N(alpha)-acetyltransferase 40-mediated histone acetylation plays an important role in ecdysone regulation of metamorphosis in the red flour beetle, Tribolium castaneum.

Histone acetylation, a crucial epigenetic modification, is governed by histone acetyltransferases (HATs), that regulate many biological processes. Functions of HATs in insects are not well understood. We identified 27 HATs and determined their functions using RNA interference (RNAi) in the model insect, Tribolium castaneum. Among HATs studied, N-alpha-acetyltransferase 40 (NAA40) knockdown caused a severe phenotype of arrested larval development. The steroid hormone, ecdysone induced NAA40 expression through its receptor, EcR (ecdysone receptor). Interestingly, ecdysone-induced NAA40 regulates EcR expression. NAA40 acetylates histone H4 protein, associated with the promoters of ecdysone response genes: EcR, E74, E75, and HR3, and causes an increase in their expression. In the absence of ecdysone and NAA40, histone H4 methylation by arginine methyltransferase 1 (ART1) suppressed the above genes. However, elevated ecdysone levels at the end of the larval period induced NAA40, promoting histone H4 acetylation and increasing the expression of ecdysone response genes. NAA40 is also required for EcR, and steroid-receptor co-activator (SRC) mediated induction of E74, E75, and HR3. These findings highlight the key role of ecdysone-induced NAA40-mediated histone acetylation in the regulation of metamorphosis.

Hypoxia delays steroid-induced developmental maturation in Drosophila by suppressing EGF signaling.

Animals often grow and develop in unpredictable environments where factors like food availability, temperature, and oxygen levels can fluctuate dramatically. To ensure proper sexual maturation into adulthood, juvenile animals need to adapt their growth and developmental rates to these fluctuating environmental conditions. Failure to do so can result in impaired maturation and incorrect body size. Here we describe a mechanism by which Drosophila larvae adapt their development in low oxygen (hypoxia). During normal development, larvae grow and increase in mass until they reach critical weight (CW), after which point a neuroendocrine circuit triggers the production of the steroid hormone ecdysone from the prothoracic gland (PG), which promotes maturation to the pupal stage. However, when raised in hypoxia (5% oxygen), larvae slow their growth and delay their maturation to the pupal stage. We find that, although hypoxia delays the attainment of CW, the maturation delay occurs mainly because of hypoxia acting late in development to suppress ecdysone production. This suppression operates through a distinct mechanism from nutrient deprivation, occurs independently of HIF-1 alpha and does not involve dilp8 or modulation of Ptth, the main neuropeptide that initiates ecdysone production in the PG. Instead, we find that hypoxia lowers the expression of the EGF ligand, spitz, and that the delay in maturation occurs due to reduced EGFR/ERK signaling in the PG. Our study sheds light on how animals can adjust their development rate in response to changing oxygen levels in their environment. Given that hypoxia is a feature of both normal physiology and many diseases, our findings have important implications for understanding how low oxygen levels may impact animal development in both normal and pathological situations.

Supreme glutathione-dependent ketosteroid isomerase in the yellow-fever transmitting mosquito Aedes aegypti.

The steroid hormone ecdysone is essential for the reproduction and survival of insects. The hormone is synthesized from dietary sterols such as cholesterol, yielding ecdysone in a series of consecutive enzymatic reactions. In the insect orders Lepidoptera and Diptera a glutathione transferase called Noppera-bo (Nobo) plays an essential, but biochemically uncharacterized, role in ecdysteroid biosynthesis. The Nobo enzyme is consequently a possible target in harmful dipterans, such as disease-carrying mosquitoes. Flavonoid compounds inhibit Nobo and have larvicidal effects in the yellow-fever transmitting mosquito Aedes aegypti, but the enzyme is functionally incompletely characterized. We here report that within a set of glutathione transferase substrates the double-bond isomerase activity with 5-androsten-3,17-dione stands out with an extraordinary specific activity of 4000 µmol min-1 mg-1. We suggest that the authentic function of Nobo is catalysis of a chemically analogous ketosteroid isomerization in ecdysone biosynthesis.

RNAi-mediated silencing of the neverland gene inhibits molting in the migratory locust, Locusta migratoria.

