/or AlCAR treatments, molting spaces began to form between the cuticle and epidermal cells, and the molting progress of the mirid was significantly improved. These composite data indicated that AlAMPK, as a phosphorylated form in the 20E pathway, plays an important role in hormonal signaling and, in short, regulating insect molting and metamorphosis by switching its phosphorylation status.