Recent studies have identified circular RNAs (circRNAs) in microbes, plants, and animals (Danan et al., 2012; Patop et al., 2019; Tan and Lim, 2021). These structurally unique RNA molecules exhibit remarkable stability, spatiotemporal specificity, and evolutionary conservation (Chen, 2016; Kristensen et al., 2019). CircRNAs have emerged as key regulators in various biological processes. Generally, they act as competing endogenous RNAs (ceRNAs) by sponging microRNAs (miRNAs) to inhibit their activity (Hansen et al., 2013; Nemeth et al., 2024) and serve as protein decoys, adaptors, and scaffolds, thereby regulating protein functions (Tay and Pek, 2017; Wu et al., 2019; Huang et al., 2020; Schreiner et al., 2020; Liu and Chen, 2022). In Drosophila melanogaster, circRNAs regulate neural development, embryogenesis, and lifespan (Westholm et al., 2014; Kramer et al., 2015; Pek et al., 2015; Pamudurti et al., 2017; Gao et al., 2020; Weigelt et al., 2020). However, the understanding of circRNA functions in non-model insect species remains limited. In Aedes albopictus, circRNA-407 functions as a sponge of miR-9a-5p, which enhances the expression of forkhead box protein L for ovarian development (Gao et al., 2023). In Bombyx mori, circRNAs display tissue-specific expression patterns in the silk glands (Gan et al., 2017). circRNAs are also predicted to be potentially involved in task allocation behaviors of the honeybee Apis mellifera (Tholken et al., 2019). In Dendrolimus punctatus, circRNAs are predicted to potentially regulate synapses and cell junctions (Zhang et al., 2020a). Interestingly, circRNAs can be spliced from the receptor gene of the toxin-producing bacterium Bacillus thuringiensis (Bt) in the cotton bollworm Helicoverpa armigera; however, their functions have not yet been experimentally elucidated (Gao et al., 2019). Despite these studies on circRNAs in insects, the role of circRNAs in insect metamorphosis and reproduction remains unclear.

Metamorphosis and high fecundity are hallmark traits of insects, driving their evolutionary success and ecological dominance (Truman, 2019). Juvenile hormone (JH), an arthropod-specific sesquiterpenoid secreted by corpora allata, plays a pivotal role both in insect metamorphosis and female reproduction. JH maintains the juvenile status by antagonizing the metamorphic action of 20-hydroxyecdysone in the larval stage, while promoting reproduction, including vitellogenesis and oogenesis in adulthood (Wyatt and Davey, 1996; Raikhel et al., 2005; Jindra et al., 2015; Roy et al., 2018; Leyria, 2024). Methoprene-tolerant (Met), a basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) transcription factor initially reported in D. melanogaster (Wilson and Fabian, 1986; Ashok et al., 1998), has been identified as the nuclear JH receptor (Charles et al., 2011; Li et al., 2011; Jindra et al., 2013). During the larval stage, Met upregulates the transcription of Krüppel-homolog 1 (Kr-h1) that inhibits the expression of pupa-specific Broad-complex (Br-C) and adult-specific Ecdysone-induced protein 93F (E93), thereby exerting the anti-metamorphic action of JH (Truman and Riddiford, 2019; Song and Zhou, 2020). In adult insects, Met plays a crucial role in previtellogenic development and vitellogenic processes (Raikhel et al., 2005; Wu et al., 2021) by upregulating the expression of genes involved in ribosome proliferation (Raikhel and Lea, 1990; Wang et al., 2017a), carbohydrate and lipid metabolism (Hou et al., 2015; Wang et al., 2017b), and cell polyploidization (Luo et al., 2017; Guo et al., 2019). As a transcription factor, Met's activity is enhanced through phosphorylation in several insect species (Liu et al., 2015; Jindra et al., 2021; Kim et al., 2021). Additionally, in Anopheles gambiae and Tribolium castaneum, Met itself is targeted by miRNAs such as miR-8, miR-14, miR-34, miR-278, and miR-92b, suggesting a potential aspect of post-transcriptional regulation. However, the specific and regulatory roles of these miRNAs remain largely unexplored (Qu et al., 2017). Aside from these advancements, the regulation of Met in JH signaling and JH-mediated insect metamorphosis and female reproduction remains elusive.

In this study, we identified two circRNAs that are derived from Met in the migratory locust Locusta migratoria. circMet1 is predominantly expressed in the cuticle of final instar nymphs, whereas circMet2 is abundant in the fat body of vitellogenic adults. Whereas circMet2 primarily supports vitellogenic development via miRNA sponging, circMet1 not only participates in vitellogenesis but also modulates nymph-to-adult metamorphosis. Our study reveals the molecular mechanism by which circMet1 and circMet2 mediate the distinct actions of JH in inhibiting larval metamorphosis and stimulating female reproduction.