7-dehydrocholesterol (7-DHC) is a key intermediate product used for biosynthesis of molting hormone. This is achieved through a series of hydroxylation reactions catalyzed by the Halloween family of cytochrome P450s. Neverland is an enzyme catalyzes the first reaction of the ecdysteroidogenic pathway, which converts dietary cholesterol into 7-DHC. However, research on the physiological function of neverland in orthopteran insects is lacking. In this study, neverland from Locusta migratoria (LmNvd) was cloned and analyzed. LmNvd was mainly expressed in the prothoracic gland and highly expressed on days 6 and 7 of fifth instar nymphs. RNAi-mediated silencing of LmNvd resulted in serious molting delays and abnormal phenotypes, which could be rescued by 7-DHC and 20-hydroxyecdysone supplementation. Hematoxylin and eosin staining results showed that RNAi-mediated silencing of LmNvd disturbed the molting process by both promoting the synthesis of new cuticle and suppressing the degradation of the old cuticle. Quantitative real-time PCR results suggested that the mRNA expression of E75 early gene and chitinase 5 gene decreased and that of chitin synthase 1 gene was markedly upregulated after knockdown of LmNvd. Our results suggest that LmNvd participates in the biosynthesis process of molting hormone, which is involved in regulating chitin synthesis and degradation in molting cycles.

RNA Interference-Mediated Suppression of Ecdysone Signaling Inhibits Choriogenesis in Two Coleoptera Species.

During choriogenesis in insects, chorion (eggshell) is formed by surrounding follicular epithelial cells in ovarioles. However, the regulatory endocrine factor(s) activating choriogenesis and the effect of chemical components on eggshell deserve further exploration. In two representative coleopterans, a coccinellid Henosepilachna vigintioctopunctata and a chrysomelid Leptinotarsa decemlineata, genes encoding the 20-hydroxyecdysone (20E) receptor heterodimer, ecdysone receptor (EcR) and ultraspiracle (USP), and two chitin biosynthesis enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP) and chitin synthase (ChS1), were highly expressed in ovaries of the young females. RNA interference (RNAi)-aided knockdown of either HvEcR or Hvusp in H. vigintioctopunctata inhibited oviposition, suppressed the expression of HvChS1, and lessened the positive signal of Calcofluor staining on the chorions, which suggests the reduction of a chitin-like substance (CLS) deposited on eggshells. Similarly, RNAi of LdEcR or Ldusp in L. decemlineata constrained oviposition, decreased the expression of LdUAP1 and LdChS1, and reduced CLS contents in the resultant ovaries. Knockdown of LdUAP1 or LdChS1 caused similar defective phenotypes, i.e., reduced oviposition and CLS contents in the L. decemlineata ovaries. These results, for the first time, indicate that 20E signaling activates choriogenesis in two coleopteran species. Moreover, our findings suggest the deposition of a CLS on the chorions.

Reconstruction of Metabolic-Protein Interaction Integrated Network of Eriocheir sinensis and Analysis of Ecdysone Synthesis.

Integrated networks have become a new interest in genome-scale network research due to their ability to comprehensively reflect and analyze the molecular processes in cells. Currently, none of the integrated networks have been reported for higher organisms. Eriocheir sinensis is a typical aquatic animal that grows through ecdysis. Ecdysone has been identified to be a crucial regulator of ecdysis, but the influence factors and regulatory mechanisms of ecdysone synthesis in E. sinensis are still unclear. In this work, the genome-scale metabolic network and protein-protein interaction network of E. sinensis were integrated to reconstruct a metabolic-protein interaction integrated network (MPIN). The MPIN was used to analyze the influence factors of ecdysone synthesis through flux variation analysis. In total, 236 integrated reactions (IRs) were found to influence the ecdysone synthesis of which 16 IRs had a significant impact. These IRs constitute three ecdysone synthesis routes. It is found that there might be alternative pathways to obtain cholesterol for ecdysone synthesis in E. sinensis instead of absorbing it directly from the feeds. The MPIN reconstructed in this work is the first integrated network for higher organisms. The analysis based on the MPIN supplies important information for the mechanism analysis of ecdysone synthesis in E. sinensis.

