Female reproductive dormancy in Drosophila is regulated by DH31-producing neurons projecting into the corpus allatum.

Female insects can enter reproductive diapause, a state of suspended egg development, to conserve energy under adverse environments. In many insects, including the fruit fly, Drosophila melanogaster, reproductive diapause, also frequently called reproductive dormancy, is induced under low-temperature and short-day conditions by the downregulation of juvenile hormone (JH) biosynthesis in the corpus allatum (CA). In this study, we demonstrate that neuropeptide Diuretic hormone 31 (DH31) produced by brain neurons that project into the CA plays an essential role in regulating reproductive dormancy by suppressing JH biosynthesis in adult D. melanogaster. The CA expresses the gene encoding the DH31 receptor, which is required for DH31-triggered elevation of intracellular cAMP in the CA. Knocking down Dh31 in these CA-projecting neurons or DH31 receptor in the CA suppresses the decrease of JH titer, normally observed under dormancy-inducing conditions, leading to abnormal yolk accumulation in the ovaries. Our findings provide the first molecular genetic evidence demonstrating that CA-projecting peptidergic neurons play an essential role in regulating reproductive dormancy by suppressing JH biosynthesis.

Functional impact of subunit composition and compensation on Drosophila melanogaster nicotinic receptors-targets of neonicotinoids.

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs) and their adverse effects on non-target insects are of serious concern. We recently found that cofactor TMX3 enables robust functional expression of insect nAChRs in Xenopus laevis oocytes and showed that neonicotinoids (imidacloprid, thiacloprid, and clothianidin) exhibited agonist actions on some nAChRs of the fruit fly (Drosophila melanogaster), honeybee (Apis mellifera) and bumblebee (Bombus terrestris) with more potent actions on the pollinator nAChRs. However, other subunits from the nAChR family remain to be explored. We show that the Dα3 subunit co-exists with Dα1, Dα2, Dß1, and Dß2 subunits in the same neurons of adult D. melanogaster, thereby expanding the possible nAChR subtypes in these cells alone from 4 to 12. The presence of Dα1 and Dα2 subunits reduced the affinity of imidacloprid, thiacloprid, and clothianidin for nAChRs expressed in Xenopus laevis oocytes, whereas the Dα3 subunit enhanced it. RNAi targeting Dα1, Dα2 or Dα3 in adults reduced expression of targeted subunits but commonly enhanced Dß3 expression. Also, Dα1 RNAi enhanced Dα7 expression, Dα2 RNAi reduced Dα1, Dα6, and Dα7 expression and Dα3 RNAi reduced Dα1 expression while enhancing Dα2 expression, respectively. In most cases, RNAi treatment of either Dα1 or Dα2 reduced neonicotinoid toxicity in larvae, but Dα2 RNAi enhanced neonicotinoid sensitivity in adults reflecting the affinity-reducing effect of Dα2. Substituting each of Dα1, Dα2, and Dα3 subunits by Dα4 or Dß3 subunit mostly increased neonicotinoid affinity and reduced efficacy. These results are important because they indicate that neonicotinoid actions involve the integrated activity of multiple nAChR subunit combinations and counsel caution in interpreting neonicotinoid actions simply in terms of toxicity.

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

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

Endocrine Regulation of Aging in the Fruit Fly Drosophila melanogaster.

The past few decades have witnessed increasing research clarifying the role of endocrine signaling in the regulation of aging in both vertebrates and invertebrates. Studies using the model organism fruit fly Drosophila melanogaster have largely advanced our understanding of evolutionarily conserved mechanisms in the endocrinology of aging and anti-aging. Mutations in single genes involved in endocrine signaling modify lifespan, as do alterations of endocrine signaling in a tissue- or cell-specific manner, highlighting a central role of endocrine signaling in coordinating the crosstalk between tissues and cells to determine the pace of aging. Here, we review the current landscape of research in D. melanogaster that offers valuable insights into the endocrine-governed mechanisms which influence lifespan and age-related physiology.

Cofactor-enabled functional expression of fruit fly, honeybee, and bumblebee nicotinic receptors reveals picomolar neonicotinoid actions.

The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.

