Circ-379-miR-3050-DNAH3/RNF8 axis modulates wing development in Aphis citricidus.

Wings are key physiological determinants of aphid dispersal and population outbreaks. Circular RNAs (circRNAs), a novel class of non-coding RNAs, regulate diverse biological processes through post-transcriptional mechanisms. However, their roles in insect wing development remain unclear. In this study, we performed RNA sequencing to identify 715 circRNAs across four developmental stages of Aphis citricidus: fourth-instar winged nymphs, winged adults, fourth-instar wingless nymphs, and wingless adults. Differential expression analysis revealed circ-379 as the only circRNA significantly upregulated during the fourth-instar winged nymphs to winged adults transition, while showing no significant change in wingless morphs, suggesting a potential role in wing development. Subsequent reverse transcription PCR (RT-PCR) validated the circular structure of circ-379 using divergent primers. Silencing of circ-379 induced wing deformities. Bioinformatic prediction, expression profiling analysis, dual-luciferase reporter assays and biotin-avidin-based RNA pull-down assays identified miR-3050 as a direct miRNA target of circ-379, which serves as a molecular sponge to bind miR-3050, thereby suppressing the expression of its target genes dynein axonemal heavy chain 3 (DNAH3) and E3 ubiquitin-protein ligase RNF8-B-like (RNF8). Overexpression of miR-3050 or RNA interference (RNAi)-mediated knockdown of DNAH3 and RNF8 also caused wing deformities. These findings indicate that circ-379 functions as a miR-3050 sponge to influence DNAH3 and RNF8 expression, thereby ensuring proper wing formation in A. citricidus. This study uncovers a circRNA-regulated molecular axis underlying wing plasticity in aphids, thereby advancing our understanding of non-coding RNA-mediated developmental regulation in insects.