Trr/COMPASS regulates photoperiodic reproductive diapause via 20-hydroxyecdysone signaling in Colaphellus bowringi

Trr/COMPASS regulates photoperiodic reproductive diapause via 20-hydroxyecdysone signaling in Colaphellus bowringi | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Trr/COMPASS regulates photoperiodic reproductive diapause via 20-hydroxyecdysone signaling in Colaphellus bowringi Xiao-Ping Wang, Zhong Tian, Shuang Guo, Yu-Lian Zhao, Kou Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8127731/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Developmental plasticity allows animals to survive harsh conditions. In insects, reproductive diapause is a classic form of plasticity, characterized by ovarian arrest. This process is typically triggered by seasonal cues such as photoperiod and governed by endocrine pathways. However, how photoperiods are converted into endocrine signals that determine reproductive fate remains unclear. The cabbage beetle ( Colaphellus bowringi ) displays a clear photoperiodic response: long-day (LD) conditions induce diapause with ovarian arrest, whereas short-day (SD) conditions promote ovarian development and reproduction. Here, we identified eight conserved subunits of the Trr/COMPASS complex and found their transcriptional levels significantly higher in the ovaries of SD females compared to LD females. Knockdown of Trr/COMPASS components caused ovarian arrest in SD females, accompanied by reduced production and signaling of 20-hydroxyecdysone (20E). We further identified serine/threonine-protein kinase polo ( plk1 ) as a key downstream effector of 20E signaling that promotes ovarian development. Trr/COMPASS-mediated H3K4me3 enrichment was found in regulatory regions of plk1 and several 20E pathway genes, facilitating their transcriptional activation. These findings reveal a photoperiod-sensitive signaling axis, Trr/COMPASS–20E– plk1 , linking environmental cues to endocrine regulation and reproductive diapause, highlighting the essential role of histone modification in developmental adaptation to seasonal changes. Biological sciences/Zoology/Entomology Biological sciences/Developmental biology Developmental plasticity Reproductive diapause COMPASS-like complex Steroid hormone serine/threonine-protein kinase polo H3K4me3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Induction Organisms in temperate regions face recurring environmental fluctuations driven by seasonal change, which can severely constrain survival and reproduction 1 . To persist, they have evolved developmental plasticity—the ability to modify developmental trajectories and produce adaptive phenotypes in response to environmental variation 2 , 3 . A prominent example is reproductive diapause, an endocrine-mediated strategy that allows insects to survive unfavorable seasons and reproduce when favorable conditions return 4 . During diapause, adults suppress reproductive investment and redirect resources toward nutrient accumulation to endure prolonged periods of resource scarcity 5 , 6 . This plasticity enables insect populations to synchronize their life cycles with predictable seasonal environment changes in resource availability 6 , 7 . Therefore, understanding the mechanisms underlying reproductive diapause is fundamental to elucidating how organisms adapt developmentally to fluctuating environments. Epigenetic modifications that respond dynamically to environmental cues are central to regulating phenotypic plasticity 8 , 9 . Among these, specific histone marks such as H3K4me3, H3K9me3, and H3K27me3 control insect development by modulating transcription of key developmental genes 10 , 11 . H3K4me3, in particular, establishes active chromatin states and ensures precise gene expression in eukaryotes 11 , 12 . Recent studies suggest that H3K4me3 plays a pivotal role in determining whether insects successfully enter environment-sensitive reproductive diapause 13 , 14 . The deposition of H3K4me3 is catalyzed by the highly conserved COMPASS (Complex Proteins Associated with Set1) family of complexes 15 . In Drosophila melanogaster , three COMPASS-like complexes have been identified, each with a unique catalytic subunit: Set1, trithorax (Trx), and trithorax-related (Trr) 15 , 16 . Our recent study showed that knocking down set1 and trr induced diapause-like ovarian arrest in reproductive Colaphellus bowringi females 14 . While set1 mediates reproductive diapause through H3K4me3-dependent regulation of juvenile hormone (JH) production in the corpora allata (CA), the primary source of JH 14 , trr knockdown did not alter H3K4me3 levels in the CA. This raises a key question: how does trr influence reproductive diapause through mechanisms independent of H3K4me3 regulation in the CA? A lack of JH is widely recognized as the primary trigger for the initiation of reproductive diapause in insects 17 . In recent years, 20-hydroxyecdysone (20E) has also emerged as a critical hormonal regulator of reproductive diapause, often acting through modulation of JH signaling 17 , 18 . For instance, reduced 20E levels have been associated with diapause entry in Locusta migratoria 19 , D. melanogaster 20 , and C. bowringi 18 , indicating that 20E production is environmentally regulated. Our previous study demonstrated that photoperiod-sensitive 20E acts through Ecdysone receptor (EcR) to activate JH biosynthesis 18 , regulate the Dpp pathway 21 , and control DNA replication 22 , highlighting its multi-faceted role in ovarian growth in the cabbage beetle. Furthermore, recent studies indicate that histone modifications such as H3K27me3, H3K27ac, and H4K20me regulate 20E signaling by directly controlling key genes in its biosynthesis and transduction pathways in Bombyx mori , D. melanogaster , and Tribolium castaneum 23 – 26 . These findings suggest extensive crosstalk between histone modifications and 20E-mediated developmental processes. Building on this evidence, we hypothesize that photoperiod regulates 20E signaling by modifying the Trr/COMPASS histone complex, thereby influencing reproductive diapause entry. To test this, we used the cabbage beetle, C. bowringi , which enters reproductive diapause under long-day (LD) conditions but remains reproductive under short-day (SD) photoperiods, showing clear ovarian differences 27 , 28 . We identified all genes encoding Trr/COMPASS subunits and found their transcripts enriched in the ovaries of SD-induced reproductive females. We demonstrated that Trr/COMPASS regulates H3K4me3 levels in the ovaries, thereby modulating 20E signaling, which in turn interacts with JH signaling to mediate reproductive diapause. Furthermore, we identified serine/threonine-protein kinase polo ( plk1 ) as a downstream target of Trr/COMPASS-dependent H3K4me3 in the ovary; importantly, plk1 expression is 20E-dependent and contributes to diapause regulation. Together, these findings uncover an ovary-driven epigenetic pathway that integrates histone modification with endocrine signaling to coordinate reproductive diapause initiation. Results Identification and transcriptional profiling of Trr/COMPASS subunit genes Trr/COMPASS is a conserved H3K4 methyltransferase complex in eukaryotes composed of eight subunits: Trr, Retinoblastoma-binding protein 5 (RbBP5), Will die slowly (Wds), Absent, small, or homeotic discs 2 (Ash2), Dpy-30-like (Dpy-30), UTX histone demethylase (UTX), PAX-interacting protein 1 (PTIP), and PAXIP1-associated glutamate-rich protein 1 (PA1) (Table S1 ). Using D. melanogaster orthologues as queries, we identified all eight subunits in C. bowringi through BLAST searches against existing transcriptome databases (PRJNA575933 and PRJNA338895) 18 , 31 . Phylogenetic analysis revealed strong evolutionary conservation among insects, with C. bowringi Trr/COMPASS subunits clustering most closely with those of other coleopterans (Fig. 1 ). The catalytic subunit Trr belongs to the KMT2C family, and domain prediction confirmed the presence of the conserved “SET” and “post-SET” domains (Fig. S1 ), similar to D. melanogaster Trr and Homo sapiens KMT2C, suggesting preserved methyltransferase activity. The shared components of COMPASS-like complexes (RbBP5, Wds, Ash2, and Dpy-30) and Trr/COMPASS-specific subunits (UTX, PTIP, and PA1) also exhibited highly conserved domain architectures across species (Fig. S1 ). This evolutionary conservation validates the accurate identification of these genes and supports a conserved role for Trr/COMPASS in transcriptional regulation through H3K4me3 in C. bowringi . To preliminarily evaluate the role of Trr/COMPASS in reproductive development, we analyzed its transcript levels across tissues and developmental stages using RT-qPCR. Heatmap analysis showed significantly higher transcript levels of all eight subunits in the ovary of SD females than in the head, fat body, or midgut (Fig. 2 A). Temporal profiling of LD and SD ovaries revealed no transcriptional differences in newly emerged females; however, by 4 days post-eclosion (PE), all eight subunits were significantly upregulated under SD conditions, with six genes ( trr , rbbp5 , wds , ash2 , utx , and pa1 ) already elevated at 2 days PE (Fig. 2 B). These photoperiod-dependent expression dynamics parallel ovarian developmental progression 29 , with higher transcript levels correlating with active ovarian maturation. Together, the tissue- and stage-specific patterns of Trr/COMPASS transcription suggest that this complex functions as a key mediator of photoperiodic control over reproductive diapause in C. bowringi . Knockdown of Trr/COMPASS components induces ovarian developmental arrest in reproductive females To investigate the role of Trr/COMPASS in photoperiod-mediated reproductive plasticity, we performed RNAi targeting its subunit genes in SD-induced reproductive females. Knockdown of trr , rbbp5 , wds , and utx markedly inhibited ovarian development, with 55.5%, 72.7%, 100%, and 52.0% of females exhibiting ovarian arrest at grades 0-III, respectively—far exceeding the 10% observed in the dsgfp control (Fig. 3 A). RT-qPCR analysis confirmed effective transcript reduction for all targeted genes ( trr , rbbp5 , wds , ash2 , dyp30 , utx , ptip , and pa1 ), with decreases ranging from 32.4% to 81.2% relative to the dsgfp control (Fig. 3 B), supporting the reliability of RNAi-induced phenotypes. For subsequent analyses, we focused on three key components: trr (core catalytic subunit), rbbp5 (a shared subunit of COMPASS-like complexes), and utx (Trr/COMPASS-specific subunit). Their knockdown significantly reduced vitellogenin deposition and ovarian size compared to gfp RNAi (Fig. 3 C, D). We also analyzed transcription of ovarian development-related genes via RT-qPCR, including vitellogenin ( vg ) and its receptor genes ( vg1 , vg2 , and vgr ) 30 , the follicle cell development-related gene fcp3c1 , and key genes of the Dpp signaling pathway ( tkv and medea ) 21 . Following knockdown of trr , rbbp5 , and utx , transcript levels of vg1 and vg2 were significantly decreased by over 60% in the fat body, while vgr , fcp3c1 , tkv , and medea were markedly reduced by 19.1–99.6% in the ovaries (Fig. 3 E, G). These results establish Trr/COMPASS as an essential regulator of photoperiod-dependent reproductive diapause in C. bowringi , acting primarily through control of ovarian development. Trr/COMPASS regulates reproductive diapause through modulation of 20E signaling Insect hormones, particularly 20E and JH, are central regulators of photoperiod-induced reproductive diapause 17 . In C. bowringi , Trr/COMPASS subunits are predominantly expressed in ovarian tissue (Fig. 2 A), consistent with tissue-specific 20E production 18 . We therefore investigated whether Trr/COMPASS regulates reproductive development via 20E signaling. RT-qPCR analysis showed that RNAi targeting trr , rbbp5 , and utx significantly downregulated the transcription of two key 20E biosynthetic genes ( spook and shade ) in the ovaries of SD females compared to the dsgfp control. (Fig. 4 A). ELISA quantification further confirmed that 20E titers decreased by 30–60% following Trr/COMPASS knockdown (Fig. 4 B). Moreover, Trr/COMPASS knockdown markedly reduced the transcript levels of the 20E receptor gene ecr and key ecdysone-response genes e74 ( ecdysone-inducible protein 74 ) and hr3 ( nuclear hormone receptor ) in ovarian tissue (Fig. 4 C). In addition, knockdown of Trr/COMPASS members significantly downregulated transcript levels of 3-hydroxy-3-methylglutaryl-CoA reductase 2 ( hmgr2 ) and juvenile hormone acid methyltransferase 1 ( jhamt1 ), as well as JHAMT1 protein abundance, in head tissues, indicating a reduction in JH signaling (Fig. S2). Together with previous findings, these results suggest that Trr/COMPASS likely regulates JH biosynthesis in the head via 20E signaling in C. bowringi 18 . Collectively, our data demonstrate that Trr/COMPASS modulates reproductive diapause through 20E signaling by coordinating the transcription of key genes involved in both 20E biosynthesis and downstream signaling. Transcriptome analysis identifies plk1 as a core downstream target of Trr/COMPASS-20E signaling axis Although 20E signaling is closely associated with photoperiod-mediated reproductive diapause and its indirect role in mediating JH biosynthesis is well established, its direct actions within the ovary remain poorly understood. We therefore hypothesized that Trr/COMPASS-mediated 20E signaling stimulates ovarian development directly by altering the transcription of key downstream genes. To identify such targets, we performed transcriptome analysis of ovarian tissues from day 2 LD female adults following 20E application (PRJNA1311033). De novo transcriptome assembly yielded 32,904 unigenes (Dataset S1), among which 6,417 (19.5%) were significantly upregulated and 9,617 (29.5%) were significantly downregulated after 20E treatment relative to the solvent control (Fig. 5 A, B). Additionally, differentially expression analysis between SD and LD females using a previously published transcriptome (PRJNA338895) identified 587 and 2,269 upregulated unigenes at 2dA and 4dA, respectively (Datasets S2 and S3). To pinpoint genes responsive to both photoperiod and 20E signaling, we intersected upregulated genes from 20E-treated ovaries with