Prothoracic Glands Research Articles

A cell surface protein controls endocrine ring gland morphogenesis and steroid production

Identification of signals for systemic adaption of hormonal regulation would help to understand the crosstalk between cells and environmental cues contributing to growth, metabolic homeostasis and development. Physiological states are controlled by precise pulsatile hormonal release, including endocrine steroids in human and ecdysteroids in insects. We show in Drosophila that regulation of genes that control biosynthesis and signaling of the steroid hormone ecdysone, a central regulator of developmental progress, depends on the extracellular matrix protein Obstructor-A (Obst-A). Ecdysone is produced by the prothoracic gland (PG), where sensory neurons projecting axons from the brain integrate stimuli for endocrine control. By defining the extracellular surface, Obst-A promotes morphogenesis and axonal growth in the PG. This process requires Obst-A-matrix reorganization by Clathrin/Wurst-mediated endocytosis. Our data identifies the extracellular matrix as essential for endocrine ring gland function, which coordinates physiology, axon morphogenesis, and developmental programs. As Obst-A and Wurst homologs are found among all arthropods, we propose that this mechanism is evolutionary conserved.

The Drosophila CCR4-NOT complex is required for cholesterol homeostasis and steroid hormone synthesis

CCR4-NOT is a highly conserved protein complex that regulates gene expression at multiple levels. In yeast, CCR4-NOT functions in transcriptional initiation, heterochromatin formation, mRNA deadenylation and other processes. The range of functions for Drosophila CCR4-NOT is less clear, except for a well-established role as a deadenylase for maternal mRNAs during early embryogenesis. We report here that CCR4-NOT has an essential function in the Drosophila prothoracic gland (PG), a tissue that predominantly produces the steroid hormone ecdysone. Interfering with the expression of the CCR4-NOT components twin, Pop2, Not1, and Not3 in a PG-specific manner resulted in larval arrest and a failure to initiate metamorphosis. Transcriptome analysis of PG-specific Pop2-RNAi samples revealed that Pop2 is required for the normal expression of ecdysone biosynthetic gene spookier (spok) as well as cholesterol homeostasis genes of the NPC2 family. Interestingly, dietary supplementation with ecdysone and its various sterol precursors showed that 7-dehydrocholesterol and cholesterol completely rescued the larval arrest phenotype, allowing Pop2-RNAi animals to reach pupal stage, and, to a low degree, even survival to adulthood, while the biologically active hormone, 20-Hydroxyecdysone (20E), was significantly less effective. Also, we present genetic evidence that CCR4-NOT has a nuclear function where CCR4-NOT-depleted cells exhibit aberrant chromatin and nucleoli structures. In summary, our findings indicate that the Drosophila CCR4-NOT complex has essential roles in the PG, where it is required for Drosophila steroid hormone production and cholesterol homeostasis, and likely has functions beyond a mere mRNA deadenylase in Drosophila.

Refining a steroidogenic model: an analysis of RNA-seq datasets from insect prothoracic glands

BackgroundThe prothoracic gland (PG), the principal steroidogenic organ of insects, has been proposed as a model for steroid hormone biosynthesis and regulation.ResultsTo validate the robustness of the model, we present an analysis of accumulated transcriptomic data from PGs of two model species, Drosophila melanogaster and Bombyx mori. We identify that the common core components of the model in both species are encoded by nine genes. Five of these are Halloween genes whose expression differs substantially between the PGs of these species.ConclusionsWe conclude that the PGs can be a model for steroid hormone synthesis and regulation within the context of mitochondrial cholesterol transport and steroid biosynthesis but beyond these core mechanisms, gene expression in insect PGs is too diverse to fit in a context-specific model and should be analysed within a species-specific framework.

Defects in the Neuroendocrine Axis Contribute to Global Development Delay in a Drosophila Model of NGLY1 Deficiency.