Transcriptome-based analysis reveals a crucial role of the 20E/HR3 pathway in the diapause of Pieris rapae.

Pieris rapae is among the most damaging pests globally, and diapause makes it highly resistant to environmental stresses, playing a crucial role in the survival and reproduction of P. rapae while exacerbating the challenges of pest management and control. However, the mechanisms of its diapause regulation remain poorly understood. This research used RNA sequencing to profile the transcriptomes of three diapause phases (induction and preparation, initiation, maintenance) and synchronous nondiapause phases in P. rapae. During each comparison phase, 759, 1045, and 4721 genes were found to be differentially expressed. Among these, seven clock genes and seven pivotal hormone synthesis and metabolism genes were identified as having differential expression patterns in diapause type and nondiapause type. The weighted gene co-expression network analysis (WGCNA) revealed the red and blue modules as pivotal for diapause initiation, while the grey module was identified to be crucial to diapause maintenance. Meanwhile, the hub genes HDAC11, METLL16D, Dyw-like, GST, and so on, were identified within these hub modules. Moreover, an ecdysone downstream nuclear receptor gene, HR3, was found to be a shared transcription factor across all three phases. RNA interference of HR3 resulted in delayed pupal development, indicating its involvement in regulating pupal dipause in P. rapae. The further hormone assays revealed that the 20-hydroxyecdysone (20E) titer in diapause type pupae was lower than that in nondiapause type pupae, which exhibited a similar trend to HR3. When 20E was injected into diapause pupae, the HR3 expression levels were improved, and the pupal diapause were broken. These results indicate that the 20E/HR3 pathway is a critical pathway for the diapause regulation of P. rapae, and perturbing this pathway by ecdysone treatment or RNAi would result in the disruption of diapause. These findings provide initial insights into the molecular mechanisms of P. rapae diapause and suggest the potential use of ecdysone analogs and HR3 RNAi pesticides, which specifically target to diapause, as a means of pest control in P. rapae.

Ecdysone-controlled nuclear receptor ERR regulates metabolic homeostasis in the disease vector mosquito Aedes aegypti.

Hematophagous mosquitoes require vertebrate blood for their reproductive cycles, making them effective vectors for transmitting dangerous human diseases. Thus, high-intensity metabolism is needed to support reproductive events of female mosquitoes. However, the regulatory mechanism linking metabolism and reproduction in mosquitoes remains largely unclear. In this study, we found that the expression of estrogen-related receptor (ERR), a nuclear receptor, is activated by the direct binding of 20-hydroxyecdysone (20E) and ecdysone receptor (EcR) to the ecdysone response element (EcRE) in the ERR promoter region during the gonadotropic cycle of Aedes aegypti (named AaERR). RNA interference (RNAi) of AaERR in female mosquitoes led to delayed development of ovaries. mRNA abundance of genes encoding key enzymes involved in carbohydrate metabolism (CM)-glucose-6-phosphate isomerase (GPI) and pyruvate kinase (PYK)-was significantly decreased in AaERR knockdown mosquitoes, while the levels of metabolites, such as glycogen, glucose, and trehalose, were elevated. The expression of fatty acid synthase (FAS) was notably downregulated, and lipid accumulation was reduced in response to AaERR depletion. Dual luciferase reporter assays and electrophoretic mobility shift assays (EMSA) determined that AaERR directly activated the expression of metabolic genes, such as GPI, PYK, and FAS, by binding to the corresponding AaERR-responsive motif in the promoter region of these genes. Our results have revealed an important role of AaERR in the regulation of metabolism during mosquito reproduction and offer a novel target for mosquito control.

Pri peptides temporally coordinate transcriptional programs during epidermal differentiation.

To achieve a highly differentiated state, cells undergo multiple transcriptional processes whose coordination and timing are not well understood. In Drosophila embryonic epidermal cells, polished-rice (Pri) smORF peptides act as temporal mediators of ecdysone to activate a transcriptional program leading to cell shape remodeling. Here, we show that the ecdysone/Pri axis concomitantly represses the transcription of a large subset of cuticle genes to ensure proper differentiation of the insect exoskeleton. The repression relies on the transcription factor Ken and persists for several days throughout early larval stages, during which a soft cuticle allows larval crawling. The onset of these cuticle genes normally awaits the end of larval stages when the rigid pupal case assembles, and their premature expression triggers abnormal sclerotization of the larval cuticle. These results uncovered a temporal switch to set up distinct structures of cuticles adapted to the animal lifestyle and which might be involved in the evolutionary history of insects.

