Prothoracic Glands Research Articles

Soul is a master control gene governing the development of the Drosophila prothoracic gland

The prothoracic gland (PG) is a major insect endocrine organ. It is the principal source of insect steroid hormones, and critical for key developmental events such as the molts, the establishment of critical weight (CW), pupation, and sexual maturation. However, little is known about the developmental processes that regulate PG morphology. In this study, we identified soul, which encodes a PG-specific basic helix-loop-helix (bHLH) transcription factor. We demonstrate that Tap, also a bHLH protein, dimerizes with Soul. Both are expressed in the developing PG. Interfering with either soul or tap function caused strikingly similar phenotypes, resulting in small and fragmented PGs, the abolishment of steroid hormone-producing gene expression, larval arrest, and a failure to undergo metamorphosis. Furthermore, both soul and tap showed expression peaks just prior to the CW checkpoint. Disrupting soul- or tap-function before, but not after, the CW checkpoint caused larval arrest, and perturbed highly similar gene cohorts, which were enriched for regulators and components of the steroid hormone biosynthesis pathway. Intriguingly, a chitin-based cuticle gene, Cpr49Ah, and a POU domain transcription factor gene, pdm3, are direct target genes of the Soul/Tap complex, and disruption of either phenocopied key aspects of soul/tap loss-of-function phenotypes. Taken together, our findings demonstrate that the Soul/Tap heterodimer resides at the top of a complex gene hierarchy that drives PG development, CW establishment, and steroid hormone production.

HIF signaling in the prothoracic gland regulates growth and development in hypoxia but not normoxia in Drosophila.

The developmental regulation of body size is a fundamental life-history characteristic that in most animals is tied to the transition from juvenile to adult form. In holometabolous insects, this transition is ostensibly initiated at the attainment of a critical weight in the final larval instar. It has been hypothesized that the size-sensing mechanism used to determine attainment of critical weight exploits oxygen limitation as a larvae grows beyond the oxygen-delivery capacity of its fixed tracheal system; that is, developmentally induced cellular hypoxia initiates the synthesis of the molting hormone ecdysone by the prothoracic gland. We tested this hypothesis in Drosophila by assaying cellular hypoxia throughout the third larval instar at 21 and 10 kPa O2, using the activity of the HIF (hypoxia inducible factor)-signaling pathway as a measure of hypoxia. While HIF signaling was elevated at low levels of environmental O2, it did not markedly increase during development at either oxygen level, and was only suppressed by hyperoxia after feeding had ceased. Further, changes in HIF signaling in the prothoracic gland alone did not alter body size or developmental time in a way that would be expected if cellular hypoxia in the prothoracic gland was part of the critical weight mechanism. Our data do show, however, that reduced HIF signaling in the prothoracic gland decreases survival and retards development at 10 kPa O2, suggesting that prothoracic HIF signaling is a necessary part of the beneficial plasticity mechanism that controls growth and development in response to low oxygen level.

The role of neuropeptide prothoracicotropic hormone (PTTH) - Torso in pyriproxyfen-induced larval-pupal abnormal metamorphosis in silkworms

The neuropeptide prothoracicotropic hormone (PTTH) plays a key role in regulating ecdysone synthesis and promoting insect metamorphosis. Pyriproxyfen is a juvenile hormone analogue. We previously reported that pyriproxyfen disrupts ecdysone secretion and inhibits larval-pupal metamorphosis in silkworms. However, the specific molecular mechanisms by which pyriproxyfen interferes with ecdysone signaling remain to be elucidated. Herein, the RNA-seq analysis on the ecdysone-secretion organ prothoracic gland (PG) was conducted following pyriproxyfen exposure. A total of 3774 differentially expressed genes (DEGs) were identified, with 1667 up-regulated and 2107 down-regulated. KEGG analysis showed that DEGs were enriched in the MAPK signaling pathway, a conserved pathway activated by PTTH binding to Torso, which regulates the ecdysone synthesis. qRT-PCR results indicated a significant up-regulation in PTTH transcription level, while the transcription levels of torso and downstream MAPK pathway genes, Ras2, Raf and ERK, were down-regulated 24 h post-pyriproxyfen treatment. Consistent with these transcriptional changes, PTTH titers in the brain also increased following pyriproxyfen treatment. These results suggest that pyriproxyfen induces abnormal metamorphosis in silkworms by impairing PTTH-Torso signaling. This study enhances our understanding of the molecular mechanisms of pyriproxyfen-induced larval-pupal abnormal metamorphosis in silkworms, and also provides insights for developing detoxification strategies for juvenile hormone analog pesticides to non-target organisms.

