Ecdysone Levels Research Articles

Knockdown of TcGluCl leads to the premature pupation of Tribolium castaneum larvae possibly by influencing the calcium-mediating hormone homeostasis

The glutamate-gated chloride channels (GluCls) are widely existed in the neural and nonneural tissues of invertebrate. In addition to play important roles in signal transduction, the GluCls also showed multiple physiological functions in insects such as participate in the juvenile hormone synthesis. In the present study, the potential roles of TcGluCl in growth and development of the red flour beetle Tribolium castaneum were explored. Knockdown of TcGluCl showed no effects on the survivability, weight growth, final pupation rate, eclosion and fecundity of T. castaneum, whereas resulted in the significant premature pupation of larvae. Inhibition of TcGluCl expression significantly changed the levels of juvenile hormone and ecdysone as well as the expressions of hormone biosynthetic genes. The increased ecdysone level and decreased juvenile hormone level were observed at the late stage of dsGluCl-treated larvae. Knockdown of TcGluCl significantly reduced the expressions of TcSTIM1 and TcOrai1, which were the primary proteins in store-operated calcium entry (SOCE) mediated Ca2+ influx mechanism. Whilst the L-glutamic acid treatment led to the increased TcOrai1 expression in T. castaneum. These findings suggested that knockdown of TcGluCl increased the ecdysone level and contributed to the premature pupation of larvae, which might be due to the reduced Ca2+ influx caused by the decreased expressions of TcSTIM1 and TcOrai1. These studies provide novel insights on the function of GluCls in insects.

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.

The Role of Cholesterol during the Ovarian Maturation and Lipid Metabolism of Female Chinese Mitten Crab (Eriocheir sinensis)

In previous study, we found that the cholesterol requirement of Eriocheir sinensis was 0.27%, to further investigate the effects of cholesterol on health status, ovarian maturation, and lipid metabolism of female Eriocheir sinensis broodstock. Two diets containing 0% and 0.25% (actually 0.05% and 0.27%) cholesterol were fed to the female crabs (average weight: 49.21 ± 0.11 g) for 4 months and sampled once a month. The results showed that the body weight (BW), survival rate (SR), meat yield (MY), condition factor (CF), hepatosomatic index (HSI), and gonadosomatic index (GSI) of Eriocheir sinensis were significantly affected by treatment time and compared with the cholesterol deficient group, supplementing cholesterol significantly increased BW, HSI, and GSI (P<0.05). In addition, long-term lack of cholesterol will lead to a significant decrease in the activity of ACP, AKP, and SOD and a significant increase in the content of MDA. The histological results showed that cholesterol significantly increased the volume of oocytes (P<0.05). Further studies found that 0.27% cholesterol significantly increased the transcription levels of vtg and vgr in hepatopancreas and ovaries, which may be the main reason for the increase of oocyte size (P<0.05). When fed with 0.27% cholesterol diet, the contents of nutrients in hepatopancreas and ovaries increased significantly, especially lipids and cholesterol (P<0.05). Through the analysis of mRNA expression level of genes related to lipid metabolism, it was found that cholesterol enhanced the transcription level of genes related to lipid synthesis and transport in hepatopancreas, thereby promoting the accumulation of lipid in the organism. Furthermore, compared with control group, the levels of juvenile hormone (JH), 17β-estradiol (E2), methyl farnesoate (MF), and ecdysone in the organism were significantly increased after feeding a diet with 0.27% cholesterol (P<0.05). In summary, supplementing an appropriate amount of cholesterol in the diet can improve the growth performance of Eriocheir sinensis broodstock, enhance the body’s antioxidant and immune system, and promote the accumulation of nutrients in the ovaries, thereby promoting ovarian maturation.

A Drosophila model targets Eiger/TNFα to alleviate obesity-related insulin resistance and macrophage infiltration.

