Cis-regulatory polymorphism at fiz ecdysone oxidase contributes to polygenic evolutionary response to malnutrition in Drosophila.

We investigate the contribution of a candidate gene, fiz (fezzik), to complex polygenic adaptation to juvenile malnutrition in Drosophila melanogaster. Experimental populations maintained for >250 generations of experimental evolution to a nutritionally poor larval diet (Selected populations) evolved several-fold lower fiz expression compared to unselected Control populations. Here we show that this divergence in fiz expression is mediated by a cis-regulatory polymorphism. This polymorphism, originally sampled from a natural population in Switzerland, is distinct from a second cis-regulatory SNP previously identified in non-African D. melanogaster populations, implying that two independent cis-regulatory variants promoting high fiz expression segregate in non-African populations. Enzymatic analyses of Fiz protein expressed in E. coli demonstrate that it has ecdysone oxidase activity acting on both ecdysone and 20-hydroxyecdysone. Four of five fiz paralogs annotated to ecdysteroid metabolism also show reduced expression in Selected larvae, implying that malnutrition-driven selection favored general downregulation of ecdysone oxidases. Finally, as an independent test of the role of fiz in poor diet adaptation, we show that fiz knockdown by RNAi results in faster larval growth on the poor diet, but at the cost of greatly reduced survival. These results imply that downregulation of fiz in Selected populations was favored by selection on the nutritionally poor diet because of its role in suppressing growth in response to nutrient shortage. However, they suggest that fiz downregulation is only adaptive in combination with other changes evolved by Selected populations, which ensure that the organism can sustain the faster growth promoted by fiz downregulation.

Timing Drosophila development through steroid hormone action.

Specifically timed pulses of the moulting hormone ecdysone are necessary for developmental progression in insects, guiding development through important milestones such as larval moults, pupation and metamorphosis. It also coordinates the acquisition of cell identities, known as cell patterning, and growth in a tissue-specific manner. In the absence of ecdysone, the ecdysone receptor heterodimer Ecdysone Receptor and Ultraspiracle represses expression of target primary response genes, which become de-repressed as the ecdysone titre rises. However, ecdysone signalling elicits both repressive and activating responses in a temporal and tissue-specific manner. To understand how ecdysone achieves such specificity, this review explores the layers of gene regulation involved in stage-appropriate ecdysone responses in Drosophila fruit flies.

Steroid receptor coactivator TAIMAN is a new modulator of insect circadian clock.

TAIMAN (TAI), the only insect ortholog of mammalian Steroid Receptor Coactivators (SRCs), is a critical modulator of ecdysone and juvenile hormone (JH) signaling pathways, which govern insect development and reproduction. The modulatory effect is mediated by JH-dependent TAI's heterodimerization with JH receptor Methoprene-tolerant and association with the Ecdysone Receptor complex. Insect hormones regulate insect physiology and development in concert with abiotic cues, such as photo- and thermoperiod. Here we tested the effects of JH and ecdysone signaling on the circadian clock by a combination of microsurgical operations, application of hormones and hormone mimics, and gene knockdowns in the linden bug Pyrrhocoris apterus males. Silencing taiman by each of three non-overlapping double-strand RNA fragments dramatically slowed the free-running period (FRP) to 27-29 hours, contrasting to 24 hours in controls. To further corroborate TAIMAN's clock modulatory function in the insect circadian clock, we performed taiman knockdown in the cockroach Blattella germanica. Although Blattella and Pyrrhocoris lineages separated ~380 mya, B. germanica taiman silencing slowed the FRP by more than 2 hours, suggesting a conserved TAI clock function in (at least) some insect groups. Interestingly, the pace of the linden bug circadian clock was neither changed by blocking JH and ecdysone synthesis, by application of the hormones or their mimics nor by the knockdown of corresponding hormone receptors. Our results promote TAI as a new circadian clock modulator, a role described for the first time in insects. We speculate that TAI participation in the clock is congruent with the mammalian SRC-2 role in orchestrating metabolism and circadian rhythms, and that TAI/SRCs might be conserved components of the circadian clock in animals.