Schlank orchestrates insect developmental transition by switching H3K27 acetylation to trimethylation in the prothoracic gland

Insect developmental transitions are precisely coordinated by ecdysone and juvenile hormone (JH). We previously revealed that accumulated H3K27 trimethylation (H3K27me3) at the locus encoding JH signal transducer Hairy is involved in the larval-pupal transition in insects, but the underlying mechanism remains to be fully defined. Here, we show in Drosophila and Bombyx that Rpd3-mediated H3K27 deacetylation in the prothoracic gland during the last larval instar promotes ecdysone biosynthesis and the larval-pupal transition by enabling H3K27me3 accumulation at the Hairy locus to induce its transcriptional repression. Importantly, we find that the homeodomain transcription factor Schlank acts to switch active H3K27 acetylation (H3K27ac) to repressive H3K27me3 at the Hairy locus by directly binding to the Hairy promoter and then recruiting the histone deacetylase Rpd3 and the histone methyltransferase PRC2 component Su(z)12 through physical interactions. Moreover, Schlank inhibits Hairy transcription to facilitate the larval-pupal transition, and the Schlank signaling cascade is suppressed by JH but regulated in a positive feedback manner by ecdysone. Together, our data uncover that Schlank mediates epigenetic reprogramming of H3K27 modifications in hormone actions during insect developmental transition.

Insect hormone PTTH regulates lifespan through temporal and spatial activation of NF-κB signaling during metamorphosis.

The prothoracicotropic hormone (PTTH) is a well-known neuropeptide that regulates insect metamorphosis (the juvenile-to-adult transition) by inducing the biosynthesis of steroid hormones. However, the role of PTTH in adult physiology and longevity is largely unexplored. Here, we show that Ptth loss-of-function mutants are long-lived and exhibit increased resistance to oxidative stress in Drosophila. Intriguingly, we find that loss of Ptth blunt age-dependent upregulation of NF-κB signaling specifically in fly hepatocytes (oenocytes). We further show that oenocyte-specific overexpression of Relish/NF-κB blocks the lifespan extension of Ptth mutants, suggesting that PTTH regulates lifespan through oenocyte-specific NF-κB signaling. Surprisingly, adult-specific knockdown of Ptth did not prolong lifespan, indicating that PTTH controls longevity through developmental programs. Indeed, knockdown of PTTH receptor Torso in prothoracic gland (PG) during fly development prolongs lifespan. To uncover the developmental processes underlying PTTH-regulated lifespan, we perform a developmental transcriptomic analysis and identify an unexpected activation of NF-κB signaling in developing oenocytes during fly metamorphosis, which is blocked in Ptth mutants. Importantly, knockdown of Relish/NF-κB specifically in oenocytes during early pupal stages significantly prolongs the lifespan of adult flies. Thus, our findings uncover an unexpected role of PTTH in controlling adult lifespan through temporal and spatial activation of NF-κB signaling in developing hepatocytes and highlight the vital role of developmental NF-κB signaling in shaping adult physiology.

Developmental analysis of Spalt function in the Drosophila prothoracic gland.

The Spalt transcriptional regulators participate in a variety of cell fate specification processes during development, regulating transcription through interactions with DNA AT-rich regions. Spalt proteins also bind to heterochromatic regions, and some of their effects require interactions with the NuRD chromatin remodeling and deacetylase complex. Most of the biological roles of Spalt proteins have been characterized in diploid cells engaged in cell proliferation. Here, we address the function of Drosophila Spalt genes in the development of a larval tissue formed by polyploid cells, the prothoracic gland, the cells of which undergo several rounds of DNA replication without mitosis during larval development. We show that prothoracic glands depleted of Spalt expression display severe changes in the size of the nucleolus, the morphology of the nuclear envelope and the disposition of the chromatin within the nucleus, leading to a failure in the synthesis of ecdysone. We propose that loss of ecdysone production in the prothoracic gland of Spalt mutants is primarily caused by defects in nuclear pore complex function that occur as a consequence of faulty interactions between heterochromatic regions and the nuclear envelope.

The people behind the papers - Douglas Terry and Kenneth Moberg.