N-glycanase 1 (NGLY1) Deficiency is a rare monogenic multi-system disorder first described in 2014. NGLY1 is evolutionarily conserved in model organisms. Here we conducted a natural history study and chemical-modifier screen on the Drosophila melanogaster NGLY1 homolog, Pngl. We generated a new fly model of NGLY1 Deficiency, engineered with a nonsense mutation in Pngl at codon 420 that results in a truncation of the C-terminal carbohydrate-binding PAW domain. Homozygous mutant animals exhibit global development delay, pupal lethality and small body size as adults. We developed a 96-well-plate, image-based, quantitative assay of Drosophila larval size for use in a screen of the 2,650-member Microsource Spectrum compound library of FDA approved drugs, bioactive tool compounds, and natural products. We found that the cholesterol-derived ecdysteroid molting hormone 20-hydroxyecdysone (20E) partially rescued the global developmental delay in mutant homozygotes. Targeted expression of a human NGLY1 transgene to tissues involved in ecdysteroidogenesis, e.g., prothoracic gland, also partially rescues global developmental delay in mutant homozygotes. Finally, the proteasome inhibitor bortezomib is a potent enhancer of global developmental delay in our fly model, evidence of a defective proteasome “bounce-back” response that is also observed in nematode and cellular models of NGLY1 Deficiency. Together, these results demonstrate the therapeutic relevance of a new fly model of NGLY1 Deficiency for drug discovery and gene modifier screens.

Dpp regulates autophagy-dependent midgut removal and signals to block ecdysone production

Animal development and homeostasis require the programmed removal of cells. Autophagy-dependent cell deletion is a unique form of cell death often involved in bulk degradation of tissues. In Drosophila the steroid hormone ecdysone controls developmental transitions and triggers the autophagy-dependent removal of the obsolete larval midgut. The production of ecdysone is exquisitely coordinated with signals from numerous organ systems to mediate the correct timing of such developmental programs. Here we report an unexpected role for the Drosophila bone morphogenetic protein/transforming growth factor β ligand, Decapentaplegic (Dpp), in the regulation of ecdysone-mediated midgut degradation. We show that blocking Dpp signaling induces premature autophagy, rapid cell death, and midgut degradation, whereas sustained Dpp signaling inhibits autophagy induction. Furthermore, Dpp signaling in the midgut prevents the expression of ecdysone responsive genes and impairs ecdysone production in the prothoracic gland. We propose that Dpp has dual roles: one within the midgut to prevent improper tissue degradation, and one in interorgan communication to coordinate ecdysone biosynthesis and developmental timing.

Calcium and cAMP directly modulate the speed of the Drosophila circadian clock

Circadian clocks impose daily periodicities to animal behavior and physiology. At their core, circadian rhythms are produced by intracellular transcriptional/translational feedback loops (TTFL). TTFLs may be altered by extracellular signals whose actions are mediated intracellularly by calcium and cAMP. In mammals these messengers act directly on TTFLs via the calcium/cAMP-dependent transcription factor, CREB. In the fruit fly, Drosophila melanogaster, calcium and cAMP also regulate the periodicity of circadian locomotor activity rhythmicity, but whether this is due to direct actions on the TTFLs themselves or are a consequence of changes induced to the complex interrelationship between different classes of central pacemaker neurons is unclear. Here we investigated this question focusing on the peripheral clock housed in the non-neuronal prothoracic gland (PG), which, together with the central pacemaker in the brain, controls the timing of adult emergence. We show that genetic manipulations that increased and decreased the levels of calcium and cAMP in the PG caused, respectively, a shortening and a lengthening of the periodicity of emergence. Importantly, knockdown of CREB in the PG caused an arrhythmic pattern of eclosion. Interestingly, the same manipulations directed at central pacemaker neurons caused arrhythmicity of eclosion and of adult locomotor activity, suggesting a common mechanism. Our results reveal that the calcium and cAMP pathways can alter the functioning of the clock itself. In the PG, these messengers, acting as outputs of the clock or as second messengers for stimuli external to the PG, could also contribute to the circadian gating of adult emergence.

