Abstract:
 Abstract: 【Aim】 Ecdysone plays important roles in wing dimorphism of the parthenogenetic aphid. In the previous study we found that five microRNAs (miRNAs) also play pivotal roles in the wing dimorphism of the pea aphid, Acyrthosiphon pisum, but whether ecdysone and miRNAs have interaction in wing differentiation of aphids is unclear. This study aims to explore the effect of ecdysone on the expression of miRNAs and their predicted target genes, and to reveal the interaction of ecdysone and miRNAs in wing differentiation of aphids. 【Methods】 The five miRNAs (Let-7, miR-92a, miR-92b, miR-92a-1-p5 and miR-277) related to the wing dimorphism of A. pisum were selected. The 2nd instar nymphs of parthenogenetic A. pisum were exposed to 0.1 mol/L ecdysone analog 20-hydroxyecdysone (20E) for 10 min and 30 min, respectively, and sampled at 48 h after treatment. The expression levels of the five miRNAs and their predicted target genes after 20E treatment were detected by qPCR. The predicted target gene of miR-92a-1-p5, flightin, was verified by dual luciferase activity assay. Finally, the expression of miR-92a-1-p5 in the 4th instar nymphs of parthenogenetic A. pisum was knocked down with nanocarrier/detergent to verify the interaction between miR-92a-1-p5 and its predicted target gene flightin. 【Results】 The expression of the five miRNAs in the 2nd instar nymphs of parthenogenetic A. pisum could be extremely significantly induced by the treatment of 0.1 mol/L 20E for 30 min. But when the 2nd instar nymphs were exposed to 0.1 mol/L 20E for 10 min, the expression levels of miR-92a-1-p5 and miR-92b extremely significantly decreased, while those of Let-7 and miR-277 increased extremely significantly compared with the control. The expression trends of Let-7 and its predicted target gene abrupt were opposite in the 2nd instar nymphs of parthenogenetic A. pisum after 20E treatment. The expression levels of wingless and Uba1, which are the predicted target genes of miR-92a and miR-277, respectively, decreased extremely significantly in the 2nd instar nymphs of parthenogenetic A. pisum exposed to 0.1 mol/L 20E for 10 min, showing the opposite trend with those of the corresponding two miRNAs. The expression level of flightin, the predicted target gene of miR92a-1-p5, decreased extremely significantly in the 2nd instar nymphs of parthenogenetic A. pisum exposed to 0.1 mol/L 20E for 30 min, exhibiting an opposite expression trend to that of miR-92a-1-p5. Dual luciferase activity assay results showed that after co-transfection of the mimics of miR-92a-1-p5 and the flightin CDS overexpression vector pmirGlO ［flightin］ the luciferase activity was extremely significantly decreased by 40% compared to the control transfected with NC mimics. Knocking down the expression of miR-92a-1-p5 in the 4th instar nymphs of parthenogenetic A. pisum extremely significantly decreased its expression by 83%, while extremely significantly enhanced the expression of its predicted target gene flightin by 48%, confirming that flightin is the target gene of miR-92a-1-p5. 【Conclusion】 Ecdysone can induce the expression of miRNAs in A. pisum. miR-92a1-p5 may be involved in wing differentiation of parthenogenetic aphids by regulating flightin gene. Nanocarrier/detergent can achieve effective miRNA interference in aphids. This study lays a foundation for further exploring the interaction of ecdysone and miRNAs in wing differentiation of parthenogenetic aphids.

Key words: Acyrthosiphon pisum, wing differentiation, ecdysone, miRNA, target gene