Obesity is associated with various metabolic disorders, such as insulin resistance and adipose tissue inflammation (ATM), characterized by macrophage infiltration into adipose cells. This study presents a new Drosophila model to investigate the mechanisms underlying these obesity-related pathologies. We employed genetic manipulation to reduce ecdysone levels to prolong the larval stage. These animals are hyperphagic and exhibit features resembling obesity in mammals, including increased lipid storage, adipocyte hypertrophy and high circulating glucose levels. Moreover, we observed significant infiltration of immune cells (hemocytes) into the fat bodies, accompanied by insulin resistance. We found that attenuation of Eiger/TNFα signaling reduced ATM and improved insulin sensitivity. Furthermore, using metformin and the antioxidants anthocyanins, we ameliorated both phenotypes. Our data highlight evolutionarily conserved mechanisms allowing the development of Drosophila models for discovering therapeutic pathways in adipose tissue immune cell infiltration and insulin resistance. Our model can also provide a platform to perform genetic screens or test the efficacy of therapeutic interventions for diseases such as obesity, type 2 diabetes and non-alcoholic fatty liver disease.

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.

An entomopathogenic fungus exploits its host humoral antibacterial immunity to minimize bacterial competition in the hemolymph

BackgroundThe insect hemolymph (blood-equivalent fluid), composed of a large number of hemocytes (blood cells) and a variety of soluble immune effectors, is hostile for pathogens including fungi. In order to survive in the insect hemocoel (body cavity), the entomopathogenic fungus (EPF) has evolved two classical coping strategies, namely evasion and suppression of the host immune reactions. However, it remains unclear whether EPF has other ways of coping with host immunity.ResultsIn this study, we demonstrated that Metarhizium rileyi (an EPF) infection by injection of blastospores into the hemocoel enhanced the plasma antibacterial activity of cotton bollworm (Helicoverpa armigera), which was partially due to the enhanced expression of antimicrobial peptides (AMPs). The early stage of M. rileyi infection induced the translocation of gut bacteria into the hemocoel, where they were subsequently cleared due to the enhanced plasma antibacterial activity. Further, we showed that the enhanced plasma antibacterial activity and AMP expression were attributable to M. rileyi but not the invasive gut bacteria (opportunistic bacteria). Elevated ecdysone (major steroid hormone in insects) levels in the hemolymph at 48 h post-M. rileyi infection might contribute to the enhanced expression of AMPs. The fungus-elicited AMPs, such as cecropin 3 or lebocin, exhibited potent inhibitory activity against the opportunistic bacteria but not against hyphal bodies. In addition, the opportunistic bacteria competed with hyphal bodies for amino acid nutrients.ConclusionsM. rileyi infection induced the translocation of gut bacteria, and then the fungi activated and exploited its host humoral antibacterial immunity to eliminate opportunistic bacteria, preventing them from competing for nutrients in the hemolymph. Unlike the classical strategies, EPF utilizes to evade or suppress host immunity, our findings reveal a novel strategy of interaction between EPF and host immunity.BbkogtQagejkWT1MfyqrGJVideo

Correlated responses in basal immune function in response to selection for fast development in Drosophila melanogaster.

Often, immunity is invoked in the context of infection, disease and injury. However, an ever alert and robust immune system is essential for maintaining good health, but resource investment into immunity needs to be traded off against allocation to other functions. In this study, we study the consequences of such a trade-off with growth by ascertaining various components of baseline innate immunity in two types of Drosophila melanogaster populations selected for fast development, in combination with either a long effective lifespan (FLJs) or a short effective lifespan (FEJs). We found that distinct immunological parameters were constitutively elevated in both, FLJs and FEJs compared to their ancestral control (JB) populations, and these constitutive elevated immunological parameters were associated with reduced insulin signalling and comparable total gut microbiota. Our results bring into focus the inter-relationship between egg to adult development time, ecdysone levels, larval gut microbiota, insulin signalling, adult reproductive longevity and immune function. We discuss how changes in selection pressures operating on life-history traits can modulate different components of immune system.