Mechanisms for Oxygen‐Mediation of Body Size

Hypoxic‐rearing reduces the adult body size of animals, but the mechanisms responsible remain poorly understood. Holometabolous insects are important models for understanding the regulation of body size. Here we focus on how hypoxic rearing reduces final body size of Drosophila melanogaster. Larval D. melanogaster approximately triple their body mass during the last larval instar. We tested the hypothesis that functional hypoxia develops over time within the instar. Later instar animals had higher whole‐body copy numbers of HIFalpha and HIFbeta RNA and greater quantities of HIFalpha protein (Westerns), providing clear evidence that HIF signaling emerges late in the instar. Later‐instar animals also had greater expression of lactate dehydrogenase genes, and higher capacities for production of lactate, though not higher levels of whole‐body lactate, suggesting that functional hypoxia is not so extreme as to trigger anaerobic metabolism. However, CO2 emission rates increased in response to hyperoxia in late but not early‐instar larvae, providing strong evidence of oxygen limitation of metabolism. Hyperoxia reduced, and hypoxia elevated the RNA copy number of prolyl hydroxylase (whole body), a downstream target of HIF‐signaling. In sum, late‐instar normoxic‐reared larvae experience functional hypoxia, and whole‐body HIF‐signaling is increased with hypoxic‐rearing. Next, to understand the link between functional hypoxia and developmental transitions, we tested whether HIF signaling in the prothoracic glands (which produce ecdysone, aka molting hormone) affects development rate or adult size, using the GAL4‐UAS system to reduce or increase HIF signaling in this endocrine gland. Knockdown of HIF signaling in the prothoracic glands strongly delayed pupariation in 10% but not 21% oxygen atmospheres. Thus HIF‐signaling in the prothoracic gland appears to be an important factor in triggering Drosophila pupariation at a smaller body size in hypoxia, but we found no evidence that this is a key pathway for of the control of molting and size in normoxia.Support or Funding InformationSupported by NSF IOS 1256745.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Torso-Like Is a Component of the Hemolymph and Regulates the Insulin Signaling Pathway in Drosophila

In Drosophila, key developmental transitions are governed by the steroid hormone ecdysone. A number of neuropeptide-activated signaling pathways control ecdysone production in response to environmental signals, including the insulin signaling pathway, which regulates ecdysone production in response to nutrition. Here, we find that the Membrane Attack Complex/Perforin-like protein Torso-like, best characterized for its role in activating the Torso receptor tyrosine kinase in early embryo patterning, also regulates the insulin signaling pathway in Drosophila We previously reported that the small body size and developmental delay phenotypes of torso-like null mutants resemble those observed when insulin signaling is reduced. Here we report that, in addition to growth defects, torso-like mutants also display metabolic and nutritional plasticity phenotypes characteristic of mutants with impaired insulin signaling. We further find that in the absence of torso-like, the expression of insulin-like peptides is increased, as is their accumulation in insulin-producing cells. Finally, we show that Torso-like is a component of the hemolymph and that it is required in the prothoracic gland to control developmental timing and body size. Taken together, our data suggest that the secretion of Torso-like from the prothoracic gland influences the activity of insulin signaling throughout the body in Drosophila.

Krüppel homolog 1 represses insect ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes

In insects, juvenile hormone (JH) and the steroid hormone ecdysone have opposing effects on regulation of the larval-pupal transition. Although increasing evidence suggests that JH represses ecdysone biosynthesis during larval development, the mechanism underlying this repression is not well understood. Here, we demonstrate that the expression of the Krüppel homolog 1 (Kr-h1), a gene encoding a transcription factor that mediates JH signaling, in ecdysone-producing organ prothoracic gland (PG) represses ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes in both Drosophila and Bombyx Application of a JH mimic on ex vivo cultured PGs from Drosophila and Bombyx larvae induces Kr-h1 expression and inhibits the transcription of steroidogenic enzymes. In addition, PG-specific knockdown of Drosophila Kr-h1 promotes-while overexpression hampers-ecdysone production and pupariation. We further find that Kr-h1 inhibits the transcription of steroidogenic enzymes by directly binding to their promoters to induce promoter DNA methylation. Finally, we show that Kr-h1 does not affect DNA replication in Drosophila PG cells and that the reduction of PG size mediated by Kr-h1 overexpression can be rescued by feeding ecdysone. Taken together, our data indicate direct and conserved Kr-h1 repression of insect ecdysone biosynthesis in response to JH stimulation, providing insights into mechanisms underlying the antagonistic roles of JH and ecdysone.