Steroid hormone signaling synchronizes cell migration machinery, adhesion and polarity to direct collective movement.

Migratory cells - either individually or in cohesive groups - are critical for spatiotemporally regulated processes such as embryonic development and wound healing. Their dysregulation is the underlying cause of formidable health problems such as congenital abnormalities and metastatic cancers. Border cell behavior during Drosophila oogenesis provides an effective model to study temporally regulated, collective cell migration in vivo. Developmental timing in flies is primarily controlled by the steroid hormone ecdysone, which acts through a well-conserved, nuclear hormone receptor complex. Ecdysone signaling determines the timing of border cell migration, but the molecular mechanisms governing this remain obscure. We found that border cell clusters expressing a dominant-negative form of ecdysone receptor extended ineffective protrusions. Additionally, these clusters had aberrant spatial distributions of E-cadherin (E-cad), apical domain markers and activated myosin that did not overlap. Remediating their expression or activity individually in clusters mutant for ecdysone signaling did not restore proper migration. We propose that ecdysone signaling synchronizes the functional distribution of E-cadherin, atypical protein kinase C (aPKC), Discs large (Dlg1) and activated myosin post-transcriptionally to coordinate adhesion, polarity and contractility and temporally control collective cell migration.

Fecundity is optimized by levels of nutrient signal-dependent expression of Dve and EcR in Drosophila male accessory gland.

Steroid hormones play various physiological roles including metabolism and reproduction. Steroid hormones in insects are ecdysteroids, and the major form in Drosophila melanogaster is ecdysone. In Drosophila males, the accessory gland is responsive to nutrient-dependent regulation of fertility/fecundity. The accessory gland is composed of two types of binucleated epithelial cells: a main cell and a secondary cell (SC). The transcription factors Defective proventriculus (Dve), Abdominal-B, and Ecdysone receptors (EcRs) are strongly expressed in adult SCs. We show that this EcR expression is regulated by parallel pathways of nutrient signaling and the Dve activity. Induction of Dve expression is also dependent on nutrient signaling, and it becomes nutrient signal-independent during a restricted period of development. Forced dve expression during the restricted period significantly increased the number of SCs. Here, we provide evidence that the level of nutrient signal-dependent Dve expression during the restricted period determines the number of SCs, and that ecdysone signaling is also crucial to optimize male fecundity through nutrient signal-dependent survival and maturation of SCs.

Eclosion muscles secrete ecdysteroids to initiate asymmetric intestinal stem cell division in Drosophila.

During organ development, tissue stem cells first expand via symmetric divisions and then switch to asymmetric divisions to minimize the time to obtain a mature tissue. In the Drosophila midgut, intestinal stem cells switch their divisions from symmetric to asymmetric at midpupal development to produce enteroendocrine cells. However, the signals that initiate this switch are unknown. Here, we identify the signal as ecdysteroids. In the presence of ecdysone, EcR and Usp promote the expression of E93 to suppress Br expression, resulting in asymmetric divisions. Surprisingly, the primary source of pupal ecdysone is not from the prothoracic gland but from dorsal internal oblique muscles (DIOMs), a group of transient skeletal muscles that are required for eclosion. Genetic analysis shows that DIOMs secrete ecdysteroids during mTOR-mediated muscle remodeling. Our findings identify sequential endocrine and mechanical roles for skeletal muscle, which ensure the timely asymmetric divisions of intestinal stem cells.

New insights into the regulation mechanism of Pacific white shrimp (Litopenaeus vannamei) hepatopancreas under 4-nonylphenol exposure using transcriptome analysis.