Sex-specific effects of propiconazole on the molting of the Chinese mitten crab (Eriocheir sinensis)

Given the inevitable exposure of Eriocheir sinensis (E. sinensis) to fungicides in rice-crab co-culture systems, understanding the potential effect of fungisides is important for practical application. Molting is a crucial development process of E. sinensis, which is regulated by endocrine system and genetic factors, and is susceptible to exogenous chemicals. However, the impact of fungicides application on the molting of E. sinensis have been rarely reported. In the present study, propiconazole, a widely used fungicide for rice disease management, was found to exert potential effects on the molting of E. sinensis at residual-related level in the rice-crab co-culture fields. After 14 days of short-term exposure to propiconazole, female crabs exhibited remarkably higher levels of hemolymph ecdysone than males. When the exposure was extended to 28 days, propiconazole markedly accelerated molt-inhibiting hormone expression by 3.3-fold, ecdysone receptor expression by 7.8-fold, and crustacean retinoid X receptor expression by 9.6-fold in male crabs, while it showed the opposite effect in females with suppressed gene expression. Propiconazole also induced the activity of N-acetylglucosaminidase in male crabs rather than females during the experiments. Our study suggests that propiconazole exerts sex-specific effects on the molting of E. sinensis. The impact of propiconazole application in the rice-crab co-culture systems remains more assessment to avoid affecting the growth of cultured E. sinensis.

Effects of Temperature on Growth, Molting, Feed Intake, and Energy Metabolism of Individually Cultured Juvenile Mud Crab Scylla paramamosain in the Recirculating Aquaculture System

An eight-week experiment was conducted to investigate the effects of temperature (20, 25, 30, and 35 °C) on growth performance, feed intake, energy metabolism, antioxidant capacity, and the stress response of juvenile Scylla paramamosain in a recirculating aquaculture system. The results showed that the survival rate of the 35 °C group was 80.36 ± 5.92%, significantly lower than that of the other three groups (100%). The high molt frequency of mud crabs was observed in high-temperature groups, accompanied by a higher ecdysone level and ecdysone receptor gene expression but lower molt inhibitory hormone gene expression. However, the molt increment (73.58 ± 2.18%), food intake, and feed conversion efficiency showed a parabolic trend, with the lowest value found in the 35 °C group. Oxygen consumption rate and ammonia excretion rate increased with the increasing temperature, and oxygen-nitrogen ratio, lactic acid, triglyceride, total cholesterol, glucose, and cortisol peaked at 35 °C. Temperature also significantly affected the antioxidant system of S. paramamosain. Crabs in the 25 °C and 30 °C had a significantly higher total antioxidant capacity and lower malondialdehyde compared with the 35 °C group (p < 0.05). Although the high temperature promoted molting, it decreased the feeding rate and growth performance, leading to oxidative stress and functional hypoxia. The quadratic function model demonstrated the optimum temperature for the specific growth rate of juvenile S. paramamosain was 28.5–29.7 °C.

Ecdysone coordinates plastic growth with robust pattern in the developing wing.

Animals develop in unpredictable, variable environments. In response to environmental change, some aspects of development adjust to generate plastic phenotypes. Other aspects of development, however, are buffered against environmental change to produce robust phenotypes. How organ development is coordinated to accommodate both plastic and robust developmental responses is poorly understood. Here, we demonstrate that the steroid hormone ecdysone coordinates both plasticity of organ size and robustness of organ pattern in the developing wings of the fruit fly Drosophila melanogaster. Using fed and starved larvae that lack prothoracic glands, which synthesize ecdysone, we show that nutrition regulates growth both via ecdysone and via an ecdysone-independent mechanism, while nutrition regulates patterning only via ecdysone. We then demonstrate that growth shows a graded response to ecdysone concentration, while patterning shows a threshold response. Collectively, these data support a model where nutritionally regulated ecdysone fluctuations confer plasticity by regulating disc growth in response to basal ecdysone levels and confer robustness by initiating patterning only once ecdysone peaks exceed a threshold concentration. This could represent a generalizable mechanism through which hormones coordinate plastic growth with robust patterning in the face of environmental change.

Ecdysone exerts biphasic control of regenerative signaling, coordinating the completion of regeneration with developmental progression

In Drosophila melanogaster, loss of regenerative capacity in wing imaginal discs coincides with an increase in systemic levels of the steroid hormone ecdysone, a key coordinator of their developmental progression. Regenerating discs release the relaxin hormone Dilp8 (Drosophila insulin-like peptide 8) to limit ecdysone synthesis and extend the regenerative period. Here, we describe how regenerating tissues produce a biphasic response to ecdysone levels: lower concentrations of ecdysone promote local and systemic regenerative signaling, whereas higher concentrations suppress regeneration through the expression of broad splice isoforms. Ecdysone also promotes the expression of wingless during both regeneration and normal development through a distinct regulatory pathway. This dual role for ecdysone explains how regeneration can still be completed successfully in dilp8- mutant larvae: higher ecdysone levels increase the regenerative activity of tissues, allowing regeneration to reach completion in a shorter time. From these observations, we propose that ecdysone hormone signaling functions to coordinate regeneration with developmental progression.