The steroid hormone ecdysone (Ec) is secreted from the prothoracic gland for growth in the developing Drosophila larva. How Ec-dependent regeneration can occur despite a drop in circulating Ec in the injured developing larvae remains unclear. In a new study in Development, Kenneth Moberg and colleagues find that injury induces local Ec synthesis at the wounded site to delay development and promote tissue repair in Drosophila. To learn more about the story behind the paper, we caught up with first author Douglas Terry and corresponding author Kenneth Moberg, Professor of Cell Biology at Emory University School of Medicine, USA.

Drosophila Evi5 is a critical regulator of intracellular iron transport via transferrin and ferritin interactions

Vesicular transport is essential for delivering cargo to intracellular destinations. Evi5 is a Rab11-GTPase-activating protein involved in endosome recycling. In humans, Evi5 is a high-risk locus for multiple sclerosis, a debilitating disease that also presents with excess iron in the CNS. In insects, the prothoracic gland (PG) requires entry of extracellular iron to synthesize steroidogenic enzyme cofactors. The mechanism of peripheral iron uptake in insect cells remains controversial. We show that Evi5-depletion in the Drosophila PG affected vesicle morphology and density, blocked endosome recycling and impaired trafficking of transferrin-1, thus disrupting heme synthesis due to reduced cellular iron concentrations. We show that ferritin delivers iron to the PG as well, and interacts physically with Evi5. Further, ferritin-injection rescued developmental delays associated with Evi5-depletion. To summarize, our findings show that Evi5 is critical for intracellular iron trafficking via transferrin-1 and ferritin, and implicate altered iron homeostasis in the etiology of multiple sclerosis.

PTTH-Torso Signaling System Controls Developmental Timing, Body Size, and Reproduction through Regulating Ecdysone Homeostasis in the Brown Planthopper, Nilaparvata lugens.

In holometabolous insects, such as Drosophila and Bombyx, prothoracicotropic hormone (PTTH) is well established to be critical in controlling developmental transitions and metamorphosis by stimulating the biosynthesis of ecdysone in the prothoracic glands (PGs). However, the physiological role of PTTH and the receptor Torso in hemimetabolous insects remains largely unexplored. In this study, homozygous PTTH- and Torso-null mutants of the brown planthopper (BPH), Nilaparvata lugens, were successfully generated by employing clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR-Cas9). Further characterization showed that both NlPTTH-/- and NlTorso-/- mutants exhibited prolonged nymphal duration and increased final adult size. Enzyme-linked immunosorbent assay (ELISA) revealed that NlPTTH-/- and NlTorso-/- mutants exhibited a significant reduction in 20-hydroxyecdysone (20E) in fifth-instar nymphs at 48 h post-ecdysis compared to Wt controls. Furthermore, our results indicated that both NlPTTH-/- and NlTorso-/- mutants had shortened lifespan, reduced female fecundity, and reduced egg hatching rates in adults. These findings suggest a conserved role for the PTTH-Torso signaling system in the regulation of developmental transitions by stimulating ecdysone biosynthesis in hemimetabolous insects.

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.

Timed receptor tyrosine kinase signaling couples the central and a peripheral circadian clock in Drosophila

Circadian clocks impose daily periodicities to behavior, physiology, and metabolism. This control is mediated by a central clock and by peripheral clocks, which are synchronized to provide the organism with a unified time through mechanisms that are not fully understood. Here, we characterized in Drosophila the cellular and molecular mechanisms involved in coupling the central clock and the peripheral clock located in the prothoracic gland (PG), which together control the circadian rhythm of emergence of adult flies. The time signal from central clock neurons is transmitted via small neuropeptide F (sNPF) to neurons that produce the neuropeptide Prothoracicotropic Hormone (PTTH), which is then translated into daily oscillations of Ca2+ concentration and PTTH levels. PTTH signaling is required at the end of metamorphosis and transmits time information to the PG through changes in the expression of the PTTH receptor tyrosine kinase (RTK), TORSO, and of ERK phosphorylation, a key component of PTTH transduction. In addition to PTTH, we demonstrate that signaling mediated by other RTKs contributes to the rhythmicity of emergence. Interestingly, the ligand to one of these receptors (Pvf2) plays an autocrine role in the PG, which may explain why both central brain and PG clocks are required for the circadian gating of emergence. Our findings show that the coupling between the central and the PG clock is unexpectedly complex and involves several RTKs that act in concert and could serve as a paradigm to understand how circadian clocks are coordinated.

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.

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.

KDM5-mediated activation of genes required for mitochondrial biology is necessary for viability in Drosophila.