4-Nonylphenol (4-NP) is one of the common endocrine-disrupting chemicals (EDCs) in estuaries and coastal zones, which can exert detrimental effects on the physiological function of aquatic organisms. However, the molecular response triggered by 4-NP remains largely unknown in Pacific white shrimp (Litopenaeus vannamei). In this study, transcriptomic analysis was performed to investigate the underlying mechanisms of 4-NP toxicity in the hepatopancreas of L. vannamei. Nine RNA-Seq libraries were generated from L. vannamei at 0 h, 24 h, and 48 h following exposure to 4-NP. Compared with 0 h vs 24 h, 962 up- and 463 down-regulated differentially expressed genes (DEGs) were identified, indicating that many genes in L. vannamei were induced to resist adverse circumstances by 4-NP exposure. In contrast, 902 up- and 1027 down-regulated DEGs were revealed in the comparison of 0 h vs 48 h, demonstrating that prolonged exposure to the stress from 4-NP resulted in more inhibited genes. To validate the accuracy of the transcriptome data, eight DEGs were selected for quantitative real-time polymerase chain reaction (qRT-PCR), which were consistent with the RNA-Seq results. Through KEGG pathway enrichment analysis, three specific pathways related to hormonal effects and endocrine function of L. vannamei were enriched significantly, including tyrosine metabolism, insect hormone biosynthesis, and melanogenesis. After 4-NP stress, genes involved in tyrosine metabolism (Tyr) and melanogenesis pathway (AC, CBP, Wnt, Frizzled, Tcf, and Ras) were induced to promote melanin pigment to help shrimp resist adverse environments. In the insect hormone biosynthesis, ALDH, CYP15A1, CYP15A1/C1, and JHE genes were activated to synthesize juvenile hormone (JH), while Spook, Phm, Sad, and CYP18A1 were induced to generate molting hormone. There is an enhanced interaction between the molting hormone and JH, with JH playing a dominant role and maintaining its "classic status quo action". Our study demonstrated that 4-NP exposure led to impairments of biological functions in L. vannamei hepatopancreas. The genes and pathways identified provide novel insights into the molecular mechanisms underlying 4-NP toxicity effects in prawns and enrich the information on the toxicity mechanism of crustaceans in response to EDCs exposure.

Ecdysone-induced microRNA miR-276a-3p controls developmental growth by targeting the insulin-like receptor in Drosophila.

Animal growth is controlled by a variety of external and internal factors during development. The steroid hormone ecdysone plays a critical role in insect development by regulating the expression of various genes. In this study, we found that fat body-specific expression of miR-276a, an ecdysone-responsive microRNA (miRNA), led to a decrease in the total mass of the larval fat body, resulting in significant growth reduction in Drosophila. Changes in miR-276a expression also affected the proliferation of Drosophila S2 cells. Furthermore, we found that the insulin-like receptor (InR) is a biologically relevant target gene regulated by miR-276a-3p. In addition, we found that miR-276a-3p is upregulated by the canonical ecdysone signalling pathway involving the ecdysone receptor and broad complex. A reduction in cell proliferation caused by ecdysone was compromised by blocking miR-276a-3p activity. Thus, our results suggest that miR-276a-3p is involved in ecdysone-mediated growth reduction by controlling InR expression in the insulin signalling pathway.

Opposing effects of ecdysone signaling regulate neuroblast proliferation to ensure coordination of brain and organism development.

Growth regulation must be robust to ensure correct final size, but also adaptative to adjust to less favorable environmental conditions. Developmental coordination between whole-organism and the brain is particularly important, as the brain is a critical organ with little adaptability. Brain growth mainly depends on neural stem cell (NSC) proliferation to generate differentiated neural cells, it is however unclear how organism developmental progression is coordinated with NSCs. Here we demonstrate that the steroid hormone ecdysone plays a multi-step, stage specific role in regulating Drosophila NSCs, the neuroblasts. We used animals that are unable to synthesize ecdysone, to show that the developmental milestone called "critical weight peak", the peak that informs the body has reached minimum viable weight to survive metamorphosis, acts a checkpoint necessary to set neuroblast cell cycle pace during larval neurogenesis. The peaks of ecdysone that occur post-critical weight are no longer required to maintain neuroblast division rate. We additionally show that in a second stage, at the onset of pupariation, ecdysone is instead required to trigger neuroblast's proliferation exit and consequently the end of neurogenesis. We demonstrate that, without this signal from ecdysone, neuroblasts lose their ability to exit proliferation. Interestingly, although these neuroblasts proliferate for a longer period, the number of differentiated neurons is smaller compared to wild-type brains, suggesting a role for ecdysone in neuron maintenance. Our study provides insights into how neural stem cells coordinate their division rate with the pace of body growth, identifying a novel coordination mechanism between animal development and NSC proliferation.

