Nuclear receptors (NRs) are ligand-regulated transcription factors that play essential roles in metamorphosis, reproduction, metabolism, immunity, and lipid signaling (Fahrbach et al., 2012, Hollman et al., 2012, Lamers et al., 2012). They mediate responses to hormonal signals such as 20-hydroxyecdysone (20E) and juvenile hormone (JH) (Gao et al., 2022). Numerous NR genes have been identified in many insect species. Based on phylogenetic analysis and sequence similarity, NRs are classified into seven subfamilies: NR0, NR1, NR2, NR3, NR4, NR5, and NR6 (Bertrand et al., 2004, Fahrbach et al., 2012). These subfamilies interact cooperatively or antagonistically to form a complex regulatory network (Bonneton et al., 2006). Nuclear receptors (NRs) are structurally conserved in insects and contain two key functional domains: a ligand-binding domain (LBD) and a zinc finger DNA-binding domain (DBD) (Fahrbach et al., 2012). The LBD binds signaling molecules like 20E and JH, inducing conformational changes that recruit co-activators or co-repressors to regulate transcription. The DBD ensures target specificity by recognizing hormone response elements (HREs) in gene promoter regions (Liu et al., 2022, Liu et al., 2015a). Currently, the research on nuclear receptor genes in insects mainly focuses on the response genes related to insect molting, including EcR, E75, HR3, E78, HR96, etc. These genes mainly belong to the NR1 subfamil (Fahrbach et al., 2012). For instance, The SfE75 gene of Sogatella furcifera activates Chitin synthase(Chs) and Chitinase(Cht) genes to promote epidermal remodeling, and RNAi silencing causes 76.61 % nymph mortality (Wang et al., 2025a). The TuE78 gene of Tetranychus urticae regulates cuticle protein deposition, and interference leads to epidermal holes and abnormal layering (Li et al., 2024). An unusual aspect of NR function in insects without a clear parallel in vertebrates is a conserved cascade of NR gene expression triggered by the liganded EcR-USP complex (Guo et al., 2018). The steroid hormone 20E triggers this cascade of gene expression through the EcR/USP receptor heterodimer (Lezzi et al., 2002). For instance, in Drosophila melanogaster, the EcR/USP complex induces a sequential activation of early-response genes (such as EcR, USP, HR3, E75, E78, and FTZ-F1), coordinating epidermal remodeling, organ dissociation, and regeneration during larval-to-pupal transition. Within this cascade, E75 is first directly induced by the 20E pulse, and its protein inhibits the activity of HR3, As E75 protein levels decline, HR3 derepresses and activates Ftz-f1 expression. Ftz-f1 then serves as a critical regulatory hub, priming tissues for subsequent 20E responses. This E75 → HR3 → Ftz-f1 regulatory axis is the common pathway for all 20E pulse responses in the pre-adult stages of fruit fly development (Riddiford, 2012). A similar cascade was defined for metamorphosis in the yellowfever mosquito, Ae. Aegypti (Margam et al., 2006) and in the tobacco hornworm, Manduca sexta (Kiyoshi et al., 2010).

Chitin, a key structural component of the insect exoskeleton and peritrophic matrix, requires a precise balance between synthesis and degradation during molting (Muthukrishnan et al., 2016, Zhu et al., 2016). NRs regulate this process by controlling chitin metabolism genes. In Manduca sexta, EcR/USP upregulates Chs to promote new cuticle formation, while HR3 suppresses chitinase (Cht) to delay old cuticle degradation (Margam et al., 2006, Zhao et al., 2018). Additionally, the antagonistic interaction between 20E and JH adds complexity to this regulatory network. In Tribolium castaneum, JH inhibits EcR/USP activity to delay molting, whereas 20E counteracts this effect by activating HR3 and E75 (Minakuchi et al., 2008). This cross-talk underscores the dynamic equilibrium necessary for successful development. Currently, most studies on nuclear receptors have focused on model insects such as Lepidoptera (Bombyx mori, Spodoptera litura) (Cheng et al., 2008, Yang et al., 2023) and Dipteran (Drosophila melanogaster, Aedes aegypti) (Bertrand et al., 2004, Cruz et al., 2009), while Coleoptera, the largest insect group, remains poorly studied. H. oblita larvae, one of the most destructive pests in China, cause significant yield and quality losses in peanuts, corn, wheat, soybeans, and potatoes (Toepfer et al., 2014, Wang et al., 2019). Chemical insecticides remain the primary method for H. oblita larvae control, but their excessive use leads to environmental pollution, insecticide resistance, and adverse effects on natural enemies (Li et al., 2021).

RNA interference (RNAi) is a sequence-specific approach for silencing target gene expression and holds promise for developing species-specific, long-term pest management strategies (Zhang et al., 2017). It has proven highly effective against Coleopteran pests (Shen et al., 2022). Our previous studies have shown that the chitin-related metabolic genes HpNAG and HpNAGK play critical roles in chitin tissue formation during epidermal development in Holotrichia parallela Motschulsky. Silencing these genes results in high lethality, suggesting their potential as RNAi targets (Zhao et al., 2022). Several RNA-based biopesticides are already commercially available. For example, in 2019, Bayer submitted an application to the US EPA for Bio Direct, an exogenous RNA-based biopesticide targeting Varroa destructor. In 2022, Greenlight Biosciences submitted a dsRNA product for controlling Leptinotarsa decemlineata. These cases highlight the potential of RNA-based biopesticides as next-generation plant protection products, supporting sustainable and eco-friendly agriculture (Taning et al., 2020). NRs are promising RNAi targets due to their central role in molting and epidermal formation. In Henosepilachna vigintioctopunctata, RNAi-mediated EcR gene silencing significantly inhibited larval molting, with 85 % of HvEcR RNAi larvae failing to develop into adults (Wu et al., 2021). However, the role of NRs in H. oblita and their suitability as RNAi targets remain to be determined.

In this study, three nuclear receptor genes (HoHR3, HoE75, and HoEcR) were identified for the first time, and their roles in larval molting and chitin metabolic pathway were examined through spatiotemporal expression analysis, RNA interference, and hormone induction experiments. This study aims to fill the gap in the functional study of Coleopteran NRs and provides a theoretical basis for developing targeted molecular strategies.