Histone-modifying proteins play important roles in the precise regulation of the transcriptional programs that coordinate development. KDM5 family proteins interact with chromatin through demethylation of H3K4me3 as well as demethylase-independent mechanisms that remain less understood. To gain fundamental insights into the transcriptional activities of KDM5 proteins, we examined the essential roles of the single Drosophila Kdm5 ortholog during development. KDM5 performs crucial functions in the larval neuroendocrine prothoracic gland, providing a model to study its role in regulating key gene expression programs. Integrating genome binding and transcriptomic data, we identify that KDM5 regulates the expression of genes required for the function and maintenance of mitochondria, and we find that loss of KDM5 causes morphological changes to mitochondria. This is key to the developmental functions of KDM5, as expression of the mitochondrial biogenesis transcription factor Ets97D, homolog of GABPα, is able to suppress the altered mitochondrial morphology as well as the lethality of Kdm5 null animals. Together, these data establish KDM5-mediated cellular functions that are important for normal development and could contribute to KDM5-linked disorders when dysregulated.

Azadirachtin Inhibits Nuclear Receptor HR3 in the Prothoracic Gland to Block Larval Ecdysis in the Fall Armyworm, Spodoptera frugiperda.

Azadirachtin has been used to control agricultural pests for a long time; however, the molecular mechanism of azadirachtin on lepidopterans is still not clear. In this study, the fourth instar larvae of fall armyworm were fed with azadirachtin, and then the ecdysis was blocked in the fourth instar larval stage (L4). The prothoracic glands (PGs) of the treated larvae were dissected for RNA sequencing to determine the effect of azadirachtin on ecdysis inhibition. Interestingly, one of the PG-enriched genes, the nuclear hormone receptor 3 (HR3), was decreased after azadirachtin treatment, which plays a critical role in the 20-hydroxyecdysone action during ecdysis. To deepen the understanding of azadirachtin on ecdysis, the HR3 was knocked out by using the CRISPR/Cas9 system, while the HR3 mutants displayed embryonic lethal phenotype; thus, the stage-specific function of HR3 during larval molting was not enabled to unfold. Hence, the siRNA was injected into the 24 h L4 larvae to knock down HR3. After 96 h, the injected larvae were blocked in the old cuticle during ecdysis which is consistent with the azadirachtin-treated larvae. Taken together, we envisioned that the inhibition of ecdysis in the fall armyworm after the azadirachtin treatment is due to an interference with the expression of HR3 in PG, resulting in larval mortality. The results in this study specified the understanding of azadirachtin on insect ecdysis and the function of HR3 in lepidopteran in vivo.

The macrophage genetic cassette inr/dtor/pvf2 is a nutritional status checkpoint for developmental timing.

A small number of signaling molecules, used reiteratively, control differentiation programs, but the mechanisms that adapt developmental timing to environmental cues are less understood. We report here that a macrophage inr/dtor/pvf2 genetic cassette is a developmental timing checkpoint in Drosophila, which either licenses or delays biosynthesis of the steroid hormone in the endocrine gland and metamorphosis according to the larval nutritional status. Insulin receptor/dTor signaling in macrophages is required and sufficient for production of the PDGF/VEGF family growth factor Pvf2, which turns on transcription of the sterol biosynthesis Halloween genes in the prothoracic gland via its receptor Pvr. In response to a starvation event or genetic manipulation, low Pvf2 signal delays steroid biosynthesis until it becomes Pvr-independent, thereby prolonging larval growth before pupariation. The significance of this developmental timing checkpoint for host fitness is illustrated by the observation that it regulates the size of the pupae and adult flies.

Signaling in cAMP-stimulated ecdysteroidogenesis in prothoracic glands of the silkworm, Bombyx mori

In the present study, we investigated downstream pathways of cyclic adenosine monophosphate (cAMP) signaling (which is related to prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis) in Bombyx mori prothoracic glands (PGs). Results showed that treatment with either dibutyryl cAMP (dbcAMP) or 1-methyl-3-isobutylxanthine (MIX) inhibited phosphorylation of adenosine 5′-monophosphate-activated protein kinase (AMPK) and activated phosphorylation of the translational repressor, 4E-binding protein (4E-BP), a marker of target of rapamycin (TOR) signaling. A chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, AICAR) increased dbcAMP-inhibited AMPK phosphorylation and blocked dbcAMP-stimulated phosphorylation of 4E-BP, indicating that inhibition of AMPK phosphorylation lies upstream of dbcAMP-stimulated TOR signaling. Treatment of PGs with dbcAMP and MIX also stimulated phosphorylation of a 37-kDa protein, as recognized by a protein kinase C (PKC) substrate antibody, indicating that cAMP activates PKC signaling. Treatment with either LY294002 or AICAR did not affect dbcAMP-stimulated phosphorylation of the PKC-dependent 37-kDa protein, indicating that cAMP-stimulated PKC signaling is not related to phosphoinositide 3-kinase (PI3K) or AMPK. In addition, dbcAMP-stimulated ecdysteroidogenesis in PGs was partially inhibited by pretreatment with either LY294002, AICAR, or calphostin C. From these results, we concluded that AMPK/TOR/4E-BP and PKC pathways are involved in ecdysteroidogenesis of PGs stimulated by cAMP signaling in B. mori.