those from SD-induced females at 2 and 4 days PE. Venn analysis based on unique KEGG Orthology (KO) identifiers revealed 143 shared KOs across the three gene sets (Fig. 5 C), corresponding to 541 unigenes in the 20E-treated transcriptome (Dataset S4). KEGG pathway enrichment of these unigenes revealed 57 pathways with diverse molecular functions (Fig. S3). Among these, 25 unigenes were mapped to three ovarian development-related pathways—“Cell cycle”, “Oocyte meiosis”, and “Progesterone-mediated oocyte maturation”—including three shared transcripts: CL3824.Contig1 , CL3824.Contig2 , and unigene12228 (Fig. 5 C). BLAST search and phylogenetic analysis confirmed that both CL3824.Contig1 and CL3824.Contig2 encode PLK1, whereas unigene12228 encodes a G2/mitotic-specific cyclin-B (CycB) (Figs. 5 D and S4). These results identify plk1 and cycb as candidate downstream effectors of the Trr/COMPASS–20E signaling axis that mediate reproductive diapause in C. bowringi . RT-qPCR analysis showed that plk1 and cycb transcripts were highly specific to the ovaries of SD females, with levels significantly higher than those in other tissues (Fig. 5 E), suggesting their essential roles in ovarian development. Application of 20E markedly upregulated plk1 and cycb transcription in the ovaries of LD females (Fig. 5 F), whereas RNAi knockdown of 20E signaling genes ( spook , shade , and ecr ) reduced their transcript levels by over 60% in the ovaries of SD females compared to the dsgfp control (Fig. 5 G). Under SD conditions, plk1 and cycb transcript levels increased approximately 3-fold and 10-fold, respectively, relative to LD females at 2 and 4 days PE (Fig. 5 H), confirming regulation by both photoperiod and 20E signaling. These expression patterns indicate that the transcription of plk1 and cycb is regulated by both photoperiod and 20E signaling. Knockdown of plk1 , but not cycb , in 2-day SD female pupae significantly inhibited yolk deposition, ovarian developmental grade, and ovary size at 4 days PE (Fig. 5 I, K). RT-qPCR analysis confirmed that RNAi significantly reduced plk1 and cycb transcript levels by 72.7% and 89.1%, respectively, in the ovaries of SD females (Fig. 5 L), validating the reliability of the RNAi-induced ovarian phenotypes. In addition, knockdown of either ecr or plk1 significantly downregulated transcript levels of ovarian development-related genes vg1 , vg2 , and fcp3c1 in SD females, whereas cycb RNAi had no significant effect (Fig. 5 M). Moreover, plk1 transcript levels decreased significantly by 63.0%, 27.3%, and 30.7% in the ovaries of SD females following knockdown of trr , rbbp5 , and utx , respectively (Fig. 5 N). In addition, a heatmap generated from a previously established transcriptome (PRJNA338895) showed a concordant high-expression trend for transcripts related to Trr/COMPASS, 20E signaling, and plk1 under SD conditions compared to LD conditions (Fig. S5 and Dataset S5). Together, these results identify plk1 as a key ovary-specific effector acting downstream of the Trr/COMPASS–20E signaling axis to regulate photoperiodic reproductive diapause in C. bowringi . Trr/COMPASS-mediated H3K4me3 governs activation of the 20E- plk1 signaling cascade Trr/COMPASS and its animal orthologues catalyze H3K4me3 deposition, a histone mark associated with euchromatin and active transcription. Western blot analysis revealed a significant 1.4-fold upregulation of H3K4me3 abundance in the ovaries of SD females compared to LD individuals (Fig. 6 A). As expected, knockdown of Trr/COMPASS subunits— trr , rbbp5 , and utx —reduced H3K4me3 levels by 47.7%, 52.3%, and 25.8%, respectively, in the ovaries of SD females (Fig. 6 B). These findings indicate that Trr/COMPASS-mediated H3K4me3 is photoperiod-sensitive and is positively associated with ovarian development. Given that H3K4me3 is typically enriched in promoter regions to activate transcription 12 , we next examined whether Trr/COMPASS-mediated H3K4me3 is enriched in the upstream regions of key genes in the 20E-EcR- plk1 signaling axis. Using two pairs of primers targeting two non-overlapping upstream regions of spook , shade , ecr , and plk1 (Fig. S6), we performed chromatin immunoprecipitation (ChIP)-qPCR with a commercial H3K4me3 antibody and a negative control antibody (IgG). ChIP-qPCR analysis showed significant enrichment of H3K4me3 at all targeted upstream regions of spook , shade , ecr , and plk1 in the ovaries of SD females compared to LD individuals, whereas the IgG control showed no significant differences (Fig. 6 C). In contrast, trr knockdown significantly reduced H3K4me3 enrichment in the upstream regions of these genes compared to the dsgfp control (Fig. 6 D). Together, our findings demonstrate that photoperiod-responsive Trr/COMPASS governs reproductive development by mediating H3K4me3 deposition in the ovary to control transcription of the 20E–EcR– plk1 signaling axis. Discussion Reproductive diapause is an evolutionarily conserved strategy that allows organisms to optimize resource use and maintain population continuity across fluctuating environments 1 , 32 . This adaptive trait reflects their ability to reprogram physiological and molecular processes in response to external cues 2 , 7 , 33 . In insects, reproductive diapause is typically induced by photoperiod and temperature, with endocrine signals directly regulating reproductive arrest or activation 4 , 17 . However, the transcriptional mechanisms that link photoperiodic cues to endocrine responses remains unclear. Using the cabbage beetle C. bowringi as a model, our study shows that Trr/COMPASS—a histone modification regulator—directly controls the 20E signaling pathway by modulating H3K4me3 levels, thereby determining female reproductive diapause. Key evidence supports this conclusion: i) Trr/COMPASS components are highly expressed in the ovaries of reproductively active females and exhibit photoperiod-sensitive transcription (Figs. 1 and 2 ). ii) Knockdown of Trr/COMPASS leads to ovarian arrest and disrupts both 20E production and downstream signaling (Figs. 3 and 4 ). iii) Trr/COMPASS-mediated H3K4me3 is enriched in upstream regulatory regions of genes within the 20E–EcR– plk1 signaling axis, activating their transcription and promoting ovarian development (Figs. 5 and 6 ). These findings establish Trr/COMPASS as a key photoperiod-responsive epigenetic regulator that links environmental signals to endocrine activity, thereby coordinating reproductive diapause initiation. H3K4me3 functions as a key regulator of gene transcription, translating environmental signals into the control of insect development 15 . In D. melanogaster , Set1/COMPASS-mediated H3K4me3 contributes to tissue development and morphogenesis 34 – 36 , underscoring its broad developmental role. In C. bowringi , we further clarify its function: the establishment of H3K4me3 is photoperiod-sensitive, driven by transcriptional activation of Trr/COMPASS in response to environmental cues. While Set1/COMPASS-mediated H3K4me3 in the CA promotes JH production 14 , we now show that Trr/COMPASS-mediated H3K4me3 in the ovaries enhances 20E biosynthesis. Integrating these results, we propose that during ovarian maturation, 20E trans-tissue activates JH signaling 18 , 37 and directly influences ovarian development via plk1 regulation. This process, initiated by photoperiod, operates through the Trr/COMPASS–H3K4me3–20E– plk1 axis in the ovary, allowing precise environmental regulation of reproductive diapause. Since many organisms rely on reproductive arrest to survive seasonal extremes, targeting this pathway could offer a viable strategy for pest control and extending the shelf life of natural enemies. Trr/COMPASS-mediated H3K4me3 as a central epigenetic regulator of photoperiodic reproductive diapause Eukaryotes share conserved H3K4 methyltransferases, homologous to the yeast Set1-centered COMPASS complex 16 . In higher insects, the core Set1-like methyltransferases have diversified into Trx and Trr, orthologous to mammalian MLL1/MLL2 and MLL3/MLL4, respectively 15 . Previous functional studies in C. bowringi showed that only set1 and trr are essential for ovarian development, while trx is dispensable 14 , indicating distinct roles for COMPASS variants. Here, we systematically identified full complement of Trr/COMPASS subunits and found that their animo acid sequences are highly conserved with orthologs in Drosophila and humans. These genes are preferentially expressed in the ovaries and are transcriptionally upregulated under SD conditions (Fig. 2 ), suggesting that their transcription is photoperiod-dependent. H3K4 methyltransferases are typically inactive without incorporation into the multi-subunit COMPASS complex 15 , 38 . RNAi knockdown of Trr/COMPASS subunits resulted in severe ovarian arrest phenotypes—both physiologically and molecularly—that phenocopied trr depletion. Although utx depletion was previously shown to promote glycogen and lipid accumulation in Culex pipiens 39 , our results reveal an additional role for utx in ovarian development in C. bowringi (Fig. 3 ). These findings from two distinct species collectively underscore the central role of UTX-mediated histone modification in reproductive diapause regulation. Importantly, the direct correlation between Trr/COMPASS activity and ovarian H3K4me3 levels establishes this histone mark as a key epigenetic switch that controls the transition between ovarian growth and arrest under changing photoperiods. This represents a previously unrecognized mechanism of reproductive diapause, reflecting species-specific adaptations of Trr/COMPASS to environmental cues. Comparable functions of other histone modifications further emphasize the evolutionary conservation of epigenetic control in oogenesis. For example, H3K9me3 is essential for oogenesis and germline stem cell maintenance in D. melanogaster 40 , 41 , while in Aedes aegypti , H3K27me3 promotes ovarian maturation 42 . In contrast, H3K27me2 levels are elevated in reproductive females compared to those in diapause 43 , indicating that its abundance can be modulated by environmental cues. These histone marks —H3K4me3, H3K9me3, and H3K27me3—act as conserved epigenetic regulators of oogenesis across metazoans 10 , 44 . In summary, our findings establish an H3K4me3-centered photoperiodic regulatory mechanism that links environmental signals to reproductive fate, illustrating how epigenetic flexibility supports adaptive developmental plasticity in fluctuating environments. 20E signaling integrates photoperiod-dependent Trr/COMPASS-H3K4me3 regulation As a classical endocrine initiator, 20E acts as a central hub that translates environmental cues into physiological and molecular responses 45 . 20E production is photoperiod-dependent and significantly influences developmental outcomes such as diapause and reproduction 18 , 37 , 46 . Upon 20E stimulation, its receptor EcR is activated and transduces signals through early-response transcription factors, including E74, E75, and HR3 47,48 . Previous studies have shown that Trr/COMPASS promotes ecr transcription by enriching H3K4me3 at its promoter, underscoring its critical role in activating 20E signaling 49 , 50 . This signaling cascade is essential for female reproductive processes, including ovarian cell differentiation, oogenesis, and vitellogenesis 51 – 53 . A key unresolved question, however, is how photoperiod controls 20E production to modulate reproductive diapause. Photoperiod signals are detected by the compound eyes, processed in the brain, and transmitted to endocrine organs—yet the terminal regulatory step governing hormone synthesis remains unclear 54 . Our study addresses this gap by demonstrating that the photoperiod-responsive Trr/COMPASS directly activates transcription of 20E synthesis genes via H3K4me3 modification, thereby promoting 20E production. This epigenetic mechanism establishes a fundamental regulatory framework for photoperiod-dependent 20E signaling. Comparable regulatory paradigms have been identified in other insect species, such as PRC2-mediated H3K27me3 in B. mori and METTL3/METTL14-mediated m 6 A RNA methylation in Bactrocera dorsalis 23 , 55 . Although these epigenetic regulators do not sense photoperiod directly, they function as key intermediaries linking environmental cues to developmental and reproductive responses 23 , 56 . The circadian clock has long been recognized as the central system for photoperiod signal processing in the brain 54 , 57 . In Rhodnius prolixus , rhythmic oscillations in 20E production during oogenesis have been observed, strongly suggesting circadian regulation 58 . Although direct evidence in adult reproduction is still limited, core clock genes such as timeless and period have indeed been correlated with photoperiod sensing and 20E production 59 . Therefore, future studies should elucidate how photoperiod signals are integrated by the ovarian Trr/COMPASS–H3K4me3–20E axis to reveal the precise mechanisms of insect reproductive diapause. plk1 is an ovary-specific downstream effector of the Trr/COMPASS-H3K4me3-20E signaling axis PLK1, a serine/threonine kinase, is a central regulator of eukaryotic cell division 60 , 61 . It functions in both mitotic organ development and meiotic gametogenesis during reproductive system formation 61 , 62 . Our study demonstrates that ovarian maturation in C. bowringi strictly depends on plk1 , and its ovary-specific transcription is precisely controlled by the Trr/COMPASS–H3K4me3–20E signaling axis. This pathway begins with photoperiodic input, which is transduced via epigenetically regulated endocrine signaling to activate key target genes, enabling precise control of tissue development. These findings establish plk1 as a critical downstream effector of 20E signaling during reproductive diapause. As a fundamental cell cycle regulator, plk1 is highly conserved in C. bowringi , showing strong homology to orthologs in other insects (Fig. S4). This suggests its role in ovarian growth is likely associated with cell cycle regulation, although direct experimental verification in non-model insects remains technically challenging. Notably, in Drosophila , PLK1 regulates germ cell division through mechanisms that mirror those in mice 63 , 64 . This functional conservation