Molecular action of larvicidal flavonoids on ecdysteroidogenic glutathione S-transferase Noppera-bo in Aedes aegypti.

BACKGROUND: Mosquito control is a crucial global issue for protecting the human community from mosquito-borne diseases. There is an urgent need for the development of selective and safe reagents for mosquito control. Flavonoids, a group of chemical substances with variable phenolic structures, such as daidzein, have been suggested as potential mosquito larvicides with less risk to the environment. However, the mode of mosquito larvicidal action of flavonoids has not been elucidated. RESULTS: Here, we report that several flavonoids, including daidzein, inhibit the activity of glutathione S-transferase Noppera-bo (Nobo), an enzyme used for the biosynthesis of the insect steroid hormone ecdysone, in the yellow fever mosquito Aedes aegypti. The crystal structure of the Nobo protein of Ae. aegypti (AeNobo) complexed with the flavonoids and its molecular dynamics simulation revealed that Glu113 forms a hydrogen bond with the flavonoid inhibitors. Consistent with this observation, substitution of Glu113 with Ala drastically reduced the inhibitory activity of the flavonoids against AeNobo. Among the identified flavonoid-type inhibitors, desmethylglycitein (4',6,7-trihydroxyisoflavone) exhibited the highest inhibitory activity in vitro. Moreover, the inhibitory activities of the flavonoids correlated with the larvicidal activity, as desmethylglycitein suppressed Ae. aegypti larval development more efficiently than daidzein. CONCLUSION: Our study demonstrates the mode of action of flavonoids on the Ae. aegypti Nobo protein at the atomic, enzymatic, and organismal levels.

Shortened lifespan induced by a high-glucose diet is associated with intestinal immune dysfunction in Drosophila sechellia.

Organisms can generally be divided into two nutritional groups: generalists that consume various types of food and specialists that consume specific types of food. However, it remains unclear how specialists adapt to only limited nutritional conditions in nature. In this study, we addressed this question by focusing on Drosophila fruit flies. The generalist Drosophila melanogaster can consume a wide variety of foods that contain high glucose levels. In contrast, the specialist Drosophila sechellia consumes only the Indian mulberry, known as noni (Morinda citrifolia), which contains relatively little glucose. We showed that the lifespan of D. sechellia was significantly shortened under a high-glucose diet, but this effect was not observed for D. melanogaster. In D. sechellia, a high-glucose diet induced disorganization of the gut epithelia and visceral muscles, which was associated with abnormal digestion and constipation. RNA-sequencing analysis revealed that many immune-responsive genes were suppressed in the gut of D. sechellia fed a high-glucose diet compared with those fed a control diet. Consistent with this difference in the expression of immune-responsive genes, high glucose-induced phenotypes were restored by the addition of tetracycline or scopoletin, a major nutritional component of noni, each of which suppresses gut bacterial growth. We propose that, in D. sechellia, a high-glucose diet impairs gut immune function, which leads to a change in gut microbiota, disorganization of the gut epithelial structure and a shortened lifespan.

An integrated approach to unravel a crucial structural property required for the function of the insect steroidogenic Halloween protein Noppera-bo.

Ecdysteroids are the principal steroid hormones essential for insect development and physiology. In the last 18 years, several enzymes responsible for ecdysteroid biosynthesis encoded by Halloween genes were identified and genetically and biochemically characterized. However, the tertiary structures of these proteins have not yet been characterized. Here, we report the results of an integrated series of in silico, in vitro, and in vivo analyses of the Halloween GST protein Noppera-bo (Nobo). We determined crystal structures of Drosophila melanogaster Nobo (DmNobo) complexed with GSH and 17ß-estradiol, a DmNobo inhibitor. 17ß-Estradiol almost fully occupied the putative ligand-binding pocket and a prominent hydrogen bond formed between 17ß-estradiol and Asp-113 of DmNobo. We found that Asp-113 is essential for 17ß-estradiol-mediated inhibition of DmNobo enzymatic activity, as 17ß-estradiol did not inhibit and physically interacted less with the D113A DmNobo variant. Asp-113 is highly conserved among Nobo proteins, but not among other GSTs, implying that this residue is important for endogenous Nobo function. Indeed, a homozygous nobo allele with the D113A substitution exhibited embryonic lethality and an undifferentiated cuticle structure, a phenocopy of complete loss-of-function nobo homozygotes. These results suggest that the nobo family of GST proteins has acquired a unique amino acid residue that appears to be essential for binding an endogenous sterol substrate to regulate ecdysteroid biosynthesis. To the best of our knowledge, ours is the first study describing the structural characteristics of insect steroidogenic Halloween proteins. Our findings provide insights relevant for applied entomology to develop insecticides that specifically inhibit ecdysteroid biosynthesis.