Effects of 20-hydroxyecdysone on UVB-induced photoaging in hairless mice

We recently reported that exposure of skin to ultraviolet B (UVB) irradiation for 2 weeks induces stress and accelerates skin aging. Interestingly, aldosterone synthase is known to be crucial in generating UVB-induced stress-related responses, suggesting that drugs that regulate its activity can be used as skin antiaging agents. Through extensive drug screening, we have identified 20-hydroxyecdysone (20E), a steroidal prohormone secreted by the prothoracic glands of insects, as a potent inhibitor of UVB-induced aging. Although 20E has been shown to exert antistress and anti-collagenase effects in vitro, its effects in vivo remain unexplored. Furthermore, the pharmacological and physiological effects of 20E on UVB-mediated photoaging are poorly understood. Therefore, in this study, we investigated the effects of 20E on aldosterone synthase and UVB-induced photoaging and skin lesions in hairless mice, focusing on the stress-related hypothalamic–pituitary–adrenal axis. We confirmed that 20E inhibited aldosterone synthase and reduced corticosterone levels. When applied to a UV-induced skin aging animal model, it ameliorated UV-induced stress and protected against the decrease in collagen levels. Importantly, when the aldosterone synthase inhibitor osilodrostat, an FDA-approved drug, was applied to the UV-induced skin aging model, the stress-reducing and antiaging effects of 20E were not observed. Thus, we conclude that 20E inhibits UVB-induced skin aging by blocking aldosterone synthase and is a potential candidate to prevent skin aging.

Genome-Wide Identification and Transcriptome-Based Expression Profile of Cuticular Protein Genes in Antheraea pernyi.

Antheraea pernyi is one of the most famous edible and silk-producing wild silkworms of Saturniidae. Structural cuticular proteins (CPs) are the primary component of insect cuticle. In this paper, the CPs in the genome of A. pernyi were identified and compared with those of the lepidopteran model species Bombyx mori, and expression patterns were analyzed based on the transcriptomic data from the larval epidermis/integument (epidermis in the following) and some non-epidermis tissues/organs of two silkworm species. A total of 217 CPs was identified in the A. pernyi genome, a comparable number to B. mori (236 CPs), with CPLCP and CPG families being the main contribution to the number difference between two silkworm species. We found more RR-2 genes expressed in the larval epidermis of fifth instar of A. pernyi than B. mori, but less RR-2 genes expressed in the prothoracic gland of A. pernyi than B. mori, which suggests that the hardness difference in the larval epidermis and prothoracic gland between the two species may be caused by the number of RR-2 genes expressed. We also revealed that, in B. mori, the number of CP genes expressed in the corpus allatum and prothoracic gland of fifth instar was higher than that in the larval epidermis. Our work provided an overall framework for functional research into the CP genes of Saturniidae.

Diverse functions of the ecdysone receptor (EcR) in the panoistic ovary of the German cockroach

Ecdysone regulates essential processes in insect life. Perhaps the most well-known of these are related to metamorphosis. However, ecdysone is also required to regulate the proliferation and differentiation of germ cells in the ovary. The role of ecdysone in insect oogenesis has been studied in depth in holometabolan species with meroistic ovaries, such as Drosophila melanogaster, while in hemimetabolan species with panoistic ovaries their functions are still poorly understood. In the present work, we studied the role of ecdysone in the ovary of the last nymphal instar of the cockroach Blattella germanica by using RNA interference to reduce the levels of the ecdysone receptor (EcR), and thereby deplete the expression of ecdysteroidogenic genes in the prothoracic gland. However, the expression of ecdysteroidogenic genes was upregulated in the ovary, resulting in cell overproliferation in the germarium, which appeared swollen. By analysing the expression of genes that respond to ecdysone, we found that when the source of 20E is the nymphal ovary, EcR appears to repress 20E-associated genes bypassing early genes signalling.