suggests that the epigenetic regulation of plk1 transcription uncovered in C. bowringi may represent a widespread mechanism across species. Therefore, elucidating the Trr/COMPASS–H3K4me3–20E– plk1 signaling axis advances our understanding of how developmental plasticity integrates environmental adaptation. Environmental regulation of developmental plasticity remains a central question in biology. Based on our findings, we propose a model in which Trr/COMPASS regulates photoperiod-dependent reproductive diapause (Fig. 7 ). Under SD conditions, elevated Trr/COMPASS activity promotes H3K4me3 enrichment at the upstream regulatory regions of 20E biosynthetic genes ( spook and shade ), the 20E receptor gene ( ecr ), and the downstream effector plk1 . This enrichment activates transcription of these targets, thereby enhancing 20E production and subsequently initiating the EcR– plk1 signaling cascade. The 20E–EcR– plk1 signaling axis further coordinates the transcription of genes involved in vitellogenesis and ovarian growth, promoting full ovarian maturation. In contrast, under LD conditions, suppression of Trr/COMPASS-mediated H3K4me3 attenuates the 20E–EcR– plk1 pathway, resulting in reduced hormone signaling and diapause-like ovarian arrest. This study reveals a previously unknown epigenetically regulated endocrine cascade that links photoperiodic cues to reproductive diapause. Future studies should focus on identifying the upstream factors that transmit environmental cues to the Trr/COMPASS–H3K4me3 signal, thereby deepening our understanding of how insects adapt to their environments. Materials and methods Experimental insects Approximately 500 adult C. bowringi beetles were collected from Xiushui County, Jiangxi Province, China (29°1’N, 114°4’E) in November 2017. The colony was maintained under laboratory conditions at 25 ± 1 °C and 70% relative humidity, with fresh radish leaves ( Raphanus sativus var. longipinnatus ) provided as food 27 . To induce reproductive development, larvae were reared under SD conditions (12 h light: 12 h dark). Reproductive females display a 4-day pre-oviposition period, marked by yolk deposition and ovarian development. In contrast, exposure to LD conditions (16 h light: 8 h dark) during larval and pupal stages triggers reproductive diapause in adults, marked by ovarian arrest during the same PE period 28,29 . Larval and pupal stages last approximately eight and four days, respectively 27 . Females were identified at the pupal stage and selected for experiments. Gene identification and sequence analysis cDNA sequences of trr , rbbp5 , wds , absent , ash2 , dpy-30 , utx , ptip , pa1 , plk1 , and cycb were obtained from previously published transcriptomes (NCBI accession No. PRJNA338895 and PRJNA575933). Protein sequences were deduced using the ExPASy translation tool (https://web.expasy.org/translate/), and domains were predicted with SMART (http://smart.embl-heidelberg.de/). For phylogenetic analysis, the amino acid sequences were aligned and rooted trees were constructed using MEGA 6.06 with 1000 bootstrap replications. Microinjection of double-stranded RNA (dsRNA) and ovarian development analysis Primers for dsRNA synthesis were designed using an online tool (https://www.flyrnai.org/cgi-bin/RNAi_find_primers.pl), with a T7 promoter sequence (5’-gcgtaatacgactcactatagg-3’) added to the 5’ end of each primer. The resulting PCR-amplified products served as templates for in vitro transcription using a T7 Transcription kit (Thermo Fisher Scientific, USA). A 467-bp fragment of the green fluorescent protein ( gfp ; GenBank accession No. AB062168.1) was used to generate control dsRNA (dsgfp). All primer sequences are provided in Table S2. For RNAi, 2-day-old female pupae were injected with 200 nL of dsRNA solution (2 μg total) into the abdominal membrane using a Nanoliter 2000 microinjector (World Precision Instruments, USA). Control beetles received an equivalent dose of dsgfp. To analyze the effect of RNAi on ovarian development, ovaries were meticulously dissected from dsRNA-injected females at 4 days PE. Ovarian development was classified into five distinct stages according to yolk deposition patterns and overall ovarian dimensions 14 . Grade 0 indicates ovaries with no detectable vitellogenin deposition; grade I–III represents ovaries with progressively increasing vitellogenin accumulation but incomplete development; grade IV denotes fully developed ovaries containing mature oocytes. Ovaries exhibiting typical developmental status were carefully dissected, gently cleaned, and documented using a stereomicroscopic imaging system (Nikon, Japan). Ovarian sizes (n = 12), including vertical length and width, were measured using ScopePhoto 3.0 software (Scopetek Opto Electric, China). Additionally, to assess the impact of RNAi on ovarian development-related genes, ovaries and fat bodies were collected from injected females at 4 days PE, and total RNA was extracted for RT-qPCR analysis. Reverse transcription-quantitative PCR ( RT-qPCR ) RT-qPCR experiments were conducted following our previously published protocol with minor adjustments 65 . Briefly, total RNA was extracted using TRIzol reagent (Takara, Japan) following the manufacturer’s protocol. RNA pellets were resuspended in RNase-free water, and purity/concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). For cDNA synthesis, 1 μg of total RNA was reverse-transcribed using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Japan). RT-qPCR was performed using Hieff qPCR SYBR Green Master Mix (Yeasen, China) and gene-specific primers (Table S3) on a CFX Connect Real-Time PCR Detection System (Bio-Rad, USA). The thermal cycling protocol consisted of an initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. The expression levels of target genes were normalized to those of rpl19 and actin1 67 and calculated using the 2 −ΔΔC T method 66 . Three biological replicates were analyzed per treatment, with each replicate comprising pooled tissue from 8–12 individuals. 20E titer measurement The 20E titer was quantified using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), as previously described 68 . Briefly, ovarian tissues were dissected from RNAi-treated female adults at 4 days PE. Each treatment group consisted of three biological replicates, with each replicate comprising pooled samples from 10–15 individuals. For sample preparation, the ovaries were homogenized in ice-cold 1 × phosphate-buffered saline (PBS) and centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was collected for 20E quantification using a commercial ELISA kit (Meimian Industrial Co., Ltd., China) according to the manufacturer’s instructions. Following the assay, absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA). The 20E concentration for each sample was calculated based on a standard curve. Western blot Total proteins were extracted from the ovarian tissues of 8–12 female adults using ice-cold RIPA buffer (Beyotime Biotechnology, China) supplemented with 1 mM protease inhibitor cocktail (Beyotime Biotechnology, China). The lysates were centrifuged, and the supernatant was collected after incubation on ice for 30 min. The total protein concentration of each sample was quantified using a BCA Protein Assay Kit (Beyotime Biotechnology, China). Equal amounts of total protein were then subjected to western blotting. Prior to electrophoresis, samples were mixed with 5 × sample loading buffer (Beyotime Biotechnology, China) and heated at 95°C for 5 min. Anti-H3K4me3 primary antibody (ABclonal, China) was used at a 1: 4,000 dilution, followed by an HRP-conjugated goat anti-rabbit IgG secondary antibody (ABclonal, China) at a 1: 8,000 dilution. Anti-H3 (PTM Biolabs, China) served as the loading control. Protein bands were detected using an ECL chemiluminescence assay kit (Beyotime Biotechnology, China) and visualized with a ChemiDoc Touch Imaging System (Bio-Rad, USA). Band intensities were quantified using ImageJ 1.46r software (National Institutes of Health, USA) and normalized to histone H3 levels. Three biological replicates were analyzed for each treatment. 20E application Stock solutions of 20E (Selleck Chemicals, USA) were prepared at 50 μg/μL in dimethyl sulfoxide (DMSO) and diluted to a working concentration of 5 μg/μL using 1× PBS. Newly emerged LD female adults were topically treated with 200 nL of the 20E working solution using a Nanoliter 2000 injector (World Precision Instruments, USA). Control beetles received an equivalent volume of solvent (DMSO diluted in 1× PBS). Ovaries were dissected at 2 and 4 days post-treatment for RNA isolation and subsequent analysis of 20E-induced transcriptional changes. The 20E application protocol followed established methods 18 . Transcriptome analysis Ovarian samples were collected from 10–15 LD females injected with 20E or a solvent control for transcriptome analysis. Each replicate consisted of three biological replicates. Total RNA was extracted, and its concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). mRNA was purified using oligo(dT)-attached magnetic beads, fragmented, and used for cDNA synthesis with random hexamer primers. The purified cDNA was subjected to end-repair, phosphorylation, and adapter ligation. Libraries were size-selected for fragments of approximately 300 bp and PCR-amplified. After concentration and quality assessment, the libraries were sequenced on the Illumina HiSeq 2500 platform (BGI-Shenzhen, China). Raw paired-end reads were filtered and trimmed using Fastp, and high-quality reads were assembled de novo using Trinity. Transcript abundance was quantified using RSEM based on fragments per kilobase per million mapped fragments (FPKM). Unigenes were classified according to fold change in FPKM as follows: upregulated (log 2 fold change > 0 and P -value < 0.001), downregulated (log 2 fold change < 0 and P -value < 0.001), and not significant ( P -value ≥ 0.001). Venn diagram analysis and KEGG enrichment analysis of the target gene set were performed using the OmicShare online platform (https://www.omicshare.com/tools/). The raw transcriptome data have been deposited in the NCBI Sequence Read Archive under accession number PRJNA1311033. ChIP-qPCR ChIP-qPCR experiment was performed using our previously established protocol for C. bowringi 14 . Ovarian tissues were collected at 4 days PE following experimental treatment for ChIP analysis. Each treatment group comprised three biological replicates, with each replicate consisting of pooled samples from 10–15 individuals. Immunoprecipitation was carried out with a Sonication ChIP Kit (ABclonal, China) in accordance with the manufacturer’s instructions. Ovarian samples were fragmented, crosslinked in 1× PBS containing a protease inhibitor (ABclonal, China) and 16% formaldehyde (Thermo Fisher Scientific, USA) for 10 min, after which the reaction was quenched with 125 mM ice-cold glycine for 5 min. After washing, samples were homogenized in cell swelling buffer (provided in the kit), and nuclei were pelleted by centrifugation. The nuclei were resuspended in ultrasonic buffer (kit-supplied) and sonicated at 4°C for 10 min to shear DNA into 100–500 bp fragments. The supernatant was incubated with protein A/G magnetic beads at 4°C for 2 h. Chromatin was subsequently eluted and purified using a DNA gel extraction kit. Two primer pairs were designed to target the upstream region of each candidate gene, with amplicon specificity and amplification efficiency (90–110%) verified beforehand (Table S3). Purified DNA templates, gene-specific primers, and Hieff qPCR SYBR Green Master Mix (Yeasen, China) were used to prepare the qPCR reaction mixture. Data analysis was conducted via the Percent Input Method 69 , with IgG serving as the negative control. Statistical analysis Statistical analyses and graphical representations were performed using SPSS 11.5 (SPSS Inc., USA) and GraphPad Prism 9.4 (GraphPad Software Inc., USA), respectively. For gene expression profiling across tissues, one-way ANOVA followed by Tukey’s HSD test (α = 0.05) was used to determine statistical significance. Data normality was assessed using the Shapiro-Wilk test, and homogeneity of variance was verified using Levene’s test before conducting ANOVA. In other experiments, significant differences were evaluated using independent-samples t -tests (* P < 0.05, ** P < 0.01). Data are presented as mean ± standard deviation. Declarations Competing interests The authors declare no competing interests. Author contributions Z.T. and S.G. contributed equally to this work. Z.T., S.G., W.L., and X.P.W. conceived and designed the study. Z.T., S.G., Y.L.Z., and K.W. conducted the experiments and performed data analysis. Z.T., S.G., Y.L.Z., K.W., and W.L. supervised the project. X.P.W. provided technical guidance and funding. Z.T. and S.G. wrote the initial draft. W.L. and X.P.W. reviewed and edited the final version. All authors reviewed and approved the final manuscript. Acknowledgements We sincerely thank Prof. Fang-Sen Xue (Jiangxi Agricultural University, China) for his invaluable assistance with C. bowringi collection. 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11:20:06","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":181952,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8127731/v1/3f64888a037f9e38a611ea1b.html"},{"id":97437319,"identity":"9653282d-5eac-4624-8afc-a1888c701da8","added_by":"auto","created_at":"2025-12-04 11:20:05","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":128319,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of Trr/COMPASS components in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. bowringi\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e Schematic representation of identified Trr/COMPASS subunits in \u003cem\u003eC. bowringi\u003c/em\u003e, including Trr, RbBP5, Wds, Ash2, Dpy-30, UTX, PTIP, and PA1. The rooted phylogenetic tree was constructed using MEGA software with neighbor-joining method. The species are as follows: \u003cem\u003eColaphellus bowringi\u003c/em\u003e, \u003cem\u003eTribolium castaneum\u003c/em\u003e, \u003cem\u003eLeptinotarsa decemlineata\u003c/em\u003e, \u003cem\u003eAnoplophora glabripennis\u003c/em\u003e, \u003cem\u003eApis mellifera\u003c/em\u003e, \u003cem\u003eBombus Impatiens\u003c/em\u003e,\u003cem\u003e Camponotus floridanus\u003c/em\u003e, \u003cem\u003eBombyx mori\u003c/em\u003e, \u003cem\u003eManduca sexta\u003c/em\u003e, \u003cem\u003eDanaus Plexippus\u003c/em\u003e,\u003cem\u003e Drosophila melanogaster\u003c/em\u003e, \u003cem\u003eMusca domestica\u003c/em\u003e, \u003cem\u003eDrosophila simulans\u003c/em\u003e, and \u003cem\u003eLucilia sericata\u003c/em\u003e. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003ewas served as the outgroup.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8127731/v1/e4ab0ab3f8a311709af435d9.jpg"},{"id":97437289,"identity":"ef0a55db-e69e-4682-aa64-b0f887a09516","added_by":"auto","created_at":"2025-12-04 11:20:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":95329,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptional profiling of Trr/COMPASS components in female \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. bowringi\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e adults. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) The transcriptional profiles of Trr/COMPASS subunit genes (\u003cem\u003etrr\u003c/em\u003e, \u003cem\u003erbbp5\u003c/em\u003e, \u003cem\u003ewds\u003c/em\u003e, \u003cem\u003eash2\u003c/em\u003e, \u003cem\u003edpy30\u003c/em\u003e, \u003cem\u003eutx\u003c/em\u003e, \u003cem\u003eptip\u003c/em\u003e, and \u003cem\u003epa1\u003c/em\u003e) were examined in the head, ovary, fat body, and midgut of SD female adults using RT-qPCR. Transcript levels among tissues were visualized using a heatmap, and statistical differences were evaluated by one-way ANOVA followed by Tukey’s HSD test (α = 0.05), with different letters indicating significant differences. (\u003cstrong\u003eB\u003c/strong\u003e) Transcript levels of \u003cem\u003etrr\u003c/em\u003e, \u003cem\u003erbbp5\u003c/em\u003e, \u003cem\u003ewds\u003c/em\u003e, \u003cem\u003eash2\u003c/em\u003e, \u003cem\u003edpy30\u003c/em\u003e, \u003cem\u003eutx\u003c/em\u003e, \u003cem\u003eptip\u003c/em\u003e, and \u003cem\u003epa1\u003c/em\u003e in the ovaries of LD and SD females were quantified by RT-qPCR. Ovarian samples were collected from adults at 0 day (0dA, eclosion), 2 days (2dA), and 4 days PE (4dA). Differences in gene expression between photoperiod treatments at each time point were assessed using independent-sample \u003cem\u003et\u003c/em\u003e-tests (ns, not significant; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8127731/v1/4978d09d0dca1eb9f166af07.jpg"},{"id":97437330,"identity":"1995aece-c4e3-4197-93ad-27628992e28d","added_by":"auto","created_at":"2025-12-04 11:20:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":148912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of RNAi-mediated knockdown of Trr/COMPASS components on the reproductive development of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. bowringi\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e adults. \u003c/strong\u003eFollowing RNAi targeting of Trr/COMPASS subunit genes (\u003cem\u003etrr\u003c/em\u003e, \u003cem\u003erbbp5\u003c/em\u003e, \u003cem\u003ewds\u003c/em\u003e, \u003cem\u003eash2\u003c/em\u003e, \u003cem\u003edpy30\u003c/em\u003e, \u003cem\u003eutx\u003c/em\u003e, \u003cem\u003eptip\u003c/em\u003e, and \u003cem\u003epa1\u003c/em\u003e) in SD female pupae, we evaluated both (\u003cstrong\u003eA\u003c/strong\u003e) ovary developmental grades (n = 23 to 27) and \u003cstrong\u003e(B)\u003c/strong\u003e knockdown efficiency in adult ovaries using RT-qPCR. Subsequent analysis after \u003cem\u003etrr\u003c/em\u003e, \u003cem\u003erbbp5\u003c/em\u003e, and \u003cem\u003eutx\u003c/em\u003e knockdown included: (\u003cstrong\u003eC\u003c/strong\u003e) ovarian developmental status, (\u003cstrong\u003eD\u003c/strong\u003e) ovary size quantification (n = 12), and (\u003cstrong\u003eE–G\u003c/strong\u003e) transcriptional changes of ovarian development-related genes (\u003cem\u003evg1\u003c/em\u003e, \u003cem\u003evg2\u003c/em\u003e, \u003cem\u003evgr\u003c/em\u003e, \u003cem\u003efcp3c1\u003c/em\u003e, \u003cem\u003etkv\u003c/em\u003e, and \u003cem\u003emedea\u003c/em\u003e) using RT-qPCR. Specifically, \u003cem\u003evg1 \u003c/em\u003eand \u003cem\u003evg2\u003c/em\u003e transcript levels were measured in the fat body, while \u003cem\u003evgr\u003c/em\u003e, \u003cem\u003efcp3c1\u003c/em\u003e, \u003cem\u003etkv\u003c/em\u003e, and \u003cem\u003emedea\u003c/em\u003e were assessed in the ovaries. Significant differences were assessed using independent-sample \u003cem\u003et\u003c/em\u003e-tests (ns, not significant; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8127731/v1/d60130ca3dc04b29506ab748.jpg"},{"id":97437386,"identity":"8add448d-ce2d-44f7-b937-0b19c8162c65","added_by":"auto","created_at":"2025-12-04 11:20:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":46334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of Trr/COMPASS components suppresses 20E signaling in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. bowringi\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e adults. \u003c/strong\u003eMicroinjection of dsRNAs targeting \u003cem\u003etrr\u003c/em\u003e, \u003cem\u003erbbp5\u003c/em\u003e, and \u003cem\u003eutx\u003c/em\u003ewas performed in SD female pupae, and tissue samples were collected at 4 days PE for subsequent analysis. (\u003cstrong\u003eA\u003c/strong\u003e) Transcript levels of 20E biosynthetic enzyme genes (\u003cem\u003espook\u003c/em\u003e and \u003cem\u003eshade\u003c/em\u003e) were analyzed by RT-qPCR. (\u003cstrong\u003eB\u003c/strong\u003e) 20E titer in the ovaries was quantified using ELISA. (\u003cstrong\u003eC\u003c/strong\u003e) Transcript abundance of the 20E receptor gene (\u003cem\u003eecr\u003c/em\u003e) and 20E-responsive genes (\u003cem\u003ee74\u003c/em\u003e and \u003cem\u003ehr3\u003c/em\u003e) was measured by RT-qPCR. Significant differences were assessed using independent-sample \u003cem\u003et\u003c/em\u003e-tests (ns, not significant; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8127731/v1/36d1f428423ec7e1f8cb994b.jpg"},{"id":97437414,"identity":"7c2f8b0c-20fe-4a66-9042-e3e131b935e5","added_by":"auto","created_at":"2025-12-04 11:20:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":120559,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome analysis identifies \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eplk1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e as a key downstream effector of the Trr/COMPASS–20E signaling axis.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) A volcano plot displays the fold-change distribution of all unigenes obtained from RNA-Seq, with bar graphs indicating the percentages of upregulated, downregulated, and nonsignificant unigenes. (\u003cstrong\u003eB\u003c/strong\u003e) Upregulated genes were extracted from three datasets: the 20E-treated transcriptome (PRJNA1311033), and our published transcriptomes of SD female adults at the 2 days and 4 days PE (vs. LD females; PRJNA338895). Venn analysis identified overlapping \u003cem\u003eunigenes\u003c/em\u003e among these groups using KEGG IDs. (\u003cstrong\u003eC\u003c/strong\u003e) KEGG enrichment analysis of overlapping genes from (B) revealed 25 unigenes from three ovarian development-related pathways, visualized in a network diagram. (\u003cstrong\u003eD\u003c/strong\u003e) Annotation for the three unigenes overlapping in the three candidate pathways from (C) is displayed individually, with two unigenes annotated to \u003cem\u003eplk1\u003c/em\u003e and one to \u003cem\u003ecycb\u003c/em\u003e. (\u003cstrong\u003eE\u003c/strong\u003e) RT-qPCR analysis of transcriptional level differences of \u003cem\u003eplk1\u003c/em\u003eand \u003cem\u003ecycb\u003c/em\u003e in the head, ovary, fat body, and midgut of SD female adults. (\u003cstrong\u003eF\u003c/strong\u003e) Changes in the transcriptional levels of \u003cem\u003eplk1\u003c/em\u003eand \u003cem\u003ecycb\u003c/em\u003e in the ovaries after 20E application and (\u003cstrong\u003eG\u003c/strong\u003e) RNAi of 20E biosynthesis and receptor genes. (\u003cstrong\u003eH\u003c/strong\u003e) RT-qPCR analysis of the transcriptional profiles of \u003cem\u003eplk1\u003c/em\u003e and \u003cem\u003ecycb\u003c/em\u003e in the ovaries of SD and LD females. Following RNAi of \u003cem\u003eplk1\u003c/em\u003e and \u003cem\u003ecycb\u003c/em\u003e, we analyzed in SD females 4 days PE: (\u003cstrong\u003eI\u003c/strong\u003e) ovarian developmental status, (\u003cstrong\u003eJ\u003c/strong\u003e) ovary developmental grades (n = 21 to 27), (\u003cstrong\u003eK\u003c/strong\u003e) ovary size measurements (n = 12), and (\u003cstrong\u003eL\u003c/strong\u003e) RNAi efficiencies in the ovaries measured by RT-qPCR. (\u003cstrong\u003eM\u003c/strong\u003e) The effects of RNAi of \u003cem\u003eecr\u003c/em\u003e, \u003cem\u003eplk1\u003c/em\u003e, and \u003cem\u003ecycb\u003c/em\u003e on the transcript levels of \u003cem\u003evg1\u003c/em\u003e and \u003cem\u003evg2\u003c/em\u003e in the fat body and \u003cem\u003evgr\u003c/em\u003e and \u003cem\u003efcp3c1\u003c/em\u003e in the ovaries of SD females. (\u003cstrong\u003eN\u003c/strong\u003e) The effects of RNAi of \u003cem\u003etrr\u003c/em\u003e, \u003cem\u003erbbp5\u003c/em\u003e, and \u003cem\u003eutx\u003c/em\u003e on the \u003cem\u003eplk1\u003c/em\u003e transcript level in the ovaries of SD females. Significant differences were assessed using independent-sample \u003cem\u003et\u003c/em\u003e-tests (ns, not significant; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8127731/v1/22870f2bfe64e62cb23f0d71.jpg"},{"id":97666844,"identity":"5f44d9ab-6588-49e6-83d8-42de9bfee8af","added_by":"auto","created_at":"2025-12-08 09:22:13","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":93392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTrr/COMPASS-mediated H3K4me3 is essential for transcriptional activation of the 20E-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eplk1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e signaling axis. \u003c/strong\u003eWestern blot analysis of H3K4me3 protein levels (\u003cstrong\u003eA\u003c/strong\u003e) under different photoperiod conditions and (\u003cstrong\u003eB\u003c/strong\u003e) following RNAi knockdown of Trr/COMPASS subunits in the ovaries of 4-day-PE adult females. The H3K4me3 protein band intensities from different treatment groups were quantified and normalized to corresponding H3 levels. ChIP-qPCR analysis of H3K4me3 enrichment in the upstream regions of (\u003cstrong\u003eC\u003c/strong\u003e) 20E biosynthetic genes (\u003cem\u003espook\u003c/em\u003e and \u003cem\u003eshade\u003c/em\u003e), 20E receptor gene (\u003cem\u003eecr\u003c/em\u003e), and \u003cem\u003eplk1\u003c/em\u003e in response to photoperiod variation, and (\u003cstrong\u003eD\u003c/strong\u003e) following \u003cem\u003etrr \u003c/em\u003eRNAi. Two primer pairs were designed to target two distinct upstream regions of \u003cem\u003espook\u003c/em\u003e, \u003cem\u003eshade\u003c/em\u003e, \u003cem\u003eecr\u003c/em\u003e, and \u003cem\u003eplk1\u003c/em\u003e, respectively. IgG served as the negative control for the H3K4me3 antibody. Significant differences were assessed using independent-sample \u003cem\u003et\u003c/em\u003e-tests (ns, not significant; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8127731/v1/0c4bf5725ecfebf223ab27c0.jpg"},{"id":97437338,"identity":"7d19d37e-8316-4432-ac85-c196fa05e933","added_by":"auto","created_at":"2025-12-04 11:20:05","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":42386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel of Trr/COMPASS-mediated H3K4me3 regulation of photoperiod-dependent reproductive diapause through the 20E–EcR–\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eplk1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e signaling axis. \u003c/strong\u003e20E, 20-hydroxyecdysone; \u003cem\u003eecr\u003c/em\u003e, \u003cem\u003eecdysone receptor\u003c/em\u003e; \u003cem\u003eplk1\u003c/em\u003e, \u003cem\u003eserine/threonine-protein kinase polo\u003c/em\u003e. \u003cem\u003espook\u003c/em\u003eand \u003cem\u003eshade\u003c/em\u003e encode key enzymes involved in 20E biosynthesis in the ovaries. Red and blue arrows represent upregulation and downregulation, respectively. Gray elements in the right panel indicate signal attenuation. Question marks denote unresolved mechanistic components.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8127731/v1/0588cf595aeaee665fe72dc5.jpg"},{"id":97677503,"identity":"c35d22bb-5cf0-4012-8dc6-f670c6e697a1","added_by":"auto","created_at":"2025-12-08 09:53:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2202528,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8127731/v1/0386ea80-5855-4412-8087-4258c208ce3a.pdf"},{"id":97437353,"identity":"c7dfe956-09a3-44bf-955e-254ab77db76d","added_by":"auto","created_at":"2025-12-04 11:20:06","extension":"xls","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12669952,"visible":true,"origin":"","legend":"Dataset S1","description":"","filename":"DatasetS1.xls","url":"https://assets-eu.researchsquare.com/files/rs-8127731/v1/e70caccfbca8bc594b2d1cc9.xls"},{"id":97437341,"identity":"a8ec6b04-6b1d-41eb-ac52-67003ed8e437","added_by":"auto","created_at":"2025-12-04 11:20:05","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":265216,"visible":true,"origin":"","legend":"\u003cp\u003eDataset S2\u003c/p\u003e","description":"","filename":"DatasetS2.xls","url":"https://assets-eu.researchsquare.com/files/rs-8127731/v1/24d2e732386fc06215675fa2.xls"},{"id":97437307,"identity":"99fbe75c-5425-4a6d-a930-25fa61164402","added_by":"auto","created_at":"2025-12-04 11:20:05","extension":"xls","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":967168,"visible":true,"origin":"","legend":"Dataset S3","description":"","filename":"DatasetS3.xls","url":"https://assets-eu.researchsquare.com/files/rs-8127731/v1/6fbef2d06104149f3c0ea733.xls"},{"id":97668486,"identity":"d2a5d097-978b-4736-8747-ce9d4e42b8c7","added_by":"auto","created_at":"2025-12-08 09:25:37","extension":"xls","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":275456,"visible":true,"origin":"","legend":"Dataset S4","description":"","filename":"DatasetS4.xls","url":"https://assets-eu.researchsquare.com/files/rs-8127731/v1/4d8f5bd9caa6cbc2bb848e3f.xls"},{"id":97437349,"identity":"229a9b29-bfed-4fa5-b7b7-53730c34d07c","added_by":"auto","created_at":"2025-12-04 11:20:06","extension":"xls","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":45568,"visible":true,"origin":"","legend":"Dataset S5","description":"","filename":"DatasetS5.xls","url":"https://assets-eu.researchsquare.com/files/rs-8127731/v1/809d06a2d7f0bf9744adc5e1.xls"},{"id":97437366,"identity":"78df22cf-3ec0-4e1f-a0c2-602cfbaf8467","added_by":"auto","created_at":"2025-12-04 11:20:06","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1758721,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8127731/v1/0c2cfacb0a83472e91bd0e69.