Starvation selection reduces and delays larval ecdysone production and signaling.

Previous studies have shown that selection for starvation resistance in Drosophila melanogaster results in delayed eclosion and increased adult fat stores. It is assumed that these traits are caused by the starvation selection pressure, but its mechanism is unknown. We found that our starvation-selected (SS) population stores more fat during larval development and has extended larval development and pupal development time. Developmental checkpoints in the third instar associated with ecdysteroid hormone pulses are increasingly delayed. The delay in the late larval period seen in the SS population is indicative of reduced and delayed ecdysone signaling. An enzyme immunoassay for ecdysteroids (with greatest affinity to the metabolically active 20-hydroxyecdysone and the α-ecdysone precursor) confirmed that the SS population had reduced and delayed hormone production compared with that of fed control (FC) flies. Feeding third instar larvae on food supplemented with α-ecdysone partially rescued the developmental delay and reduced subsequent adult starvation resistance. This work suggests that starvation selection causes reduced and delayed production of ecdysteroids in the larval stage and affects the developmental delay phenotype that contributes to subsequent adult fat storage and starvation resistance.

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.

Segmental expression of two ecdysone pathway genes during embryogenesis of hemimetabolous insects.

Signaling networks are redeployed across different developmental times and places to generate phenotypic diversity from a limited genetic toolkit. Hormone signaling networks in particular have well-studied roles in multiple developmental processes. In insects, the ecdysone pathway controls critical events in late embryogenesis and throughout post-embryonic development. While this pathway has not been shown to function in the earliest stage of embryonic development in the model insect Drosophila melanogaster, one component of the network, the nuclear receptor E75A, is necessary for proper segment generation in the milkweed bug Oncopeltus fasciatus. Published expression data from several other species suggests possible conservation of this role across hundreds of millions of years of insect evolution. Previous work also demonstrates a second nuclear receptor in the ecdysone pathway, Ftz-F1, plays a role in segmentation in multiple insect species. Here we report tightly linked expression patterns of ftz-F1 and E75A in two hemimetabolous insect species, the German cockroach Blattella germanica and the two-spotted cricket Gryllus bimaculatus. In both species, the genes are expressed segmentally in adjacent cells, but they are never co-expressed. Using parental RNAi, we show the two genes have distinct roles in early embryogenesis. E75A appears necessary for abdominal segmentation in B. germanica, while ftz-F1 is essential for proper germband formation. Our results suggest that the ecdysone network is critical for early embryogenesis in hemimetabolous insects.

[The Drosophila CG9890 Protein is Involved in the Regulation of Ecdysone-Dependent Transcription].

Previously we showed that the CG9890 protein, which has zinc finger domains, interacts with ENY2-containing complexes and is localized mainly on the promoters of active genes. The CG9890 protein is involved in the regulation of the expression of some of the genes on the promoters of which it is located, and among these genes there are genes for the ecdysone cascade. In this work, the role of the CG9890 protein in the regulation of ecdysone-dependent inducible transcription was studied. For this, 12 ecdysone-dependent genes on the promoters of which the CG9890 protein is localized were identified. Their activation was studied after the addition of 20-hydroxyecdysone to cells, both in normal conditions and after RNA interference of CG9890. The expression of ecdysone-dependent genes is significantly increased in response to the treatment of cells with ecdysone, in contrast to the control genes. Moreover, in the cell line after RNA interference CG9890, the transcription of 8 out of 12 genes was significantly higher than in the control line. Thus, the CG9890 protein is involved in the regulation of transcription of ecdysone-dependent genes, and, in most cases, acts as a repressor.

Cuticular protein genes showing peaks at different stages are probably regulated by different ecdysone responsive transcription factors during larval-pupal transformation.