A polydnavirus-encoded ANK protein has a negative impact on steroidogenesis and development

Polydnaviruses (PDV) are viral symbionts associated with ichneumonid and braconid wasps parasitizing moth larvae, which are able to disrupt the host immune response and development, as well as a number of other physiological pathways. The immunosuppressive role of PDV has been more intensely investigated, while very little is known about the PDV-encoded factors disrupting host development. Here we address this research issue by further expanding the functional analysis of ankyrin genes encoded by the bracovirus associated with Toxoneuron nigriceps (Hymenoptera, Braconidae). In a previous study, using Drosophila melanogaster as experimental model system, we demonstrated the negative impact of TnBVank1 impairing the ecdysone biosynthesis by altering endocytic traffic in prothoracic gland cells. With a similar approach here we demonstrate that another member of the viral ank gene family, TnBVank3, does also contribute to the disruption of ecdysone biosynthesis, but with a completely different mechanism. We show that its expression in Drosophila prothoracic gland (PG) blocks the larval-pupal transition by impairing the expression of steroidogenic genes. Furthermore, we found that TnBVank3 affects the expression of genes involved in the insulin/TOR signaling and the constitutive activation of the insulin pathway in the PG rescues the pupariation impairment. Collectively, our data demonstrate that TnBVANK3 acts as a virulence factor by exerting a synergistic and non-overlapping function with TnBVANK1 to disrupt the ecdysone biosynthesis.

Genome-Wide Screen for New Components of the Drosophila melanogaster Torso Receptor Tyrosine Kinase Pathway.

Patterning of the Drosophila embryonic termini by the Torso (Tor) receptor pathway has long served as a valuable paradigm for understanding how receptor tyrosine kinase signaling is controlled. However, the mechanisms that underpin the control of Tor signaling remain to be fully understood. In particular, it is unclear how the Perforin-like protein Torso-like (Tsl) localizes Tor activity to the embryonic termini. To shed light on this, together with other aspects of Tor pathway function, we conducted a genome-wide screen to identify new pathway components that operate downstream of Tsl. Using a set of molecularly defined chromosomal deficiencies, we screened for suppressors of ligand-dependent Tor signaling induced by unrestricted Tsl expression. This approach yielded 59 genomic suppressor regions, 11 of which we mapped to the causative gene, and a further 29 that were mapped to <15 genes. Of the identified genes, six represent previously unknown regulators of embryonic Tor signaling. These include twins (tws), which encodes an integral subunit of the protein phosphatase 2A complex, and α-tubulin at 84B (αTub84B), a major constituent of the microtubule network, suggesting that these may play an important part in terminal patterning. Together, these data comprise a valuable resource for the discovery of new Tor pathway components. Many of these may also be required for other roles of Tor in development, such as in the larval prothoracic gland where Tor signaling controls the initiation of metamorphosis.

Ecdysteroidogenesis and development in Heliothis virescens (Lepidoptera: Noctuidae): Focus on PTTH-stimulated pathways

Post-embryonic development and molting in insects are regulated by endocrine changes, including prothoracicotropic hormone (PTTH)-stimulated ecdysone secretion by the prothoracic glands (PGs). In Lepidoptera, two pathways are potentially involved in PTTH-stimulated ecdysteroidogenesis, mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/protein kinase B/target of rapamycin (PI3K/Akt/TOR). We investigated the potential roles of both these pathways in Heliothis virescens ecdysteroidogenesis. We identified putative proteins belonging to MAPK and PI3K/Akt/TOR signaling cascades, using transcriptomic analyses of PGs from last (fifth) instar larvae. Using western blots, we measured the phosphorylation of 4E-BP and S6K proteins, the main targets of TOR, following the in vitro exposure of PGs to brain extract containing PTTH (hereafter referred to as PTTH) and/or the inhibitors of MAPK (U0126), PI3K (LY294002) or TOR (rapamycin). Next, we measured ecdysone production, under the same experimental conditions, by enzyme immunoassay (EIA). We found that in Heliothis virescens last instar larvae, both pathways modulated PTTH-stimulated ecdysteroidogenesis. Finally, we analyzed the post-embryonic development of third and fourth instar larvae fed on diet supplemented with rapamycin, in order to better understand the role of the TOR pathway in larval growth. When rapamycin was added to the diet of larvae, the onset of molting was delayed, the growth rate was reduced and abnormally small larvae/pupae with high mortality rates resulted. In larvae fed on diet supplemented with rapamycin, the growth of PGs was suppressed, and ecdysone production and secretion were inhibited. Overall, the in vivo and in vitro results demonstrated that, similarly to Bombyx mori, MAPK and PI3K/Akt/TOR pathways are involved in PTTH signaling-stimulated ecdysteroidogenesis, and indicated the important role of TOR protein in H. virescens systemic growth.

Stimulation of JNK Phosphorylation by the PTTH in Prothoracic Glands of the Silkworm, Bombyx mori.

