The competing endogenous RNAlnc94641–miR957‐3p mediates male fertility in Zeugodacus cucurbitae Coquillett

BACKGROUND: Insect spermatogenesis is a complex process. Numerous genes are involved in sperm motility, which is crucial for male fertility. Few long non-coding RNAs (lncRNAs) in the testis regulate insect spermatogenesis. We previously identified 364 testis-enriched lncRNAs in the globally invasive pest Zeugodacus cucurbitae Coquillett. One of these lncRNAs, lnc94641, is abundantly expressed in the testis; however, its role in spermatogenesis remains unknown. RESULTS: Suppression of lnc94641 expression led to a 60% decrease in spermatozoa count and a 29% decrease in offspring hatchability. A microRNA (miRNA), miR-957-3p, was experimentally demonstrated to bind to lnc94641 competitively. miR-957-3p overexpression recapitulated reproductive defect phenotypes similar to those caused by lnc94641 knockdown. Furthermore, target gene predictions combined with quantitative reverse transcription PCR, RNA pull-down, and dual luciferase reporter assays confirmed that miR-957-3p targets voltage-gated potassium channel 5 (VGKC5) and odorant receptor 85c (OR85c), elucidating a functional lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) regulatory axis. Fluorescence in situ hybridization (FISH) assays demonstrated the co-localization of lnc94641, miR-957-3p, and VGKC5/OR85c in the mature and transformed regions of the testes. Suppression of VGKC5/OR85c expression resulted in a 68% and 50% decrease in spermatozoa number and an 18% and 21% decrease in offspring hatchability, mirroring the phenotype observed with lnc94641-silencing, thereby reinforcing the mechanistic coherence of this regulatory network. CONCLUSION: These results revealed a ceRNA axis mediated by 'lnc94641-miR957-3p-VGKC5/OR85c' involved in spermatogenesis that impairs male fertility in the melon fly. Molecular perturbations (lncRNA knockdown or miRNA overexpression) consistently impair sperm production and offspring viability by dysregulating ion channels and chemosensory genes. This mechanistically resolved pathway, centered on the core components VGKC5 and OR85c, revealed conserved reproductive vulnerabilities that could enable the targeted genetic control of this agricultural pest. © 2025 Society of Chemical Industry.