Molecular evolution and function analysis of circRNA-407 in mosquitoes

Vector mosquitoes (Diptera: Culicidae) are capable of transmitting multiple critical human pathogens, posing persistent public health risks globally. Following blood ingestion, female mosquitoes initiate ovarian development and undergo repeated blood feeding and oviposition cycles throughout their lifespan. These biological traits serve as the foundation for the transmission of mosquito-borne diseases and significantly contribute to the global expansion of mosquito populations. A comprehensive understanding of the molecular regulatory mechanism in ovarian development post-blood meal is essential for development of novel strategies for mosquito population control. Circular RNA (circRNA) represents a single-stranded RNA molecule characterized by their covalently closed circular structure. They play the critical biological roles through diverse gene regulatory mechanisms, while the molecular structure and function exhibit relative cross-species conservation. In our previous study, we identified a highly expressed circRNA-407 in the ovaries of Aedes albopictus females. This circRNA was found to regulate ovarian development by specifically binding to miR-9a, thereby influencing the expression of its downstream target gene Forkhead Box L (FoxL). However, the prevalence of circRNA-407 across mosquito species and the evolutionary conservation of its functional role remain to be further elucidated. In this study, we conducted gene structure and molecular evolutionary analyses of circRNA-407 and its host gene. Homologous sequences of circRNA-407 were detected in multiple mosquito species using RT-PCR, and the conservation of the circRNA-407/miR-9a/FoxL regulatory axis was assessed through bioinformatic analysis. Our RT-PCR and Sanger sequencing results first confirmed the conservation of circRNA-407 within the Culicidae family. Molecular evolution and gene structure analyses revealed that circRNA-407 comprised four conserved exons derived from its host gene, with evolutionary origins tracing back to a common ancestor of Diptera. Bioinformatics analysis further demonstrated that both circRNA-407 and FoxL in major vector mosquitoes harbor conserved miR-9a binding sites, suggesting that the circRNA-407/miR-9a/FoxL regulatory axis governing ovarian development is evolutionarily conserved. These findings elucidate the evolutionary origin and conserved characteristics of a critical circRNA molecule involved in mosquito reproduction, providing a theoretical foundation for targeting such molecules for population management.