In this study, phosphorylation of c-Jun N-terminal kinase (JNK) by the prothoracicotropic hormone (PTTH) was investigated in prothoracic glands (PGs) of the silkworm, Bombyx mori. Results showed that JNK phosphorylation was stimulated by the PTTH in time- and dose-dependent manners. In vitro activation of JNK phosphorylation in PGs by the PTTH was also confirmed in an in vivo experiment, in which a PTTH injection greatly increased JNK phosphorylation in PGs of day-6 last instar larvae. JNK phosphorylation caused by PTTH stimulation was greatly inhibited by U73122, a potent and specific inhibitor of phospholipase C (PLC) and an increase in JNK phosphorylation was also detected when PGs were treated with agents (either A23187 or thapsigargin) that directly elevated the intracellular Ca2+ concentration, thereby indicating involvement of PLC and Ca2+. Pretreatment with an inhibitor (U0126) of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) and an inhibitor (LY294002) of phosphoinositide 3-kinase (PI3K) failed to significantly inhibit PTTH-stimulated JNK phosphorylation, indicating that ERK and PI3K were not related to JNK. We further investigated the effect of modulation of the redox state on JNK phosphorylation. In the presence of either an antioxidant (N-acetylcysteine, NAC) or diphenylene iodonium (DPI), PTTH-stimulated JNK phosphorylation was blocked. The JNK kinase inhibitor, SP600125, markedly inhibited PTTH-stimulated JNK phosphorylation and ecdysteroid synthesis. The kinase assay of JNK in PGs confirmed its stimulation by PTTH and inhibition by SP600125. Moreover, PTTH treatment did not affect JNK or Jun mRNA expressions. Based on these findings, we concluded that PTTH stimulates JNK phosphorylation in Ca2+- and PLC-dependent manners and that the redox-regulated JNK signaling pathway is involved in PTTH-stimulated ecdysteroid synthesis in B. mori PGs.

Cholesterol internalization and metabolism in insect prothoracic gland, a steroidogenic organ, via lipoproteins

Dietary sterols including cholesterol and phytosterols are essential substrates for insect steroid hormone (ecdysteroid) synthesis in the prothoracic glands (PGs). In the silkworm Bombyx mori, one of the model species of insects, the steroidogenesis has been well demonstrated that cholesterol biotransformation into ecdysone in the PG cells. Because insects lack the ability to synthesize cellular sterol de novo, lipoprotein, lipophorin (Lp), has been thought to be the major cholesterol supply source; however, details of cholesterol behavior from Lp to the PG cells has not been analyzed till date. In this report, we developed Lp incorporation method using labeled cholesterols such as 22-NBD-cholesterol and cholesterol-25,26,26,26,27,27,27-d7 (cholesterol-d7), and analyzed the internalization and metabolism of cholesterol in PGs in vitro using the silkworm Bombyx mori. The internalization of cholesterol was visualized using 22-NBD-cholesterol. PGs showed an enriched cellular 22-NBD-cholesterol signal, which dissociated from the Lp localizing at the close area of cell membrane. The distribution pattern observed in the PGs was different from other tissues such as the brain, fat body, and Malpighian tubules, suggesting that the internalization of cholesterol in the PGs was distinct from other tissues. The metabolism of cholesterol was traced using LC-MS/MS methods to detect cholesterol-d7, 7-dehydrocholesterol-d7 (an expected intermediate metabolite), and the final product ecdysone-d6. 7-Dehydrocholesterol-d7 and ecdysone-d6 were detected in the PG culture incubated with labeled Lp, showing that the cholesterol of Lp was utilized for ecdysone synthesis in the PGs. Our results reveal the distinct behavior of cholesterol in the PGs, with the first direct evidence of biochemical fate of lipoprotein cholesterol in insect steroidogenic organ. This will aid in the understanding of the involvement of lipoprotein cholesterol in steroid hormone synthesis in insects.

Cooperative Control of Ecdysone Biosynthesis in Drosophila by Transcription Factors Séance, Ouija Board, and Molting Defective.