A population of neurons that produce hugin and express the diuretic hormone 44 receptor gene projects to the corpora allata in Drosophila melanogaster.

The corpora allata (CA) are essential endocrine organs that biosynthesize and secrete the sesquiterpenoid hormone, namely juvenile hormone (JH), to regulate a wide variety of developmental and physiological events in insects. CA are directly innervated with neurons in many insect species, implying the innervations to be important for regulating JH biosynthesis. Although this is also true for the model organism Drosophila melanogaster, neurotransmitters produced in the CA-projecting neurons are yet to be identified. In this study on D. melanogaster, we aimed to demonstrate that a subset of neurons producing the neuropeptide hugin, the invertebrate counterpart of the vertebrate neuromedin U, directly projects to the adult CA. A synaptic vesicle marker in the hugin neurons was observed at their axon termini located on the CA, which were immunolabeled with a newly-generated antibody to the JH biosynthesis enzyme JH acid O-methyltransferase. We also found the CA-projecting hugin neurons to likely express a gene encoding the specific receptor for diuretic hormone 44 (Dh44). Moreover, our data suggest that the CA-projecting hugin neurons have synaptic connections with the upstream neurons producing Dh44. Unexpectedly, the inhibition of CA-projecting hugin neurons did not significantly alter the expression levels of the JH-inducible gene Krüppel-homolog 1, which implies that the CA-projecting neurons are not involved in JH biosynthesis but rather in other known biological processes. This is the first study to identify a specific neurotransmitter of the CA-projecting neurons in D. melanogaster, and to anatomically characterize a neuronal pathway of the CA-projecting neurons and their upstream neurons.

Midgut-derived neuropeptide F controls germline stem cell proliferation in a mating-dependent manner.

Stem cell maintenance is established by neighboring niche cells that promote stem cell self-renewal. However, it is poorly understood how stem cell activity is regulated by systemic, tissue-extrinsic signals in response to environmental cues and changes in physiological status. Here, we show that neuropeptide F (NPF) signaling plays an important role in the pathway regulating mating-induced germline stem cell (GSC) proliferation in the fruit fly Drosophila melanogaster. NPF expressed in enteroendocrine cells (EECs) of the midgut is released in response to the seminal-fluid protein sex peptide (SP) upon mating. This midgut-derived NPF controls mating-induced GSC proliferation via ovarian NPF receptor (NPFR) activity, which modulates bone morphogenetic protein (BMP) signaling levels in GSCs. Our study provides a molecular mechanism that describes how a gut-derived systemic factor couples stem cell behavior to physiological status, such as mating, through interorgan communication.

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.

Assembly of insect hormone enthusiasts at Nasu Highland, Japan: Report of the 3rd International Insect Hormone (21st Ecdysone) Workshop.

The 3rd International Insect Hormone (21st Ecdysone) Workshop (IIHW2017) was held in July 2017 at Nasu Highland, Japan. In the 40 years of the workshop's history, this was the first to be held in an Asian country. A total of 109 insect hormone researchers from 18 countries (62 overseas and 47 domestic participants) attended IIHW2017. During the workshop, all participants stayed on-site at the venue's hotel; this was ideal for fostering communication between participants, in particular, interactions between principal investigators and young scientists. The workshop featured one keynote, 64 oral, and 35 poster presentations spanning molecular biology, cell biology, developmental biology, neurobiology, chemical biology, physiology, and ecology of insect hormones, including ecdysteroids, juvenile hormones, and a variety of neuropeptides. The workshop provided an ideal platform for discussing insect hormone biology using not only the typical genetic model insect, the fruit fly Drosophila, but also a diversity of interesting insects, such as the silkworm, the red flour beetle, the cricket, the dragonfly, the social ant, the bloodsucking tick, and so on. The participants succeeded in sharing the latest knowledge in a wide range of insect hormone research fields and in joining active and constructive scientific discussions.

