The MicroRNA Ame-Bantam-3p Controls Larval Pupal Development by Targeting the Multiple Epidermal Growth Factor-like Domains 8 Gene ( megf8 ) in the Honeybee, Apis mellifera .

20-Hydroxyecdysone (20E) plays an essential role in coordinating developmental transitions in insects through responsive protein-coding genes and microRNAs (miRNAs). However, the interplay between 20E and miRNAs during insect metamorphosis is unknown. In this study, using small RNA sequencing, a comparative miRNA transcriptomic analysis in different development stages, and 20E treatment, we identified ame-bantam-3p as a key candidate miRNA involved in honeybee metamorphosis. Target prediction and in vitro dual-luciferase assays confirmed that ame-bantam-3p interacts with the coding region of the megf8 gene and promotes its expression. Meanwhile, temporal expression analysis revealed that the expression of ame-bantam-3p is higher in the larval stage than in prepupal and pupal stages, and that this expression pattern is similar to that of megf8 . In vivo, we found that the mRNA level of megf8 was significantly increased after the injection of ame-bantam-3p agomir. A 20E feeding assay showed that 20E downregulated the expression of both ame-bantam-3p and its target gene megf8 on larval days five, six, and seven. Meanwhile, the injection of ame-bantam-3p agomir also reduced the 20E titer, as well as the transcript levels of essential ecdysteroid synthesis genes, including Dib, Phm, Sad, and Nvd . The transcript levels of 20E cascade genes, including EcRA , ECRB1 , USP , E75 , E93 , and Br-c , were also significantly decreased after ame-bantam-3p agomir injection. However, ame-bantam-3p antagomir injection and dsmegf8 injection showed the opposite effect to ame-bantam-3p agomir injection. Ame-bantam-3p agomir treatment ultimately led to mortality and the failure of larval pupation by inhibiting ecdysteroid synthesis and the 20E signaling pathway. However, the expression of 20E signaling-related genes was significantly increased after megf8 knockdown, and larvae injected with dsmegf8 showed early pupation. Combined, our results indicate that ame-bantam-3p is involved in the 20E signaling pathway through positively regulating its target gene megf8 and is indispensable for larval-pupal development in the honeybee. These findings may enhance our understanding of the relationship between 20E signaling and small RNAs during honeybee development.