Ecdysteroids are steroid hormones that control many aspects of development and physiology. During larval development, ecdysone is synthesized in an endocrine organ called the prothoracic gland through a series of ecdysteroidogenic enzymes encoded by the Halloween genes. The expression of the Halloween genes is highly restricted and dynamic, indicating that their spatiotemporal regulation is mediated by their tight transcriptional control. In this study, we report that three zinc finger-associated domain (ZAD)-C2H2 zinc finger transcription factors—Séance (Séan), Ouija board (Ouib), and Molting defective (Mld)—cooperatively control ecdysone biosynthesis in the fruit fly Drosophila melanogaster. Séan and Ouib act in cooperation with Mld to positively regulate the transcription of neverland and spookier, respectively, two Halloween genes. Remarkably, loss-of-function mutations in séan, ouib, or mld can be rescued by the expression of neverland, spookier, or both, respectively. These results suggest that the three transcription factors have distinct roles in coordinating the expression of just two genes in Drosophila. Given that neverland and spookier are located in constitutive heterochromatin, Séan, Ouib, and Mld represent the first example of a transcription factor subset that regulates genes located in constitutive heterochromatin.

Ecdysone oxidase and 3-dehydroecdysone-3β-reductase contribute to the synthesis of ecdysone during early embryonic development of the silkworm.

Maternal ecdysteroids regulate a variety of cellular processes during early embryonic development of insects, yet little is known about the genes involved in the biosynthesis of these hormones. In this study, we found that ecdysone oxidase (EO) gene, which encodes an enzyme to catalyze ecdysone (or 20-hydroxyecdysone, 20E) to 3-dehydroecdysone (3DE), was highly expressed in the mature ovaries of the domestic silkworm, Bombyx mori. B. mori EO (BmEO) was localized in the cytoplasm around the yolk granules of oocyte. Furthermore, the down-regulated expression of the BmEO gene using RNA interference could not affect normal development of the female silkworm, but lower the 20E titer and hatching rate of its offspring. Rescue experiments by injecting the product (3DE) of BmEO can significantly elevate the 20E level and hatching rate of the BmEO RNAi offspring. Meanwhile, during embryonic stage, the down-regulating expression of 3DE-3β-reductase, which can reduce 3DE into ecdysone, also lowered the 20E titer. Taken together, our results prove that 3DE can be synthesized from ecdysone in maternal ovary yolk granules, and then the maternal 3DE is converted into active ecdysone during the early embryonic development of offspring. Thus, our findings reveal a new pathway to explain the origin of high 20E level before the formation the prothoracic gland in the silkworm.

Antagonistic actions of juvenile hormone and 20-hydroxyecdysone within the ring gland determine developmental transitions in Drosophila

In both vertebrates and insects, developmental transition from the juvenile stage to adulthood is regulated by steroid hormones. In insects, the steroid hormone, 20-hydroxyecdysone (20E), elicits metamorphosis, thus promoting this transition, while the sesquiterpenoid juvenile hormone (JH) antagonizes 20E signaling to prevent precocious metamorphosis during the larval stages. However, not much is known about the mechanisms involved in cross-talk between these two hormones. In this study, we discovered that in the ring gland (RG) of Drosophila larvae, JH and 20E control each other's biosynthesis. JH induces expression of a Krüppel-like transcription factor gene Kr-h1 in the prothoracic gland (PG), a portion of the RG that produces the 20E precursor ecdysone. By reducing both steroidogenesis autoregulation and PG size, high levels of Kr-h1 in the PG inhibit ecdysteriod biosynthesis, thus maintaining juvenile status. JH biosynthesis is prevented by 20E in the corpus allatum, the other portion of the RG that produces JH, to ensure the occurrence of metamorphosis. Hence, antagonistic actions of JH and 20E within the RG determine developmental transitions in Drosophila Our study proposes a mechanism of cross-talk between the two major hormones in the regulation of insect metamorphosis.

Dual Roles of Glutathione in Ecdysone Biosynthesis and Antioxidant Function During Larval Development in Drosophila.