Mating-Induced Increase in Germline Stem Cells via the Neuroendocrine System in Female Drosophila.

Mating and gametogenesis are two essential components of animal reproduction. Gametogenesis must be modulated by the need for gametes, yet little is known of how mating, a process that utilizes gametes, may modulate the process of gametogenesis. Here, we report that mating stimulates female germline stem cell (GSC) proliferation in Drosophila melanogaster. Mating-induced increase in GSC number is not simply owing to the indirect effect of emission of stored eggs, but rather is stimulated by a male-derived Sex Peptide (SP) and its receptor SPR, the components of a canonical neuronal pathway that induces a post-mating behavioral switch in females. We show that ecdysteroid, the major insect steroid hormone, regulates mating-induced GSC proliferation independently of insulin signaling. Ovarian ecdysteroid level increases after mating and transmits its signal directly through the ecdysone receptor expressed in the ovarian niche to increase the number of GSCs. Impairment of ovarian ecdysteroid biosynthesis disrupts mating-induced increase in GSCs as well as egg production. Importantly, feeding of ecdysteroid rescues the decrease in GSC number caused by impairment of neuronal SP signaling. Our study illustrates how female GSC activity is coordinately regulated by the neuroendocrine system to sustain reproductive success in response to mating.

Autocrine regulation of ecdysone synthesis by ß3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis.

In Drosophila, pulsed production of the steroid hormone ecdysone plays a pivotal role in developmental transitions such as metamorphosis. Ecdysone production is regulated in the prothoracic gland (PG) by prothoracicotropic hormone (PTTH) and insulin-like peptides (Ilps). Here, we show that monoaminergic autocrine regulation of ecdysone biosynthesis in the PG is essential for metamorphosis. PG-specific knockdown of a monoamine G protein-coupled receptor, ß3-octopamine receptor (Octß3R), resulted in arrested metamorphosis due to lack of ecdysone. Knockdown of tyramine biosynthesis genes expressed in the PG caused similar defects in ecdysone production and metamorphosis. Moreover, PTTH and Ilps signaling were impaired by Octß3R knockdown in the PG, and activation of these signaling pathways rescued the defect in metamorphosis. Thus, monoaminergic autocrine signaling in the PG regulates ecdysone biogenesis in a coordinated fashion on activation by PTTH and Ilps. We propose that monoaminergic autocrine signaling acts downstream of a body size checkpoint that allows metamorphosis to occur when nutrients are sufficiently abundant.

The Drosophila Zinc Finger Transcription Factor Ouija Board Controls Ecdysteroid Biosynthesis through Specific Regulation of spookier.

Steroid hormones are crucial for many biological events in multicellular organisms. In insects, the principal steroid hormones are ecdysteroids, which play essential roles in regulating molting and metamorphosis. During larval and pupal development, ecdysteroids are synthesized in the prothoracic gland (PG) from dietary cholesterol via a series of hydroxylation and oxidation steps. The expression of all but one of the known ecdysteroid biosynthetic enzymes is restricted to the PG, but the transcriptional regulatory networks responsible for generating such exquisite tissue-specific regulation is only beginning to be elucidated. Here, we report identification and characterization of the C2H2-type zinc finger transcription factor Ouija board (Ouib) necessary for ecdysteroid production in the PG in the fruit fly Drosophila melanogaster. Expression of ouib is predominantly limited to the PG, and genetic null mutants of ouib result in larval developmental arrest that can be rescued by administrating an active ecdysteroid. Interestingly, ouib mutant animals exhibit a strong reduction in the expression of one ecdysteroid biosynthetic enzyme, spookier. Using a cell culture-based luciferase reporter assay, Ouib protein stimulates transcription of spok by binding to a specific ~15 bp response element in the spok PG enhancer element. Most remarkable, the developmental arrest phenotype of ouib mutants is rescued by over-expression of a functionally-equivalent paralog of spookier. These observations imply that the main biological function of Ouib is to specifically regulate spookier transcription during Drosophila development.