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Trr/COMPASS regulates photoperiodic reproductive diapause via 20-hydroxyecdysone signaling in Colaphellus bowringi","fulltext":[{"header":"Induction","content":"\u003cp\u003eOrganisms in temperate regions face recurring environmental fluctuations driven by seasonal change, which can severely constrain survival and reproduction\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. To persist, they have evolved developmental plasticity\u0026mdash;the ability to modify developmental trajectories and produce adaptive phenotypes in response to environmental variation\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. A prominent example is reproductive diapause, an endocrine-mediated strategy that allows insects to survive unfavorable seasons and reproduce when favorable conditions return\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. During diapause, adults suppress reproductive investment and redirect resources toward nutrient accumulation to endure prolonged periods of resource scarcity\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. This plasticity enables insect populations to synchronize their life cycles with predictable seasonal environment changes in resource availability\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Therefore, understanding the mechanisms underlying reproductive diapause is fundamental to elucidating how organisms adapt developmentally to fluctuating environments.\u003c/p\u003e\u003cp\u003eEpigenetic modifications that respond dynamically to environmental cues are central to regulating phenotypic plasticity\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Among these, specific histone marks such as H3K4me3, H3K9me3, and H3K27me3 control insect development by modulating transcription of key developmental genes\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. H3K4me3, in particular, establishes active chromatin states and ensures precise gene expression in eukaryotes\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Recent studies suggest that H3K4me3 plays a pivotal role in determining whether insects successfully enter environment-sensitive reproductive diapause\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The deposition of H3K4me3 is catalyzed by the highly conserved COMPASS (Complex Proteins Associated with Set1) family of complexes\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eDrosophila melanogaster\u003c/em\u003e, three COMPASS-like complexes have been identified, each with a unique catalytic subunit: Set1, trithorax (Trx), and trithorax-related (Trr)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Our recent study showed that knocking down \u003cem\u003eset1\u003c/em\u003e and \u003cem\u003etrr\u003c/em\u003e induced diapause-like ovarian arrest in reproductive \u003cem\u003eColaphellus bowringi\u003c/em\u003e females\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. While \u003cem\u003eset1\u003c/em\u003e mediates reproductive diapause through H3K4me3-dependent regulation of juvenile hormone (JH) production in the corpora allata (CA), the primary source of JH\u003csup\u003e14\u003c/sup\u003e, \u003cem\u003etrr\u003c/em\u003e knockdown did not alter H3K4me3 levels in the CA. This raises a key question: how does \u003cem\u003etrr\u003c/em\u003e influence reproductive diapause through mechanisms independent of H3K4me3 regulation in the CA?\u003c/p\u003e\u003cp\u003eA lack of JH is widely recognized as the primary trigger for the initiation of reproductive diapause in insects\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In recent years, 20-hydroxyecdysone (20E) has also emerged as a critical hormonal regulator of reproductive diapause, often acting through modulation of JH signaling\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. For instance, reduced 20E levels have been associated with diapause entry in \u003cem\u003eLocusta migratoria\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eD. melanogaster\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eC. bowringi\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, indicating that 20E production is environmentally regulated. Our previous study demonstrated that photoperiod-sensitive 20E acts through Ecdysone receptor (EcR) to activate JH biosynthesis\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, regulate the Dpp pathway\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and control DNA replication\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, highlighting its multi-faceted role in ovarian growth in the cabbage beetle. Furthermore, recent studies indicate that histone modifications such as H3K27me3, H3K27ac, and H4K20me regulate 20E signaling by directly controlling key genes in its biosynthesis and transduction pathways in \u003cem\u003eBombyx mori\u003c/em\u003e, \u003cem\u003eD. melanogaster\u003c/em\u003e, and \u003cem\u003eTribolium castaneum\u003c/em\u003e\u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. These findings suggest extensive crosstalk between histone modifications and 20E-mediated developmental processes. Building on this evidence, we hypothesize that photoperiod regulates 20E signaling by modifying the Trr/COMPASS histone complex, thereby influencing reproductive diapause entry.\u003c/p\u003e\u003cp\u003eTo test this, we used the cabbage beetle, \u003cem\u003eC. bowringi\u003c/em\u003e, which enters reproductive diapause under long-day (LD) conditions but remains reproductive under short-day (SD) photoperiods, showing clear ovarian differences\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. We identified all genes encoding Trr/COMPASS subunits and found their transcripts enriched in the ovaries of SD-induced reproductive females. We demonstrated that Trr/COMPASS regulates H3K4me3 levels in the ovaries, thereby modulating 20E signaling, which in turn interacts with JH signaling to mediate reproductive diapause. Furthermore, we identified serine/threonine-protein kinase polo (\u003cem\u003eplk1\u003c/em\u003e) as a downstream target of Trr/COMPASS-dependent H3K4me3 in the ovary; importantly, \u003cem\u003eplk1\u003c/em\u003e expression is 20E-dependent and contributes to diapause regulation. Together, these findings uncover an ovary-driven epigenetic pathway that integrates histone modification with endocrine signaling to coordinate reproductive diapause initiation.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eIdentification and transcriptional profiling of Trr/COMPASS subunit genes\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eTrr/COMPASS is a conserved H3K4 methyltransferase complex in eukaryotes composed of eight subunits: Trr, Retinoblastoma-binding protein 5 (RbBP5), Will die slowly (Wds), Absent, small, or homeotic discs 2 (Ash2), Dpy-30-like (Dpy-30), UTX histone demethylase (UTX), PAX-interacting protein 1 (PTIP), and PAXIP1-associated glutamate-rich protein 1 (PA1) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Using \u003cem\u003eD. melanogaster\u003c/em\u003e orthologues as queries, we identified all eight subunits in \u003cem\u003eC. bowringi\u003c/em\u003e through BLAST searches against existing transcriptome databases (PRJNA575933 and PRJNA338895)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Phylogenetic analysis revealed strong evolutionary conservation among insects, with \u003cem\u003eC. bowringi\u003c/em\u003e Trr/COMPASS subunits clustering most closely with those of other coleopterans (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The catalytic subunit Trr belongs to the KMT2C family, and domain prediction confirmed the presence of the conserved \u0026ldquo;SET\u0026rdquo; and \u0026ldquo;post-SET\u0026rdquo; domains (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), similar to \u003cem\u003eD. melanogaster\u003c/em\u003e Trr and \u003cem\u003eHomo sapiens\u003c/em\u003e KMT2C, suggesting preserved methyltransferase activity. The shared components of COMPASS-like complexes (RbBP5, Wds, Ash2, and Dpy-30) and Trr/COMPASS-specific subunits (UTX, PTIP, and PA1) also exhibited highly conserved domain architectures across species (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This evolutionary conservation validates the accurate identification of these genes and supports a conserved role for Trr/COMPASS in transcriptional regulation through H3K4me3 in \u003cem\u003eC. bowringi\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo preliminarily evaluate the role of Trr/COMPASS in reproductive development, we analyzed its transcript levels across tissues and developmental stages using RT-qPCR. Heatmap analysis showed significantly higher transcript levels of all eight subunits in the ovary of SD females than in the head, fat body, or midgut (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Temporal profiling of LD and SD ovaries revealed no transcriptional differences in newly emerged females; however, by 4 days post-eclosion (PE), all eight subunits were significantly upregulated under SD conditions, with six genes (\u003cem\u003etrr\u003c/em\u003e, \u003cem\u003erbbp5\u003c/em\u003e, \u003cem\u003ewds\u003c/em\u003e, \u003cem\u003eash2\u003c/em\u003e, \u003cem\u003eutx\u003c/em\u003e, and \u003cem\u003epa1\u003c/em\u003e) already elevated at 2 days PE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These photoperiod-dependent expression dynamics parallel ovarian developmental progression\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, with higher transcript levels correlating with active ovarian maturation. Together, the tissue- and stage-specific patterns of Trr/COMPASS transcription suggest that this complex functions as a key mediator of photoperiodic control over reproductive diapause in \u003cem\u003eC. bowringi\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eKnockdown of Trr/COMPASS components induces ovarian developmental arrest in reproductive females\u003c/h3\u003e\n\u003cp\u003eTo investigate the role of Trr/COMPASS in photoperiod-mediated reproductive plasticity, we performed RNAi targeting its subunit genes in SD-induced reproductive females. Knockdown of \u003cem\u003etrr\u003c/em\u003e, \u003cem\u003erbbp5\u003c/em\u003e, \u003cem\u003ewds\u003c/em\u003e, and \u003cem\u003eutx\u003c/em\u003e markedly inhibited ovarian development, with 55.5%, 72.7%, 100%, and 52.0% of females exhibiting ovarian arrest at grades 0-III, respectively\u0026mdash;far exceeding the 10% observed in the dsgfp control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). RT-qPCR analysis confirmed effective transcript reduction for all targeted genes (\u003cem\u003etrr\u003c/em\u003e, \u003cem\u003erbbp5\u003c/em\u003e, \u003cem\u003ewds\u003c/em\u003e, \u003cem\u003eash2\u003c/em\u003e, \u003cem\u003edyp30\u003c/em\u003e, \u003cem\u003eutx\u003c/em\u003e, \u003cem\u003eptip\u003c/em\u003e, and \u003cem\u003epa1\u003c/em\u003e), with decreases ranging from 32.4% to 81.2% relative to the dsgfp control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), supporting the reliability of RNAi-induced phenotypes. For subsequent analyses, we focused on three key components: \u003cem\u003etrr\u003c/em\u003e (core catalytic subunit), \u003cem\u003erbbp5\u003c/em\u003e (a shared subunit of COMPASS-like complexes), and \u003cem\u003eutx\u003c/em\u003e (Trr/COMPASS-specific subunit). Their knockdown significantly reduced vitellogenin deposition and ovarian size compared to \u003cem\u003egfp\u003c/em\u003e RNAi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). We also analyzed transcription of ovarian development-related genes via RT-qPCR, including \u003cem\u003evitellogenin\u003c/em\u003e (\u003cem\u003evg\u003c/em\u003e) and its receptor genes (\u003cem\u003evg1\u003c/em\u003e, \u003cem\u003evg2\u003c/em\u003e, and \u003cem\u003evgr\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, the follicle cell development-related gene \u003cem\u003efcp3c1\u003c/em\u003e, and key genes of the Dpp signaling pathway (\u003cem\u003etkv\u003c/em\u003e and \u003cem\u003emedea\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Following knockdown of \u003cem\u003etrr\u003c/em\u003e, \u003cem\u003erbbp5\u003c/em\u003e, and \u003cem\u003eutx\u003c/em\u003e, transcript levels of \u003cem\u003evg1\u003c/em\u003e and \u003cem\u003evg2\u003c/em\u003e were significantly decreased by over 60% in the fat body, while \u003cem\u003evgr\u003c/em\u003e, \u003cem\u003efcp3c1\u003c/em\u003e, \u003cem\u003etkv\u003c/em\u003e, and \u003cem\u003emedea\u003c/em\u003e were markedly reduced by 19.1\u0026ndash;99.6% in the ovaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, G). These results establish Trr/COMPASS as an essential regulator of photoperiod-dependent reproductive diapause in \u003cem\u003eC. bowringi\u003c/em\u003e, acting primarily through control of ovarian development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eTrr/COMPASS regulates reproductive diapause through modulation of 20E signaling\u003c/h3\u003e\n\u003cp\u003eInsect hormones, particularly 20E and JH, are central regulators of photoperiod-induced reproductive diapause\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eC. bowringi\u003c/em\u003e, Trr/COMPASS subunits are predominantly expressed in ovarian tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), consistent with tissue-specific 20E production\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. We therefore investigated whether Trr/COMPASS regulates reproductive development via 20E signaling. RT-qPCR analysis showed that RNAi targeting \u003cem\u003etrr\u003c/em\u003e, \u003cem\u003erbbp5\u003c/em\u003e, and \u003cem\u003eutx\u003c/em\u003e significantly downregulated the transcription of two key 20E biosynthetic genes (\u003cem\u003espook\u003c/em\u003e and \u003cem\u003eshade\u003c/em\u003e) in the ovaries of SD females compared to the \u003cem\u003edsgfp\u003c/em\u003e control. