miR-let-7 Targeting ZcCTL-S1 to Regulate Reproductive Development in Zeugodacus cucurbitae

The melon fly, Zeugodacus cucurbitae (Coquillett), is recognized as a globally significant quarantine pest, and it ranks among the most destructive insect species infesting cucurbit and solanaceous crops. However, the molecular mechanisms governing reproductive regulation in female Z. cucurbitae remain poorly characterized, particularly those underlying the reproductive processes mediated by microRNAs (miRNAs). In this study, we firstly identified the ovary-specific gene ZcCTL-S1 in Z. cucurbitae via transcriptomic analysis, and subsequently predicted its targeted miRNAs using bioinformatics approaches. Among these miRNAs, overexpression or inhibition of miR-971-1 and miR-let-7 led to corresponding inverse changes in the transcriptional level of ZcCTL-S1. Notably, only miR-let-7 displayed markedly elevated expression levels in Z. cucurbitae ovaries. Further analyses confirmed that miR-let-7 exhibited a direct targeting relationship with ZcCTL-S1, via a combinatorial approach involving in vivo RNA immunoprecipitation, in vitro dual-luciferase reporter assays, and site-directed mutagenesis techniques. Phenotypic analyses showed that both knockdown of ZcCTL-S1 and overexpression of miR-let-7 significantly inhibited egg hatchability, ultimately compromising the female reproductive capacity of Z. cucurbitae. Collectively, these findings identify a novel miRNA-gene regulatory module in the reproductive development of Z. cucurbitae, and provide novel insights for the development of gene- or miRNA-based pest control strategies.