Transcriptional control of steroid biosynthesis genes in the Drosophila prothoracic gland by ventral veins lacking and knirps.

Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni), have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH) signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld), controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development.

Transcriptional regulation of insect steroid hormone biosynthesis and its role in controlling timing of molting and metamorphosis.

The developmental transition from juvenile to adult is often accompanied by many systemic changes in morphology, metabolism, and reproduction. Curiously, both mammalian puberty and insect metamorphosis are triggered by a pulse of steroid hormones, which can harmonize gene expression profiles in the body and thus orchestrate drastic biological changes. However, understanding of how the timing of steroid hormone biosynthesis is regulated at the molecular level is poor. The principal insect steroid hormone, ecdysteroid, is biosynthesized from dietary cholesterol in the specialized endocrine organ called the prothoracic gland. The periodic pulses of ecdysteroid titers determine the timing of molting and metamorphosis. To date, at least nine families of ecdysteroidogenic enzyme genes have been identified. Expression levels of these genes correlate well with ecdysteroid titers, indicating that the transcriptional regulatory network plays a critical role in regulating the ecdysteroid biosynthesis pathway. In this article, we summarize the transcriptional regulation of ecdysteroid biosynthesis. We first describe the development of prothoracic gland cells during Drosophila embryogenesis, and then provide an overview of the transcription factors that act in ecdysteroid biosynthesis and signaling. We also discuss the external signaling pathways that target these transcriptional regulators. Furthermore, we describe conserved and/or diverse aspects of steroid hormone biosynthesis in insect species as well as vertebrates.

Precocious metamorphosis in the juvenile hormone-deficient mutant of the silkworm, Bombyx mori.

Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several "moltinism" mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval-larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval-pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH-deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis.

Enzymes for ecdysteroid biosynthesis: their biological functions in insects and beyond.

Steroid hormones are responsible for the coordinated regulation of many aspects of biological processes in multicellular organisms. Since the last century, many studies have identified and characterized steroidogenic enzymes in vertebrates, including mammals. However, much less is known about invertebrate steroidogenic enzymes. In the last 15 years, a number of steroidogenic enzymes and their functions have been characterized in ecdysozoan animals, especially in the fruit fly Drosophila melanogaster. In this review, we summarize the latest knowledge of enzymes crucial for synthesizing ecdysteroids, the principal insect steroid hormones. We also discuss the functional conservation and diversity of ecdysteroidogenic enzymes in other insects and even non-insect species, such as nematodes, vertebrates, and lower eukaryotes.

The intestinal stem cell/enteroblast-GAL4 driver, escargot-GAL4, also manipulates gene expression in the juvenile hormone-synthesizing organ of Drosophila melanogaster.

Intestinal stem cells (ISCs) of the fruit fly, Drosophila melanogaster, offer an excellent genetic model to explore homeostatic roles of ISCs in animal physiology. Among available genetic tools, the escargot (esg)-GAL4 driver, expressing the yeast transcription factor gene, GAL4, under control of the esg gene promoter, has contributed significantly to ISC studies. This driver facilitates activation of genes of interest in proximity to a GAL4-binding element, Upstream Activating Sequence, in ISCs and progenitor enteroblasts (EBs). While esg-GAL4 has been considered an ISC/EB-specific driver, recent studies have shown that esg-GAL4 is also active in other tissues, such as neurons and ovaries. Therefore, the ISC/EB specificity of esg-GAL4 is questionable. In this study, we reveal esg-GAL4 expression in the corpus allatum (CA), responsible for juvenile hormone (JH) production. When driving the oncogenic gene, RasV12, esg-GAL4 induces overgrowth in ISCs/EBs as reported, but also increases CA cell number and size. Consistent with this observation, animals alter expression of JH-response genes. Our data show that esg-GAL4-driven gene manipulation can systemically influence JH-mediated animal physiology, arguing for cautious use of esg-GAL4 as a "specific" ISC/EB driver to examine ISC/EB-mediated animal physiology.