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). ELISA quantification further confirmed that 20E titers decreased by 30\u0026ndash;60% following Trr/COMPASS knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Moreover, Trr/COMPASS knockdown markedly reduced the transcript levels of the 20E receptor gene \u003cem\u003eecr\u003c/em\u003e and key ecdysone-response genes \u003cem\u003ee74\u003c/em\u003e (\u003cem\u003eecdysone-inducible protein 74\u003c/em\u003e) and \u003cem\u003ehr3\u003c/em\u003e (\u003cem\u003enuclear hormone receptor\u003c/em\u003e) in ovarian tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In addition, knockdown of Trr/COMPASS members significantly downregulated transcript levels of \u003cem\u003e3-hydroxy-3-methylglutaryl-CoA reductase 2\u003c/em\u003e (\u003cem\u003ehmgr2\u003c/em\u003e) and \u003cem\u003ejuvenile hormone acid methyltransferase 1\u003c/em\u003e (\u003cem\u003ejhamt1\u003c/em\u003e), as well as JHAMT1 protein abundance, in head tissues, indicating a reduction in JH signaling (Fig. S2). Together with previous findings, these results suggest that Trr/COMPASS likely regulates JH biosynthesis in the head via 20E signaling in \u003cem\u003eC. bowringi\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Collectively, our data demonstrate that Trr/COMPASS modulates reproductive diapause through 20E signaling by coordinating the transcription of key genes involved in both 20E biosynthesis and downstream signaling.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptome analysis identifies\u003c/b\u003e \u003cb\u003eplk1\u003c/b\u003e \u003cb\u003eas a core downstream target of Trr/COMPASS-20E signaling axis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAlthough 20E signaling is closely associated with photoperiod-mediated reproductive diapause and its indirect role in mediating JH biosynthesis is well established, its direct actions within the ovary remain poorly understood. We therefore hypothesized that Trr/COMPASS-mediated 20E signaling stimulates ovarian development directly by altering the transcription of key downstream genes. To identify such targets, we performed transcriptome analysis of ovarian tissues from day 2 LD female adults following 20E application (PRJNA1311033). \u003cem\u003eDe novo\u003c/em\u003e transcriptome assembly yielded 32,904 unigenes (Dataset S1), among which 6,417 (19.5%) were significantly upregulated and 9,617 (29.5%) were significantly downregulated after 20E treatment relative to the solvent control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Additionally, differentially expression analysis between SD and LD females using a previously published transcriptome (PRJNA338895) identified 587 and 2,269 upregulated unigenes at 2dA and 4dA, respectively (Datasets S2 and S3). To pinpoint genes responsive to both photoperiod and 20E signaling, we intersected upregulated genes from 20E-treated ovaries with those from SD-induced females at 2 and 4 days PE. Venn analysis based on unique KEGG Orthology (KO) identifiers revealed 143 shared KOs across the three gene sets (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), corresponding to 541 unigenes in the 20E-treated transcriptome (Dataset S4). KEGG pathway enrichment of these unigenes revealed 57 pathways with diverse molecular functions (Fig. S3). Among these, 25 unigenes were mapped to three ovarian development-related pathways\u0026mdash;\u0026ldquo;Cell cycle\u0026rdquo;, \u0026ldquo;Oocyte meiosis\u0026rdquo;, and \u0026ldquo;Progesterone-mediated oocyte maturation\u0026rdquo;\u0026mdash;including three shared transcripts: \u003cem\u003eCL3824.Contig1\u003c/em\u003e, \u003cem\u003eCL3824.Contig2\u003c/em\u003e, and \u003cem\u003eunigene12228\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). BLAST search and phylogenetic analysis confirmed that both \u003cem\u003eCL3824.Contig1\u003c/em\u003e and \u003cem\u003eCL3824.Contig2\u003c/em\u003e encode PLK1, whereas \u003cem\u003eunigene12228\u003c/em\u003e encodes a G2/mitotic-specific cyclin-B (CycB) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and S4). These results identify \u003cem\u003eplk1\u003c/em\u003e and \u003cem\u003ecycb\u003c/em\u003e as candidate downstream effectors of the Trr/COMPASS\u0026ndash;20E signaling axis that mediate reproductive diapause in \u003cem\u003eC. bowringi\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRT-qPCR analysis showed that \u003cem\u003eplk1\u003c/em\u003e and \u003cem\u003ecycb\u003c/em\u003e transcripts were highly specific to the ovaries of SD females, with levels significantly higher than those in other tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), suggesting their essential roles in ovarian development. Application of 20E markedly upregulated \u003cem\u003eplk1\u003c/em\u003e and \u003cem\u003ecycb\u003c/em\u003e transcription in the ovaries of LD females (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), whereas RNAi knockdown of 20E signaling genes (\u003cem\u003espook\u003c/em\u003e, \u003cem\u003eshade\u003c/em\u003e, and \u003cem\u003eecr\u003c/em\u003e) reduced their transcript levels by over 60% in the ovaries of SD females compared to the dsgfp control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). Under SD conditions, \u003cem\u003eplk1\u003c/em\u003e and \u003cem\u003ecycb\u003c/em\u003e transcript levels increased approximately 3-fold and 10-fold, respectively, relative to LD females at 2 and 4 days PE (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH), confirming regulation by both photoperiod and 20E signaling. These expression patterns indicate that the transcription of \u003cem\u003eplk1\u003c/em\u003e and \u003cem\u003ecycb\u003c/em\u003e is regulated by both photoperiod and 20E signaling. Knockdown of \u003cem\u003eplk1\u003c/em\u003e, but not \u003cem\u003ecycb\u003c/em\u003e, in 2-day SD female pupae significantly inhibited yolk deposition, ovarian developmental grade, and ovary size at 4 days PE (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, K). RT-qPCR analysis confirmed that RNAi significantly reduced \u003cem\u003eplk1\u003c/em\u003e and \u003cem\u003ecycb\u003c/em\u003e transcript levels by 72.7% and 89.1%, respectively, in the ovaries of SD females (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL), validating the reliability of the RNAi-induced ovarian phenotypes. In addition, knockdown of either \u003cem\u003eecr\u003c/em\u003e or \u003cem\u003eplk1\u003c/em\u003e significantly downregulated transcript levels of ovarian development-related genes \u003cem\u003evg1\u003c/em\u003e, \u003cem\u003evg2\u003c/em\u003e, and \u003cem\u003efcp3c1\u003c/em\u003e in SD females, whereas \u003cem\u003ecycb\u003c/em\u003e RNAi had no significant effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). Moreover, \u003cem\u003eplk1\u003c/em\u003e transcript levels decreased significantly by 63.0%, 27.3%, and 30.7% in the ovaries of SD females following knockdown of \u003cem\u003etrr\u003c/em\u003e, \u003cem\u003erbbp5\u003c/em\u003e, and \u003cem\u003eutx\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN). In addition, a heatmap generated from a previously established transcriptome (PRJNA338895) showed a concordant high-expression trend for transcripts related to Trr/COMPASS, 20E signaling, and \u003cem\u003eplk1\u003c/em\u003e under SD conditions compared to LD conditions (Fig. S5 and Dataset S5). Together, these results identify \u003cem\u003eplk1\u003c/em\u003e as a key ovary-specific effector acting downstream of the Trr/COMPASS\u0026ndash;20E signaling axis to regulate photoperiodic reproductive diapause in \u003cem\u003eC. bowringi\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTrr/COMPASS-mediated H3K4me3 governs activation of the 20E-\u003c/b\u003e\u003cb\u003eplk1\u003c/b\u003e \u003cb\u003esignaling cascade\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTrr/COMPASS and its animal orthologues catalyze H3K4me3 deposition, a histone mark associated with euchromatin and active transcription. Western blot analysis revealed a significant 1.4-fold upregulation of H3K4me3 abundance in the ovaries of SD females compared to LD individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). As expected, knockdown of Trr/COMPASS subunits\u0026mdash;\u003cem\u003etrr\u003c/em\u003e, \u003cem\u003erbbp5\u003c/em\u003e, and \u003cem\u003eutx\u003c/em\u003e\u0026mdash;reduced H3K4me3 levels by 47.7%, 52.3%, and 25.8%, respectively, in the ovaries of SD females (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These findings indicate that Trr/COMPASS-mediated H3K4me3 is photoperiod-sensitive and is positively associated with ovarian development. Given that H3K4me3 is typically enriched in promoter regions to activate transcription\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, we next examined whether Trr/COMPASS-mediated H3K4me3 is enriched in the upstream regions of key genes in the 20E-EcR-\u003cem\u003eplk1\u003c/em\u003e signaling axis. Using two pairs of primers targeting two non-overlapping upstream regions of \u003cem\u003espook\u003c/em\u003e, \u003cem\u003eshade\u003c/em\u003e, \u003cem\u003eecr\u003c/em\u003e, and \u003cem\u003eplk1\u003c/em\u003e (Fig. S6), we performed chromatin immunoprecipitation (ChIP)-qPCR with a commercial H3K4me3 antibody and a negative control antibody (IgG). ChIP-qPCR analysis showed significant enrichment of H3K4me3 at all targeted upstream regions of \u003cem\u003espook\u003c/em\u003e, \u003cem\u003eshade\u003c/em\u003e, \u003cem\u003eecr\u003c/em\u003e, and \u003cem\u003eplk1\u003c/em\u003e in the ovaries of SD females compared to LD individuals, whereas the IgG control showed no significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In contrast, \u003cem\u003etrr\u003c/em\u003e knockdown significantly reduced H3K4me3 enrichment in the upstream regions of these genes compared to the dsgfp control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Together, our findings demonstrate that photoperiod-responsive Trr/COMPASS governs reproductive development by mediating H3K4me3 deposition in the ovary to control transcription of the 20E\u0026ndash;EcR\u0026ndash;\u003cem\u003eplk1\u003c/em\u003e signaling axis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eReproductive diapause is an evolutionarily conserved strategy that allows organisms to optimize resource use and maintain population continuity across fluctuating environments\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. This adaptive trait reflects their ability to reprogram physiological and molecular processes in response to external cues\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In insects, reproductive diapause is typically induced by photoperiod and temperature, with endocrine signals directly regulating reproductive arrest or activation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, the transcriptional mechanisms that link photoperiodic cues to endocrine responses remains unclear. Using the cabbage beetle \u003cem\u003eC. bowringi\u003c/em\u003e as a model, our study shows that Trr/COMPASS\u0026mdash;a histone modification regulator\u0026mdash;directly controls the 20E signaling pathway by modulating H3K4me3 levels, thereby determining female reproductive diapause. Key evidence supports this conclusion: i) Trr/COMPASS components are highly expressed in the ovaries of reproductively active females and exhibit photoperiod-sensitive transcription (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). ii) Knockdown of Trr/COMPASS leads to ovarian arrest and disrupts both 20E production and downstream signaling (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). iii) Trr/COMPASS-mediated H3K4me3 is enriched in upstream regulatory regions of genes within the 20E\u0026ndash;EcR\u0026ndash;\u003cem\u003eplk1\u003c/em\u003e signaling axis, activating their transcription and promoting ovarian development (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These findings establish Trr/COMPASS as a key photoperiod-responsive epigenetic regulator that links environmental signals to endocrine activity, thereby coordinating reproductive diapause initiation.\u003c/p\u003e\u003cp\u003eH3K4me3 functions as a key regulator of gene transcription, translating environmental signals into the control of insect development\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eD. melanogaster\u003c/em\u003e, Set1/COMPASS-mediated H3K4me3 contributes to tissue development and morphogenesis\u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, underscoring its broad developmental role. In \u003cem\u003eC. bowringi\u003c/em\u003e, we further clarify its function: the establishment of H3K4me3 is photoperiod-sensitive, driven by transcriptional activation of Trr/COMPASS in response to environmental cues. While Set1/COMPASS-mediated H3K4me3 in the CA promotes JH production\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, we now show that Trr/COMPASS-mediated H3K4me3 in the ovaries enhances 20E biosynthesis. Integrating these results, we propose that during ovarian maturation, 20E trans-tissue activates JH signaling\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and directly influences ovarian development via \u003cem\u003eplk1\u003c/em\u003e regulation. This process, initiated by photoperiod, operates through the Trr/COMPASS\u0026ndash;H3K4me3\u0026ndash;20E\u0026ndash;\u003cem\u003eplk1\u003c/em\u003e axis in the ovary, allowing precise environmental regulation of reproductive diapause. Since many organisms rely on reproductive arrest to survive seasonal extremes, targeting this pathway could offer a viable strategy for pest control and extending the shelf life of natural enemies.