We aimed to explain the reason and function of the successive expression of ecdysone-responsive transcription factors (ERTFs) and related cuticular protein (CP) genes during transformation from larva to pupa. The regulation of the expression of CP genes by ERTFs was examined by in vitro wing disc culture and reporter assay using a gene gun transduction system. Two CP genes that showed expression peaks at different stages-BmorCPG12 at W3L and BmorCPH2 at P0 stage-were selected and examined. Reporter constructs conveying putative BHR3, ßFTZ-F1, BHR39, and E74A binding sites of BmorCPG12 and BmorCPH2 showed promoter activity when introduced into wing discs. In the present study, we showed the functioning of the putative BHR3 and E74A binding sites, together with putative ßFTZ-F1 binding sites, on the activation of CP genes, and different ERTF binding sites functioned in one CP gene. From these, we conclude that BHR3, ßFTZ-F1, and E74A that are successively expressed bring about the successive expression of CP genes, resulting in insect metamorphosis. In addition to this, reporter constructs conveying putative BHR39 binding sites of BmorCPG12 and BmorCPH2 showed negative regulation.

Co-option of immune effectors by the hormonal signalling system triggering metamorphosis in Drosophila melanogaster.

Insect metamorphosis is triggered by the production, secretion and degradation of 20-hydroxyecdysone (ecdysone). In addition to its role in developmental regulation, increasing evidence suggests that ecdysone is involved in innate immunity processes, such as phagocytosis and the induction of antimicrobial peptide (AMP) production. AMP regulation includes systemic responses as well as local responses at surface epithelia that contact with the external environment. At pupariation, Drosophila melanogaster increases dramatically the expression of three AMP genes, drosomycin (drs), drosomycin-like 2 (drsl2) and drosomycin-like 5 (drsl5). We show that the systemic action of drs at pupariation is dependent on ecdysone signalling in the fat body and operates via the ecdysone downstream target, Broad. In parallel, ecdysone also regulates local responses, specifically through the activation of drsl2 expression in the gut. Finally, we confirm the relevance of this ecdysone dependent AMP expression for the control of bacterial load by showing that flies lacking drs expression in the fat body have higher bacterial persistence over metamorphosis. In contrast, local responses may be redundant with the systemic effect of drs since reduction of ecdysone signalling or of drsl2 expression has no measurable negative effect on bacterial load control in the pupa. Together, our data emphasize the importance of the association between ecdysone signalling and immunity using in vivo studies and establish a new role for ecdysone at pupariation, which impacts developmental success by regulating the immune system in a stage-dependent manner. We speculate that this co-option of immune effectors by the hormonal system may constitute an anticipatory mechanism to control bacterial numbers in the pupa, at the core of metamorphosis evolution.

Combined Transcriptomic Analysis and RNA Interference Reveal the Effects of Methoxyfenozide on Ecdysone Signaling Pathway of Spodoptera exigua.

Spodoptera exigua is a worldwide pest afflicting edible vegetables and has developed varying levels of resistance to insecticides. Methoxyfenozide (MET), an ecdysteroid agonist, is effective against lepidopteran pests such as S. exigua. However, the mechanism of MET to S. exigua remains unclear. In this study, we analyzed the expression patterns of genes related to the ecdysone signaling pathway in transcriptome data treated with sublethal doses of MET and analyzed how expression levels of key genes affect the toxicity of MET on S. exigua. Our results demonstrated that 2639 genes were up-regulated and 2512 genes were down-regulated in S. exigua treated with LC30 of MET. Of these, 15 genes were involved in the ecdysone signaling pathway. qPCR results demonstrated that ecdysone receptor A (EcRA) expression levels significantly increased in S. exigua when treated with different doses of MET, and that the RNAi-mediated silencing of EcRA significantly increased mortality to 55.43% at 72 h when L3 S. exigua larvae were exposed to MET at the LC30 dose. Additionally, knocking down EcRA suppressed the most genes expressed in the ecdysone signaling pathway. The combination of MET and dsEcRA affected the expression of E74 and enhanced the expression of TREA. These results demonstrate that the adverse effects of sublethal MET disturb the ecdysone signaling pathway in S. exigua, and EcRA is closely related to MET toxic effect. This study increases our collective understanding of the mechanisms of MET in insect pests.