Multiple microRNAs control ecdysone signaling in the midgut of Spodoptera litura

Metamorphosis is one of the most important physiological processes in insects. It is regulated by a serial of ecdysone cascade genes. Recently, lots of microRNAs (miRNAs) were investigated in insects; however, their function in metamorphosis is largely unknown. In the present study, the dynamics of a small RNA population was investigated by RNA sequencing from the midgut of a lepidopteran pest Spodoptera litura during larval-pupal metamorphosis. A total of 101 miRNAs were identified, and 75 miRNAs were differentially expressed during the metamorphic process. The relationship between these differentially expressed miRNAs and 12 ecdysone cascade genes was analyzed by four classical software programs, and a multiple-to-multiple regulatory network was found to exist between these miRNAs and their targets. Among them, miR-14-3p and its two targets (EcR and E75) were chosen for further validation. MiR-14-3p had higher expression level in the 6th instar larvae as compared with either the prepupae or pupae, which was opposite to that of both EcR and E75, two ecdysone cascade genes. Luciferase reporter assay confirmed that both EcR and E75 were regulated by miR-14-3p. Interestingly, the 3' untranslated regions are nearly identical to each other among different transcript variants of the ecdysone cascade genes, including EcR, USP, E75, E74, E78, E93, Hr3, Hr4, Hr39, Krh1 and Ftzf1. Thus, different transcript variants of one ecdysone cascade gene could be regulated by the same miRNA. The above data suggest that the ecdysone signaling pathway is under the tight control of miRNA. These findings expand our understanding of the mechanism of insect metamorphosis and may also provide a novel possibility for the control of pest insects in the future.