Ecdysteroids, including the biologically active hormone 20-hydroxyecdysone (20E), play essential roles in controlling many developmental and physiological events in insects. Ecdysteroid biosynthesis is achieved by a series of specialized enzymes encoded by the Halloween genes. Recently, a new class of Halloween gene, noppera-bo (nobo), encoding a glutathione S-transferase (GST) in dipteran and lepidopteran species, has been identified and characterized. GSTs are well known to conjugate substrates with the reduced form of glutathione (GSH), a bioactive tripeptide composed of glutamate, cysteine, and glycine. We hypothesized that GSH itself is required for ecdysteroid biosynthesis. However, the role of GSH in steroid hormone biosynthesis has not been examined in any organisms. Here, we report phenotypic analysis of a complete loss-of-function mutant in the γ-glutamylcysteine synthetase catalytic subunit (Gclc) gene in the fruit fly Drosophila melanogaster. Gclc encodes the evolutionarily conserved catalytic component of the enzyme that conjugates glutamate and cysteine in the GSH biosynthesis pathway. Complete Gclc loss-of-function leads to drastic GSH deficiency in the larval body fluid. Gclc mutant animals show a larval-arrest phenotype. Ecdysteroid titer in Gclc mutant larvae decreases, and the larval-arrest phenotype is rescued by oral administration of 20E or cholesterol. Moreover, Gclc mutant animals exhibit abnormal lipid deposition in the prothoracic gland, a steroidogenic organ during larval development. All of these phenotypes are reminiscent to nobo loss-of-function animals. On the other hand, Gclc mutant larvae also exhibit a significant reduction in antioxidant capacity. Consistent with this phenotype, Gclc mutant larvae are more sensitive to oxidative stress response as compared to wild-type. Nevertheless, the ecdysteroid biosynthesis defect in Gclc mutant animals is not associated with loss of antioxidant function. Our data raise the unexpected hypothesis that a primary role of GSH in early D. melanogaster larval development is ecdysteroid biosynthesis, independent from the antioxidant role of GSH.

Leptinotarsa hormone receptor 4 (HR4) tunes ecdysteroidogenesis and mediates 20-hydroxyecdysone signaling during larval-pupal metamorphosis

Hormone receptor 4 (HR4) is involved in the regulation of 20-hydroxyecdysone (20E) biosynthesis and the mediation of 20E signaling during larval-pupal transition in a holometabolan Drosophila melanogaster, whereas it acts as a repressor in 20E-responsive transcriptional cascade in a hemimetabolan, Blattella germanica. Here we characterized two HR4 splicing variants, LdHR4X1 and LdHR4X2, in a coleopteran Leptinotarsa decemlineata. LdHR4X1 was highly expressed in the prothoracic gland and epidermis while LdHR4X2 was abundantly transcribed in the nervous system. In vivo results showed that both prothoracicotropic hormone and 20E pathways transcriptionally regulated LdHR4, in an isoform-dependent pattern. RNA interference of LdHR4 at the final (fourth) larval instar, in contrast to the second- and third-instar periods, enhanced the expression of two ecdysteroidogenesis genes, increased 20E titer, upregulated transcription of five 20E-response genes, and reduced the mRNA level of Fushi tarazu-factor 1 (FTZ-F1). As a result, the fourth-instar LdHR4 RNAi larvae exhibited accelerated development and reduced body weight. Moreover, knockdown of LdHR4 at the fourth instar resulted in larval lethality and impaired pupation. Feeding of pyriproxyfen (a mimic of juvenile hormone) or silencing of a juvenile hormone degrading enzyme gene restored the normal course of ecdysteroidogenesis, duration of larval development, and body weight in fourth-instar LdHR4 RNAi larvae. The treatment partially suppressed the larval mortality but not the failure to pupate. The dual role of HR4 during larval-pupal metamorphosis appears to be evolutionarily conserved among holometabolans.

Functional morphology and neuronal innervation of the prothoracic defence gland in Timema

AbstractTimema is the most basal genus of Phasmatodea and the sister group to the remaining stick and leaf‐insects (Euphasmatodea). An autapomorphy of all phasmids is the paired prothoracic exocrine defence glands. In this study, the anatomy and innervation of the defence glands in Timema petita and Timema chumash are described and compared with the data on Euphasmatodea. In all phasmids, the glands consist of a cuticular epithelium, a secretory epithelium and muscular fibres that compress the lumen. In Timematodea, the muscular part of the gland is less developed than in Euphasmatodea and the ejection of the defence secretion depends on the dorsal longitudinal neck muscles. On the neuroanatomical level, Timema petita and Timema chumash lack neurons that are involved in the independent contraction of the gland in euphasmids. In both studied species of Timema, neck muscles play an active role in the gland function which is not observed in any other phasmid. Considering the basal position of this genus, this supports the hypothesis that in euphasmids, the muscular part of the gland evolved from the dorsal longitudinal neck muscles. Additionally, the same nerves that innervate the dorsal longitudinal neck muscles in all Polyneoptera also innervate the defence glands in phasmids.