\u003c/p\u003e\n\u003ch3\u003e\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cb\u003eTrr/COMPASS-mediated H3K4me3 as a central epigenetic regulator of photoperiodic reproductive diapause\u003c/b\u003e\u003c/div\u003e\u003cp\u003eEukaryotes share conserved H3K4 methyltransferases, homologous to the yeast Set1-centered COMPASS complex\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In higher insects, the core Set1-like methyltransferases have diversified into Trx and Trr, orthologous to mammalian MLL1/MLL2 and MLL3/MLL4, respectively\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Previous functional studies in \u003cem\u003eC. bowringi\u003c/em\u003e showed that only \u003cem\u003eset1\u003c/em\u003e and \u003cem\u003etrr\u003c/em\u003e are essential for ovarian development, while \u003cem\u003etrx\u003c/em\u003e is dispensable\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, indicating distinct roles for COMPASS variants. Here, we systematically identified full complement of Trr/COMPASS subunits and found that their animo acid sequences are highly conserved with orthologs in \u003cem\u003eDrosophila\u003c/em\u003e and humans. These genes are preferentially expressed in the ovaries and are transcriptionally upregulated under SD conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), suggesting that their transcription is photoperiod-dependent. H3K4 methyltransferases are typically inactive without incorporation into the multi-subunit COMPASS complex\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. RNAi knockdown of Trr/COMPASS subunits resulted in severe ovarian arrest phenotypes\u0026mdash;both physiologically and molecularly\u0026mdash;that phenocopied \u003cem\u003etrr\u003c/em\u003e depletion. Although \u003cem\u003eutx\u003c/em\u003e depletion was previously shown to promote glycogen and lipid accumulation in \u003cem\u003eCulex pipiens\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, our results reveal an additional role for \u003cem\u003eutx\u003c/em\u003e in ovarian development in \u003cem\u003eC. bowringi\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These findings from two distinct species collectively underscore the central role of UTX-mediated histone modification in reproductive diapause regulation. Importantly, the direct correlation between Trr/COMPASS activity and ovarian H3K4me3 levels establishes this histone mark as a key epigenetic switch that controls the transition between ovarian growth and arrest under changing photoperiods. This represents a previously unrecognized mechanism of reproductive diapause, reflecting species-specific adaptations of Trr/COMPASS to environmental cues.\u003c/p\u003e\u003cp\u003eComparable functions of other histone modifications further emphasize the evolutionary conservation of epigenetic control in oogenesis. For example, H3K9me3 is essential for oogenesis and germline stem cell maintenance in \u003cem\u003eD. melanogaster\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, while in \u003cem\u003eAedes aegypti\u003c/em\u003e, H3K27me3 promotes ovarian maturation\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In contrast, H3K27me2 levels are elevated in reproductive females compared to those in diapause\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, indicating that its abundance can be modulated by environmental cues. These histone marks \u0026mdash;H3K4me3, H3K9me3, and H3K27me3\u0026mdash;act as conserved epigenetic regulators of oogenesis across metazoans\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In summary, our findings establish an H3K4me3-centered photoperiodic regulatory mechanism that links environmental signals to reproductive fate, illustrating how epigenetic flexibility supports adaptive developmental plasticity in fluctuating environments.\u003c/p\u003e\u003cp\u003e\u003cb\u003e20E signaling integrates photoperiod-dependent Trr/COMPASS-H3K4me3 regulation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs a classical endocrine initiator, 20E acts as a central hub that translates environmental cues into physiological and molecular responses\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. 20E production is photoperiod-dependent and significantly influences developmental outcomes such as diapause and reproduction\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Upon 20E stimulation, its receptor EcR is activated and transduces signals through early-response transcription factors, including E74, E75, and HR3\u003csup\u003e47,48\u003c/sup\u003e. Previous studies have shown that Trr/COMPASS promotes \u003cem\u003eecr\u003c/em\u003e transcription by enriching H3K4me3 at its promoter, underscoring its critical role in activating 20E signaling\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. This signaling cascade is essential for female reproductive processes, including ovarian cell differentiation, oogenesis, and vitellogenesis\u003csup\u003e\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. A key unresolved question, however, is how photoperiod controls 20E production to modulate reproductive diapause. Photoperiod signals are detected by the compound eyes, processed in the brain, and transmitted to endocrine organs\u0026mdash;yet the terminal regulatory step governing hormone synthesis remains unclear\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Our study addresses this gap by demonstrating that the photoperiod-responsive Trr/COMPASS directly activates transcription of 20E synthesis genes via H3K4me3 modification, thereby promoting 20E production. This epigenetic mechanism establishes a fundamental regulatory framework for photoperiod-dependent 20E signaling. Comparable regulatory paradigms have been identified in other insect species, such as PRC2-mediated H3K27me3 in \u003cem\u003eB. mori\u003c/em\u003e and METTL3/METTL14-mediated m\u003csup\u003e6\u003c/sup\u003eA RNA methylation in \u003cem\u003eBactrocera dorsalis\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Although these epigenetic regulators do not sense photoperiod directly, they function as key intermediaries linking environmental cues to developmental and reproductive responses\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The circadian clock has long been recognized as the central system for photoperiod signal processing in the brain\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. In \u003cem\u003eRhodnius prolixus\u003c/em\u003e, rhythmic oscillations in 20E production during oogenesis have been observed, strongly suggesting circadian regulation\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Although direct evidence in adult reproduction is still limited, core clock genes such as \u003cem\u003etimeless\u003c/em\u003e and \u003cem\u003eperiod\u003c/em\u003e have indeed been correlated with photoperiod sensing and 20E production\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Therefore, future studies should elucidate how photoperiod signals are integrated by the ovarian Trr/COMPASS\u0026ndash;H3K4me3\u0026ndash;20E axis to reveal the precise mechanisms of insect reproductive diapause.\u003c/p\u003e\u003cp\u003e\u003cb\u003eplk1\u003c/b\u003e \u003cb\u003eis an ovary-specific downstream effector of the Trr/COMPASS-H3K4me3-20E signaling axis\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePLK1, a serine/threonine kinase, is a central regulator of eukaryotic cell division\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. It functions in both mitotic organ development and meiotic gametogenesis during reproductive system formation\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Our study demonstrates that ovarian maturation in \u003cem\u003eC. bowringi\u003c/em\u003e strictly depends on \u003cem\u003eplk1\u003c/em\u003e, and its ovary-specific transcription is precisely controlled by the Trr/COMPASS\u0026ndash;H3K4me3\u0026ndash;20E signaling axis. This pathway begins with photoperiodic input, which is transduced via epigenetically regulated endocrine signaling to activate key target genes, enabling precise control of tissue development. These findings establish \u003cem\u003eplk1\u003c/em\u003e as a critical downstream effector of 20E signaling during reproductive diapause. As a fundamental cell cycle regulator, \u003cem\u003eplk1\u003c/em\u003e is highly conserved in \u003cem\u003eC. bowringi\u003c/em\u003e, showing strong homology to orthologs in other insects (Fig. S4). This suggests its role in ovarian growth is likely associated with cell cycle regulation, although direct experimental verification in non-model insects remains technically challenging. Notably, in \u003cem\u003eDrosophila\u003c/em\u003e, PLK1 regulates germ cell division through mechanisms that mirror those in mice\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. This functional conservation suggests that the epigenetic regulation of \u003cem\u003eplk1\u003c/em\u003e transcription uncovered in \u003cem\u003eC. bowringi\u003c/em\u003e may represent a widespread mechanism across species. Therefore, elucidating the Trr/COMPASS\u0026ndash;H3K4me3\u0026ndash;20E\u0026ndash;\u003cem\u003eplk1\u003c/em\u003e signaling axis advances our understanding of how developmental plasticity integrates environmental adaptation.\u003c/p\u003e\u003cp\u003eEnvironmental regulation of developmental plasticity remains a central question in biology. Based on our findings, we propose a model in which Trr/COMPASS regulates photoperiod-dependent reproductive diapause (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Under SD conditions, elevated Trr/COMPASS activity promotes H3K4me3 enrichment at the upstream regulatory regions of 20E biosynthetic genes (\u003cem\u003espook\u003c/em\u003e and \u003cem\u003eshade\u003c/em\u003e), the 20E receptor gene (\u003cem\u003eecr\u003c/em\u003e), and the downstream effector \u003cem\u003eplk1\u003c/em\u003e. This enrichment activates transcription of these targets, thereby enhancing 20E production and subsequently initiating the EcR\u0026ndash;\u003cem\u003eplk1\u003c/em\u003e signaling cascade. The 20E\u0026ndash;EcR\u0026ndash;\u003cem\u003eplk1\u003c/em\u003e signaling axis further coordinates the transcription of genes involved in vitellogenesis and ovarian growth, promoting full ovarian maturation. In contrast, under LD conditions, suppression of Trr/COMPASS-mediated H3K4me3 attenuates the 20E\u0026ndash;EcR\u0026ndash;\u003cem\u003eplk1\u003c/em\u003e pathway, resulting in reduced hormone signaling and diapause-like ovarian arrest. This study reveals a previously unknown epigenetically regulated endocrine cascade that links photoperiodic cues to reproductive diapause. Future studies should focus on identifying the upstream factors that transmit environmental cues to the Trr/COMPASS\u0026ndash;H3K4me3 signal, thereby deepening our understanding of how insects adapt to their environments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eExperimental insects\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproximately 500 adult \u003cem\u003eC. bowringi\u003c/em\u003e beetles were collected from Xiushui County, Jiangxi Province, China (29\u0026deg;1\u0026rsquo;N, 114\u0026deg;4\u0026rsquo;E) in November 2017. The colony was maintained under laboratory conditions at 25 \u0026plusmn; 1 \u0026deg;C and 70% relative humidity, with fresh radish leaves (\u003cem\u003eRaphanus sativus\u003c/em\u003e var. \u003cem\u003elongipinnatus\u003c/em\u003e) provided as food\u003csup\u003e27\u003c/sup\u003e. To induce reproductive development, larvae were reared under SD conditions (12 h light: 12 h dark). Reproductive females display a 4-day pre-oviposition period, marked by yolk deposition and ovarian development. In contrast, exposure to LD conditions (16 h light: 8 h dark) during larval and pupal stages triggers reproductive diapause in adults, marked by ovarian arrest during the same PE period\u003csup\u003e28,29\u003c/sup\u003e. Larval and pupal stages last approximately eight and four days, respectively\u003csup\u003e27\u003c/sup\u003e. Females were identified at the pupal stage and selected for experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene identification and sequence analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ecDNA sequences of \u003cem\u003etrr\u003c/em\u003e,\u0026nbsp;\u003cem\u003erbbp5\u003c/em\u003e,\u0026nbsp;\u003cem\u003ewds\u003c/em\u003e,\u0026nbsp;\u003cem\u003eabsent\u003c/em\u003e,\u003cem\u003e\u0026nbsp;ash2\u003c/em\u003e,\u0026nbsp;\u003cem\u003edpy-30\u003c/em\u003e,\u0026nbsp;\u003cem\u003eutx\u003c/em\u003e,\u0026nbsp;\u003cem\u003eptip\u003c/em\u003e,\u0026nbsp;\u003cem\u003epa1\u003c/em\u003e,\u0026nbsp;\u003cem\u003eplk1\u003c/em\u003e,\u0026nbsp;and\u0026nbsp;\u003cem\u003ecycb\u003c/em\u003e were obtained from previously published transcriptomes (NCBI accession No. PRJNA338895 and PRJNA575933). Protein sequences were deduced using the ExPASy translation tool (https://web.expasy.org/translate/), and domains were predicted with SMART (http://smart.embl-heidelberg.de/). For phylogenetic analysis, the amino acid sequences were aligned and rooted trees were constructed using MEGA 6.06 with 1000 bootstrap replications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroinjection of double-stranded RNA (dsRNA) and ovarian development analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimers for dsRNA synthesis were designed using an online tool (https://www.flyrnai.org/cgi-bin/RNAi_find_primers.pl), with a T7 promoter sequence (5\u0026rsquo;-gcgtaatacgactcactatagg-3\u0026rsquo;) added to the 5\u0026rsquo; end of each primer. The resulting PCR-amplified products served as templates for in vitro transcription using a T7 Transcription kit (Thermo Fisher Scientific, USA). A 467-bp fragment of the green fluorescent protein (\u003cem\u003egfp\u003c/em\u003e; GenBank accession No. AB062168.1) was used to generate control dsRNA (dsgfp). All primer sequences are provided in Table S2. For RNAi, 2-day-old female pupae were injected with 200 nL of dsRNA solution (2 \u0026mu;g total) into the abdominal membrane using a Nanoliter 2000 microinjector (World Precision Instruments, USA). Control beetles received an equivalent dose of dsgfp.