Tank bottom area influences the growth, molting, stress response, and antioxidant capacity of juvenile mud crab Scylla paramamosain

An 8-week trial was performed to determine the effects of different tank bottom areas (TBA) on growth performance, stress response, and antioxidant capacity of juvenile mud crab Scylla paramamosain. Four treatments in triplicates with different k-value (k = A/CW2, where A is the bottom area (mm2) of the tank, and CW is the carapace width (mm) of the juvenile crab), i.e., k-value = 4.42 (K4), 17.67 (K18), 39.76 (K40), and 70.68 (K70), were used in this experiment. The results showed no significant difference in survival rate (83.33–96.67%) was found, but the K40 group had the best growth performance among all the treatments (P < 0.05). With the decrease of TBA, the level of ecdysone and ecdysone receptor (EcR) gene expression decreased, while the molt inhibitory hormone (MIH) gene expression increased. As a result, prolonged molting intervals and reduced weight gain rates were observed in K4 and K18. Higher cortisol, lactic acid, and lower glycogen content were also observed in K4 (P < 0.05), accompanied by higher phosphofructokinase (PFK) and pyruvate kinase (PK) enzyme activities (P < 0.05). On the contrary, lower catalase (CAT) and higher malondialdehyde (MDA) were observed in the K4 and K18 groups. The results demonstrated that TBA significantly impacted the growth performance of S. paramamosain, and the effects could be mediated through hormonal, glycometabolism, and antioxidant pathways. Notably, the optimal TBA was estimated to be 29.23–59.19 (k-value) according to the quadratic polynomial regression analysis of WGR, and the oversized tank could not give extra benefits in terms of growth performance. Therefore, TBA should be deliberated when designing crab culture facilities.

The circadian clock gates Drosophila adult emergence by controlling the timecourse of metamorphosis

The daily rhythm of adult emergence of holometabolous insects is one of the first circadian rhythms to be studied. In these insects, the circadian clock imposes a daily pattern of emergence by allowing or stimulating eclosion during certain windows of time and inhibiting emergence during others, a process that has been described as "gating." Although the circadian rhythm of insect emergence provided many of the key concepts of chronobiology, little progress has been made in understanding the bases of the gating process itself, although the term "gating" suggests that it is separate from the developmental process of metamorphosis. Here, we follow the progression through the final stages of Drosophila adult development with single-animal resolution and show that the circadian clock imposes a daily rhythmicity to the pattern of emergence by controlling when the insect initiates the final steps of metamorphosis itself. Circadian rhythmicity of emergence depends on the coupling between the central clock located in the brain and a peripheral clock located in the prothoracic gland (PG), an endocrine gland whose only known function is the production of the molting hormone, ecdysone. Here, we show that the clock exerts its action by regulating not the levels of ecdysone but that of its actions mediated by the ecdysone receptor. Our findings may also provide insights for understanding the mechanisms by which the daily rhythms of glucocorticoids are produced in mammals, which result from the coupling between the central clock in the suprachiasmatic nucleus and a peripheral clock located in the suprarenal gland.

Transcriptome analysis reveals TOR signalling-mediated plant flush shoots governing Diaphorina citri Kuwayama oviposition.