Cold-stored mulberry leaves affect antioxidant system and silk proteins of silkworm (Bombyx mori) larva.

BACKGROUND: Cold storage has been widely used to maintain the quality of vegetables, but whether eating cold-stored vegetables affects health remains unknown. RESULTS: This study used silkworms as an animal model to evaluate the effects of nutrient changes in cold-stored mulberry leaves (CSML) on health. Compared with fresh mulberry leaves (FML), CSML contained lower vitamin C, soluble sugars and proteins, and higher H2 O2 , suggesting decreased antioxidant ability and nutrition. The CSML did not obviously affect larval survival rate, body weight or dry matter rate, cocoon shape, weight and size, or final rates of cluster and cocooning relative to the FML, suggesting CSML did not alter overall growth and development. However, the CSML increased the initial rates of cluster and cocooning and upregulated BmRpd3, suggesting CSML shortened larval lifespan and enhanced senescence. CSML upregulated BmNOX4, downregulated BmCAT, BmSOD and BmGSH-Px and increased H2 O2 in silkworms, suggesting CSML caused oxidative stress. CSML upregulated ecdysone biosynthesis and inactivation genes and elevated ecdysone concentration in silkworms, suggesting that CSML affected hormone homeostasis. CSML upregulated apoptosis-related genes, downregulated sericin and silk fibroin genes and decreased sericin content rate in silkworms, suggesting oxidative stress and protein deficiency. CONCLUSION: Cold storage reduced nutrition and antioxidant capability of mulberry leaves. CSML did not influence growth and development of silkworm larva, but affected health by causing oxidative stress and reducing protein synthesis. The findings show that the ingredient changes in CSML had negative effects on health of silkworms. © 2023 Society of Chemical Industry.

Bruceine D Acts as a Potential Insecticide by Antagonizing 20E-EcR/USP Signal Transduction.

Bruceine D (BD) is an effective insecticidal compound found in the Chinese herb Brucea javanica (L.) Merr. BD inhibits the growth and metamorphosis of Plutella xylostella and Drosophila melanogaster; however, its target protein and the molecular mechanism of insecticidal activity remain unclear. In this study, proteins with high affinity for BD were screened using surface plasmon resonance and high-performance liquid chromatography coupled with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, revealing the ecdysone receptor (EcR) is the main target of BD. In vivo results showed that BD inhibited insect growth and metamorphosis through inhibition of the expression of 20E response genes. In vitro dual luciferase and enhanced green fluorescent protein (EGFP) fluorescence experiments indicated that BD suppressed the transcriptional activation activity of EcR by blocking the ecdysone response element (EcRE)-triggered transcriptional cascade, suggesting that BD inhibits the formation of the 20E-EcR-USP-EcRE complex. Moreover, molecular docking demonstrated that BD bound well to EcR. Elucidating the insecticidal mechanism of BD will be helpful in the development of green pesticides to control pests.

Rbf/E2F1 control growth and endoreplication via steroid-independent Ecdysone Receptor signalling in Drosophila prostate-like secondary cells.