\u003c/p\u003e\n\u003cp\u003eTo analyze the effect of RNAi on ovarian development, ovaries were meticulously dissected from dsRNA-injected females at 4 days PE. Ovarian development was classified into five distinct stages according to yolk deposition patterns and overall ovarian dimensions\u003csup\u003e14\u003c/sup\u003e. Grade 0 indicates ovaries with no detectable vitellogenin deposition; grade I\u0026ndash;III represents ovaries with progressively increasing vitellogenin accumulation but incomplete development; grade IV denotes fully developed ovaries containing mature oocytes. Ovaries exhibiting typical developmental status were carefully dissected, gently cleaned, and documented using a stereomicroscopic imaging system (Nikon, Japan). Ovarian sizes (n = 12), including vertical length and width, were measured using ScopePhoto 3.0 software (Scopetek Opto Electric, China). Additionally, to assess the impact of RNAi on ovarian development-related genes, ovaries and fat bodies were collected from injected females at 4 days PE, and total RNA was extracted for RT-qPCR analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReverse transcription-quantitative PCR (\u003c/strong\u003e\u003cstrong\u003eRT-qPCR\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT-qPCR experiments were conducted following our previously published protocol with minor adjustments\u003csup\u003e65\u003c/sup\u003e. Briefly, total RNA was extracted using TRIzol reagent (Takara, Japan) following the manufacturer\u0026rsquo;s protocol. RNA pellets were resuspended in RNase-free water, and purity/concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). For cDNA synthesis, 1 \u0026mu;g of total RNA was reverse-transcribed using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Japan). RT-qPCR was performed using Hieff qPCR SYBR Green Master Mix (Yeasen, China) and gene-specific primers (Table S3) on a CFX Connect Real-Time PCR Detection System (Bio-Rad, USA). The thermal cycling protocol consisted of an initial denaturation at 95\u0026deg;C for 30 s, followed by 40 cycles of 95\u0026deg;C for 10 s and 60\u0026deg;C for 30 s. The expression levels of target genes were normalized to those of \u003cem\u003erpl19\u003c/em\u003e and \u003cem\u003eactin1\u003c/em\u003e\u003csup\u003e67\u003c/sup\u003e and\u0026nbsp;calculated using the 2\u003cem\u003e\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;C\u003c/sup\u003e\u003c/em\u003e\u003cem\u003eT\u003c/em\u003e method\u003csup\u003e66\u003c/sup\u003e.\u0026nbsp;Three biological replicates\u0026nbsp;were analyzed per treatment, with each replicate comprising pooled tissue from 8\u0026ndash;12 individuals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e20E titer measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 20E titer was quantified using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), as previously described\u003csup\u003e68\u003c/sup\u003e. Briefly, ovarian tissues were dissected from RNAi-treated female adults at 4 days PE. Each treatment group consisted of three biological replicates, with each replicate comprising pooled samples from 10\u0026ndash;15 individuals. For sample preparation, the ovaries were homogenized in ice-cold 1 \u0026times; phosphate-buffered saline (PBS) and centrifuged at 10,000 rpm for 10 min at 4 \u0026deg;C. The supernatant was collected for 20E quantification using a commercial ELISA kit (Meimian Industrial Co., Ltd., China) according to the manufacturer\u0026rsquo;s instructions. Following the assay, absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA). The 20E concentration for each sample was calculated based on a standard curve.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal proteins were extracted from the ovarian tissues of 8\u0026ndash;12 female adults using ice-cold RIPA buffer (Beyotime Biotechnology, China) supplemented with 1 mM protease inhibitor cocktail (Beyotime Biotechnology, China). The lysates were centrifuged, and the supernatant was collected after incubation on ice for 30 min. The total protein concentration of each sample was quantified using a BCA Protein Assay Kit (Beyotime Biotechnology, China). Equal amounts of total protein were then subjected to western blotting. Prior to electrophoresis, samples were mixed with 5 \u0026times; sample loading buffer (Beyotime Biotechnology, China) and heated at 95\u0026deg;C for 5 min. Anti-H3K4me3 primary antibody (ABclonal, China) was used at a 1: 4,000 dilution, followed by an HRP-conjugated goat anti-rabbit IgG secondary antibody (ABclonal, China) at a 1: 8,000 dilution. Anti-H3 (PTM Biolabs, China) served as the loading control. Protein bands were detected using an ECL chemiluminescence assay kit (Beyotime Biotechnology, China) and visualized with a ChemiDoc Touch Imaging System (Bio-Rad, USA). Band intensities were quantified using ImageJ 1.46r software (National Institutes of Health, USA) and normalized to histone H3 levels. Three biological replicates were analyzed for each treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e20E application\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStock solutions of 20E (Selleck Chemicals, USA) were prepared at 50 \u0026mu;g/\u0026mu;L in dimethyl sulfoxide (DMSO) and diluted to a working concentration of 5 \u0026mu;g/\u0026mu;L using 1\u0026times; PBS. Newly emerged LD female adults were topically treated with 200 nL of the 20E working solution using a Nanoliter 2000 injector (World Precision Instruments, USA). Control beetles received an equivalent volume of solvent (DMSO diluted in 1\u0026times; PBS). Ovaries were dissected at 2 and 4 days post-treatment for RNA isolation and subsequent analysis of 20E-induced transcriptional changes. The 20E application protocol followed established methods\u003csup\u003e18\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptome analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvarian samples were collected from 10\u0026ndash;15 LD females injected with 20E or a solvent control for transcriptome analysis. Each replicate consisted of three biological replicates. Total RNA was extracted, and its concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). mRNA was purified using oligo(dT)-attached magnetic beads, fragmented, and used for cDNA synthesis with random hexamer primers. The purified cDNA was subjected to end-repair, phosphorylation, and adapter ligation. Libraries were size-selected for fragments of approximately 300 bp and PCR-amplified. After concentration and quality assessment, the libraries were sequenced on the Illumina HiSeq 2500 platform (BGI-Shenzhen, China). Raw paired-end reads were filtered and trimmed using Fastp, and high-quality reads were assembled de novo using Trinity. Transcript abundance was quantified using RSEM based on fragments per kilobase per million mapped fragments (FPKM). Unigenes were classified according to fold change in FPKM as follows: upregulated (log\u003csub\u003e2\u003c/sub\u003e fold change \u0026gt; 0 and \u003cem\u003eP\u003c/em\u003e-value \u0026lt; 0.001), downregulated (log\u003csub\u003e2\u003c/sub\u003e fold change \u0026lt; 0 and \u003cem\u003eP\u003c/em\u003e-value \u0026lt; 0.001), and not significant (\u003cem\u003eP\u003c/em\u003e-value \u0026ge; 0.001). Venn diagram analysis and KEGG enrichment analysis of the target gene set were performed using the OmicShare online platform (https://www.omicshare.com/tools/). The raw transcriptome data have been deposited in the NCBI Sequence Read Archive under accession number PRJNA1311033.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChIP-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChIP-qPCR experiment was performed using our previously established protocol for \u003cem\u003eC. bowringi\u003c/em\u003e\u003csup\u003e14\u003c/sup\u003e. Ovarian tissues were collected at 4 days PE following experimental treatment for ChIP analysis. Each treatment group comprised three biological replicates, with each replicate consisting of pooled samples from 10\u0026ndash;15 individuals. Immunoprecipitation was carried out with a Sonication ChIP Kit (ABclonal, China) in accordance with the manufacturer\u0026rsquo;s instructions. Ovarian samples were fragmented, crosslinked in 1\u0026times; PBS containing a protease inhibitor (ABclonal, China) and 16% formaldehyde (Thermo Fisher Scientific, USA) for 10 min, after which the reaction was quenched with 125 mM ice-cold glycine for 5 min. After washing, samples were homogenized in cell swelling buffer (provided in the kit), and nuclei were pelleted by centrifugation. The nuclei were resuspended in ultrasonic buffer (kit-supplied) and sonicated at 4\u0026deg;C for 10 min to shear DNA into 100\u0026ndash;500 bp fragments. The supernatant was incubated with protein A/G magnetic beads at 4\u0026deg;C for 2 h. Chromatin was subsequently eluted and purified using a DNA gel extraction kit. Two primer pairs were designed to target the upstream region of each candidate gene, with amplicon specificity and amplification efficiency (90\u0026ndash;110%) verified beforehand (Table S3). Purified DNA templates, gene-specific primers, and Hieff qPCR SYBR Green Master Mix (Yeasen, China) were used to prepare the qPCR reaction mixture. Data analysis was conducted via the Percent Input Method\u003csup\u003e69\u003c/sup\u003e, with IgG serving as the negative control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses and graphical representations were performed using SPSS 11.5 (SPSS Inc., USA) and GraphPad Prism 9.4 (GraphPad Software Inc., USA), respectively. For gene expression profiling across tissues, one-way ANOVA followed by Tukey\u0026rsquo;s HSD test (\u0026alpha; = 0.05) was used to determine statistical significance. Data normality was assessed using the Shapiro-Wilk test, and homogeneity of variance was verified using Levene\u0026rsquo;s test before conducting ANOVA. In other experiments, significant differences were evaluated using independent-samples \u003cem\u003et\u003c/em\u003e-tests (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). Data are presented as mean \u0026plusmn; standard deviation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eZ.T. and S.G. contributed equally to this work. Z.T., S.G., W.L., and X.P.W. conceived and designed the study. Z.T., S.G., Y.L.Z., and K.W. conducted the experiments and performed data analysis. Z.T., S.G., Y.L.Z., K.W., and W.L. supervised the project. X.P.W. provided technical guidance and funding. Z.T. and S.G. wrote the initial draft. W.L. and X.P.W. reviewed and edited the final version. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eWe sincerely thank Prof. Fang-Sen Xue (Jiangxi Agricultural University, China) for his invaluable assistance with \u003cem\u003eC. bowringi\u003c/em\u003e collection. We also thank Dr. Binjie Xu (Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine, China) for providing instruments and technical support. This study was supported by grants from the National Natural Science Foundation of China (Grant Nos. 32472543 and 31972268).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available within the article file and its supplementary information. The raw RNA-Seq data generated in this study have been deposited in the Sequence Read Archive database of the National Center for Biotechnology Information under accession number PRJNA1311033, and are publicly accessible at https://www.ncbi.nlm.nih.gov/sra.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTauber, M.J., Tauber, C.A., Masaki, S.: Seasonal Adaptations of Insects. 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Genetics. \u003cb\u003e163\u003c/b\u003e, 1221\u0026ndash;1225 (2003)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Developmental plasticity, Reproductive diapause, COMPASS-like complex, Steroid hormone, serine/threonine-protein kinase polo, H3K4me3","lastPublishedDoi":"10.21203/rs.3.rs-8127731/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8127731/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDevelopmental plasticity allows animals to survive harsh conditions. In insects, reproductive diapause is a classic form of plasticity, characterized by ovarian arrest. This process is typically triggered by seasonal cues such as photoperiod and governed by endocrine pathways. However, how photoperiods are converted into endocrine signals that determine reproductive fate remains unclear. The cabbage beetle (\u003cem\u003eColaphellus bowringi\u003c/em\u003e) displays a clear photoperiodic response: long-day (LD) conditions induce diapause with ovarian arrest, whereas short-day (SD) conditions promote ovarian development and reproduction. Here, we identified eight conserved subunits of the Trr/COMPASS complex and found their transcriptional levels significantly higher in the ovaries of SD females compared to LD females. Knockdown of Trr/COMPASS components caused ovarian arrest in SD females, accompanied by reduced production and signaling of 20-hydroxyecdysone (20E). We further identified \u003cem\u003eserine/threonine-protein kinase polo\u003c/em\u003e (\u003cem\u003eplk1\u003c/em\u003e) as a key downstream effector of 20E signaling that promotes ovarian development. Trr/COMPASS-mediated H3K4me3 enrichment was found in regulatory regions of \u003cem\u003eplk1\u003c/em\u003eand several 20E pathway genes, facilitating their transcriptional activation. These findings reveal a photoperiod-sensitive signaling axis, Trr/COMPASS–20E–\u003cem\u003eplk1\u003c/em\u003e, linking environmental cues to endocrine regulation and reproductive diapause, highlighting the essential role of histone modification in developmental adaptation to seasonal changes.\u003c/p\u003e","manuscriptTitle":"Trr/COMPASS regulates photoperiodic reproductive diapause via 20-hydroxyecdysone signaling in Colaphellus bowringi","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 11:19:59","doi":"10.21203/rs.3.rs-8127731/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b5c92262-f6c2-441e-a6e1-6630cace7ce1","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58983148,"name":"Biological sciences/Zoology/Entomology"},{"id":58983149,"name":"Biological sciences/Developmental biology"}],"tags":[],"updatedAt":"2026-01-12T23:35:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-04 11:19:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8127731","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8127731","identity":"rs-8127731","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}