Asian Citrus Psyllid (ACP), Diaphorina citri, is a key vector transmitting the causative agent of Huanglongbing (HLB) disease. Population growth of ACP is evident after feeding on plant flush shoots, as they only oviposit here. However, the underlying mechanism as to why flush shoots govern oviposition is unclear. This study compares the fecundity and ovarian morphology of ACP between young flush and mature leaves. Furthermore, the transcriptome of mated females infesting Murraya paniculata was analysed. Finally, the gene of the key Target of Rapamycin (TOR) signalling pathway was silenced by RNAi. Results indicated that flush shoot feeding activated the development of the psyllids ovary and therefore induced oviposition. A total of 126 and 2794 differentially expressed genes were detected at 1 and 5 days, respectively, after pest infestation of flush shoots compared to mature leaves. Many genes are involved in protein metabolism, Mitogen-Activated Protein Kinase (MAPK) signalling pathway, hormone synthesis, and TOR signalling pathway: all thought to activate reproduction. Silencing of the positive regulator gene DcRheb in the TOR pathway resulted in lower levels of ecdysone and juvenile hormone and decreased vitellogenin synthesis, further disrupting reproductive ability. This study enhances understanding of the molecular mechanism underlying ACP's reproductive strategy.

Ecdysone-dependent feedback regulation of prothoracicotropic hormone controls the timing of developmental maturation.

ABSTRACTThe activation of a neuroendocrine system that induces a surge in steroid production is a conserved initiator of the juvenile-to-adult transition in many animals. The trigger for maturation is the secretion of brain-derived neuropeptides, yet the mechanisms controlling the timely onset of this event remain ill-defined. Here, we show that a regulatory feedback circuit controlling the Drosophila neuropeptide Prothoracicotropic hormone (PTTH) triggers maturation onset. We identify the Ecdysone Receptor (EcR) in the PTTH-expressing neurons (PTTHn) as a regulator of developmental maturation onset. Loss of EcR in these PTTHn impairs PTTH signaling, which delays maturation. We find that the steroid ecdysone dose-dependently affects Ptth transcription, promoting its expression at lower concentrations and inhibiting it at higher concentrations. Our findings indicate the existence of a feedback circuit in which rising ecdysone levels trigger, via EcR activity in the PTTHn, the PTTH surge that generates the maturation-inducing ecdysone peak toward the end of larval development. Because steroid feedback is also known to control the vertebrate maturation-inducing hypothalamic-pituitary-gonadal axis, our findings suggest an overall conservation of the feedback-regulatory neuroendocrine circuitry that controls the timing of maturation initiation.

Egfr Signaling Is a Major Regulator of Ecdysone Biosynthesis in the Drosophila Prothoracic Gland.

Understanding the mechanisms that determine final body size of animals is a central question in biology. In animals with determinate growth, such as mammals or insects, the size at which the immature organism transforms into the adult defines the final body size, as adult individuals do not grow [1]. In Drosophila, the growth period ends when the immature larva undergoes the metamorphic transition to develop the mature adult [2]. This metamorphic transition is triggered by a sharp increase of the steroid ecdysone, synthetized in the prothoracic gland (PG), that occurs at the end of the third instar larvae (L3) [3-6]. It is widely accepted that ecdysone biosynthesis in Drosophila is mainly induced by the activation of tyrosine kinase (RTK) Torso by the prothoracicotropic hormone (Ptth) produced into two pairs of neurosecretory cells that project their axons onto the PG [7, 8]. However, the fact that neither Ptth nor torso-null mutant animals arrest larval development but only present a delay in the larva-pupa transition [9-11] mandates for a reconsideration of the conventional model. Here, we show that Egfr signaling, rather than Ptth/torso, is the major contributor of ecdysone biosynthesis in Drosophila. We found that Egfr signaling is activated in the PG in an autocrine mode by the EGF ligands spitz and vein, which in turn are regulated by the levels of ecdysone. This regulatory positive feedback loop ensures the production of ecdysone to trigger metamorphosis by a progressive Egfr-dependent activation of MAPK/ERK pathway, thus determining the animal final body size.

Mating induces switch from hormone-dependent to hormone-independent steroid receptor-mediated growth in Drosophila secondary cells.