In prostate cancer, loss of the tumour suppressor gene, Retinoblastoma (Rb), and consequent activation of transcription factor E2F1 typically occurs at a late-stage of tumour progression. It appears to regulate a switch to an androgen-independent form of cancer, castration-resistant prostate cancer (CRPC), which frequently still requires androgen receptor (AR) signalling. We have previously shown that upon mating, binucleate secondary cells (SCs) of the Drosophila melanogaster male accessory gland (AG), which share some similarities with prostate epithelial cells, switch their growth regulation from a steroid-dependent to a steroid-independent form of Ecdysone Receptor (EcR) control. This physiological change induces genome endoreplication and allows SCs to rapidly replenish their secretory compartments, even when ecdysone levels are low because the male has not previously been exposed to females. Here, we test whether the Drosophila Rb homologue, Rbf, and E2F1 regulate this switch. Surprisingly, we find that excess Rbf activity reversibly suppresses binucleation in adult SCs. We also demonstrate that Rbf, E2F1 and the cell cycle regulators, Cyclin D (CycD) and Cyclin E (CycE), are key regulators of mating-dependent SC endoreplication, as well as SC growth in both virgin and mated males. Importantly, we show that the CycD/Rbf/E2F1 axis requires the EcR, but not ecdysone, to trigger CycE-dependent endoreplication and endoreplication-associated growth in SCs, mirroring changes seen in CRPC. Furthermore, Bone Morphogenetic Protein (BMP) signalling, mediated by the BMP ligand Decapentaplegic (Dpp), intersects with CycD/Rbf/E2F1 signalling to drive endoreplication in these fly cells. Overall, our work reveals a signalling switch, which permits rapid growth of SCs and increased secretion after mating, independently of previous exposure to females. The changes observed share mechanistic parallels with the pathological switch to hormone-independent AR signalling seen in CRPC, suggesting that the latter may reflect the dysregulation of a currently unidentified physiological process.

20-hydroxyecdysone Upregulates Ecdysone Receptor (ECR) Gene to Promote Pupation in the Honeybee, Apis mellifera Ligustica.

A heterodimeric complex of two nuclear receptors, the ecdysone receptor (ECR) and ultraspiracle (USP), transduces 20-hydroxyecdysone (20E) signaling to modulate insect growth and development. Here, we aimed to determine the relationship between ECR and 20E during larval metamorphosis and also the specific roles of ECR during larval-adult transition in Apis mellifera. We found that ECR gene expression peaked in the 7-day-old larvae, then decreased gradually from the pupae stage. 20E slowly reduced food consumption and then induced starvation, resulting in small-sized adults. In addition, 20E induced ECR expression to regulate larval development time. Double-stranded RNAs (dsRNAs) were prepared using common dsECR as templates. After dsECR injection, larval transition to the pupal stage was delayed, and 80% of the larvae showed prolonged pupation beyond 18 h. Moreover, the mRNA levels of shd, sro, nvd, and spo, and ecdysteroid titers were significantly decreased in ECR RNAi larvae compared with those in GFP RNAi control larvae. ECR RNAi disrupted 20E signaling during larval metamorphosis. We performed rescuing experiments by injecting 20E in ECR RNAi larvae and found that the mRNA levels of ECR, USP, E75, E93, and Br-c were not restored. 20E induced apoptosis in the fat body during larval pupation, while RNAi knockdown of ECR genes reduced apoptosis. We concluded that 20E induced ECR to modulate 20E signaling to promote honeybee pupation. These results assist our understanding of the complicated molecular mechanisms of insect metamorphosis.

Exorista sorbillans (Diptera: Tachinidae) parasitism shortens host larvae growth duration by regulating ecdysone and juvenile hormone titers in Bombyx mori (Lepidoptera: Bombycidae).

The tachinid fly, Exorista sorbillans, is a notorious ovolarviparous endoparasitoid of the silkworm, Bombyx mori, causing severe damage to silkworm cocoon industry. Silkworm larvae show typically precocious wandering behavior after being parasitized by E. sorbillans; however, the underlying molecular mechanism remains unexplored. Herein, we investigated the changes in the levels of 20-hydroxyecdysone (20E) and juvenile hormone (JH) titer, and they both increased in the hemolymph of parasitized silkworms. Furthermore, we verified the expression patterns of related genes, which showed an upregulation of 20E signaling and biosynthesis genes but a significant downregulation of ecdysone oxidase (EO), a 20E inactivation enzyme, in parasitized silkworms. In addition, related genes of the JH signaling were activated in parasitized silkworms, while related genes of the JH degradation pathway were suppressed, resulting in an increase in JH titer. Notably, the precocious wandering behavior of parasitized silkworms was partly recoverable by silencing the transcriptions of BmCYP302A1 or BmCYP307A1 genes. Our findings suggest that the developmental duration of silkworm post parasitism could be shortened by regulation of 20E and JH titers, which may help silkworm to resist the E. sorbillans infestation. These findings provide a basis for deeper insight into the interplay between silkworms and E. sorbillans and may serve as a reference for the development of a novel approach to control silkworm myiasis.