Male reproductive glands like the mammalian prostate and the paired Drosophila melanogaster accessory glands secrete seminal fluid components that enhance fecundity. In humans, the prostate, stimulated by environmentally regulated endocrine and local androgens, grows throughout adult life. We previously showed that in fly accessory glands, secondary cells (SCs) and their nuclei also grow in adults, a process enhanced by mating and controlled by bone morphogenetic protein (BMP) signalling. Here, we demonstrate that BMP-mediated SC growth is dependent on the receptor for the developmental steroid ecdysone, whose concentration is reported to reflect sociosexual experience in adults. BMP signalling appears to regulate ecdysone receptor (EcR) levels via one or more mechanisms involving the EcR’s N terminus or the RNA sequence that encodes it. Nuclear growth in virgin males is dependent on ecdysone, some of which is synthesised in SCs. However, mating induces additional BMP-mediated nuclear growth via a cell type–specific form of hormone-independent EcR signalling, which drives genome endoreplication in a subset of adult SCs. Switching to hormone-independent endoreplication after mating allows growth and secretion to be hyperactivated independently of ecdysone levels in SCs, permitting more rapid replenishment of the accessory gland luminal contents. Our data suggest mechanistic parallels between this physiological, behaviour-induced signalling switch and altered pathological signalling associated with prostate cancer progression.

Activated Ras/JNK driven Dilp8 in imaginal discs adversely affects organismal homeostasis during early pupal stage in Drosophila, a new checkpoint for development.

Dilp8-mediated inhibition of ecdysone synthesis and pupation in holometabolous insects maintains developmental homeostasis through stringent control of timing and strength of molting signals. We examined reasons for normal pupation but early pupal death observed in certain cases. Overexpression of activated Ras in developing eye/wing discs inhibited Ptth expression in brain via upregulated JNK signaling mediated Dilp8 secretion from imaginal discs, which inhibited ecdysone synthesis in prothoracic gland after pupariation, leading to death of ~25- to 30-hour-old pupae. Inhibition of elevated Ras signaling completely rescued early pupal death while post-pupation administration of ecdysone to organisms with elevated Ras signaling in eye discs partially rescued their early pupal death. Unlike the earlier known Dilp8 action in delaying pupation, hyperactivated Ras mediated elevation of pJNK signaling in imaginal discs caused Dilp8 secretion after pupariation. Ectopic expression of certain other transgene causing pupal lethality similarly enhanced pJNK and early pupal Dilp8 levels. Suboptimal ecdysone levels after 8 hours of pupation prevented the early pupal metamorphic changes and caused organismal death. Our results reveal early pupal stage as a novel Dilp8 mediated post-pupariation checkpoint and provide further evidence for interorgan signaling during development, wherein a peripheral tissue influences the CNS driven endocrine function.

EGFR Signaling is the Major Regulator of Ecdysone Biosynthesis in the Drosophila Prothoracic Gland

Understanding the mechanisms that determine the final body size of animals is a central question in biology. In insects, the level of the steroid hormone ecdysone dictates the end of the growth period by triggering metamorphosis. This process is thought to be induced by the activation of Ptth/torso signaling in the prothoracic gland (PG). Here we show that Egfr signaling, rather than Ptth/torso, is the major contributor of ecdysone biosynthesis in Drosophila. Egfr signaling is activated in the PG in an autocrine mode by the EGF ligands spitz and vein, which in turn are regulated by the level of ecdysone. This regulatory positive feedback loop ensures the production of ecdysone to trigger metamorphosis by a progressive Egfr-dependent activation of the MAPK/ERK pathway, thus determining the animal final body size.

Functional analysis of Aarf domain-containing kinase 1 in Drosophila melanogaster.

The ADCK proteins are predicted mitochondrial kinases. Most studies of these proteins have focused on the Abc1/Coq8 subfamily, which contributes to Coenzyme Q biosynthesis. In contrast, little is known about ADCK1 despite its evolutionary conservation in yeast, Drosophila, Caenorhabditis elegans and mammals. We show that Drosophila ADCK1 mutants die as second instar larvae with double mouth hooks and tracheal breaks. Tissue-specific genetic rescue and RNAi studies show that ADCK1 is necessary and sufficient in the trachea for larval viability. In addition, tracheal-rescued ADCK1 mutant adults have reduced lifespan, are developmentally delayed, have reduced body size, and normal levels of basic metabolites. The larval lethality and double mouth hooks seen in ADCK1 mutants are often associated with reduced levels of the steroid hormone ecdysone, suggesting that this gene could contribute to controlling ecdysone levels or bioavailability. Similarly, the tracheal defects in these animals could arise from defects in intracellular lipid trafficking. These studies of ADCK1 provide a new context to define the physiological functions of this poorly understood member of the ADCK family of predicted mitochondrial proteins.