Ecdysone signaling mediates the trade-off between immunity and reproduction via suppression of amyloids in the mosquito Aedes aegypti.

The balance between immunity and reproduction is essential for many key physiological functions. We report that to maintain an optimal fertility, 20-hydroxyecdysone (20E) and the ecdysone receptor (EcR) downregulate the immune deficiency (IMD) pathway during the post blood meal phase (PBM) of the Aedes aegypti reproductive cycle. RNA interference-mediated depletion of EcR elicited an increased expression of the IMD pathway components, and these mosquitoes were more resistant to infection by Gram-negative bacteria. Moreover, 20E and EcR recruit Pirk-like, the mosquito ortholog of Drosophila melanogaster Pirk. CRISPR-Cas9 knockout of Pirk-like has shown that it represses the IMD pathway by interfering with IMD-mediated formation of amyloid aggregates. 20E and EcR disruption of the amyloid formation is pivotal for maintaining normal yolk protein production and fertility. Additionally, 20E and its receptor EcR directly induce Pirk-like to interfere with cRHIM-mediated formation of amyloid. Our study highlights the vital role of 20E in governing the trade-off between immunity and reproduction. Pirk-like might be a potential target for new methods to control mosquito reproduction and pathogen transmission.

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.

Subunit P60 of phosphatidylinositol 3-kinase promotes cell proliferation or apoptosis depending on its phosphorylation status.

The regulatory subunits (P60 in insects, P85 in mammals) determine the activation of the catalytic subunits P110 in phosphatidylinositol 3-kinases (PI3Ks) in the insulin pathway for cell proliferation and body growth. However, the regulatory subunits also promote apoptosis via an unclear regulatory mechanism. Using Helicoverpa armigera, an agricultural pest, we showed that H. armigera P60 (HaP60) was phosphorylated under insulin-like peptides (ILPs) regulation at larval growth stages and played roles in the insulin/ insulin-like growth factor (IGF) signaling (IIS) to determine HaP110 phosphorylation and cell membrane translocation; whereas, HaP60 was dephosphorylated and its expression increased under steroid hormone 20-hydroxyecdysone (20E) regulation during metamorphosis. Protein tyrosine phosphatase non-receptor type 6 (HaPTPN6, also named tyrosine-protein phosphatase corkscrew-like isoform X1 in the genome) was upregulated by 20E to dephosphorylate HaP60 and HaP110. 20E blocked HaP60 and HaP110 translocation to the cell membrane and reduced their interaction. The phosphorylated HaP60 mediated a cascade of protein phosphorylation and forkhead box protein O (HaFOXO) cytosol localization in the IIS to promote cell proliferation. However, 20E, via G protein-coupled-receptor-, ecdysone receptor-, and HaFOXO signaling axis, upregulated HaP60 expression, and the non-phosphorylated HaP60 interacted with phosphatase and tensin homolog (HaPTEN) to induce apoptosis. RNA interference-mediated knockdown of HaP60 and HaP110 in larvae repressed larval growth and apoptosis. Thus, HaP60 plays dual functions to promote cell proliferation and apoptosis by changing its phosphorylation status under ILPs and 20E regulation, respectively.

The steroid hormone 20-hydroxyecdysone counteracts insulin signaling via insulin receptor dephosphorylation.

The insulin receptor (INSR) binds insulin to promote body growth and maintain normal blood glucose levels. While it is known that steroid hormones such as estrogen and 20-hydroxyecdysone counteract insulin function, the molecular mechanisms responsible for this attenuation remain unclear. In the present study, using the agricultural pest lepidopteran Helicoverpa armigera as a model, we proposed that the steroid hormone 20-hydroxyecdysone (20E) induces dephosphorylation of INSR to counteract insulin function. We observed high expression and phosphorylation of INSR during larval feeding stages that decreased during metamorphosis. Insulin upregulated INSR expression and phosphorylation, whereas 20E repressed INSR expression and induced INSR dephosphorylation in vivo. Protein tyrosine phosphatase 1B (PTP1B, encoded by Ptpn1) dephosphorylated INSR in vivo. PTEN (phosphatase and tensin homolog deleted on chromosome 10) was critical for 20E-induced INSR dephosphorylation by maintaining the transcription factor Forkhead box O (FoxO) in the nucleus, where FoxO promoted Ptpn1 expression and repressed Insr expression. Knockdown of Ptpn1 using RNA interference maintained INSR phosphorylation, increased 20E production, and accelerated pupation. RNA interference of Insr in larvae repressed larval growth, decreased 20E production, delayed pupation, and accumulated hemolymph glucose levels. Taken together, these results suggest that a high 20E titer counteracts the insulin pathway by dephosphorylating INSR to stop larval growth and accumulate glucose in the hemolymph.

A mating-induced reproductive gene promotes Anopheles tolerance to Plasmodium falciparum infection.

Anopheles mosquitoes have transmitted Plasmodium parasites for millions of years, yet it remains unclear whether they suffer fitness costs to infection. Here we report that the fecundity of virgin and mated females of two important vectors-Anopheles gambiae and Anopheles stephensi-is not affected by infection with Plasmodium falciparum, demonstrating that these human malaria parasites do not inflict this reproductive cost on their natural mosquito hosts. Additionally, parasite development is not impacted by mating status. However, in field studies using different P. falciparum isolates in Anopheles coluzzii, we find that Mating-Induced Stimulator of Oogenesis (MISO), a female reproductive gene strongly induced after mating by the sexual transfer of the steroid hormone 20-hydroxyecdysone (20E), protects females from incurring fecundity costs to infection. MISO-silenced females produce fewer eggs as they become increasingly infected with P. falciparum, while parasite development is not impacted by this gene silencing. Interestingly, previous work had shown that sexual transfer of 20E has specifically evolved in Cellia species of the Anopheles genus, driving the co-adaptation of MISO. Our data therefore suggest that evolution of male-female sexual interactions may have promoted Anopheles tolerance to P. falciparum infection in the Cellia subgenus, which comprises the most important malaria vectors.

The role of 20E biosynthesis relative gene Shadow in the reproduction of the predatory mirid bug, Cyrtorhinus lividipennis (Hemiptera: Miridae).

Cytorhinus lividipennis is a natural enemy of rice planthoppers and leafhoppers. Improving the fecundity of C. lividipennis will be helpful to improve its control effect on pests. However, little is known about the hormonal regulatory mechanism of reproduction in C. lividipennis. In the current study, we examined the role of 20-hydroxyecdysone (20E) biosynthesis relative gene Shadow in the reproduction of C. lividipennis. The complementary DNA sequence of ClSad is 2018 -bp in length with an open reading frame of 1398-bp encoding 465 amino acid residues. ClSad was readily detected in nymphal and adult stages, and highly expressed in the adult stage. ClSad was highly expressed in the midgut and ovaries of adult females. Moreover, RNA interference-mediated knockdown of ClSad reduced the 20E titers and ClVg transcript level, resulting in fewer fully developed eggs and a decrease in the number of eggs laid by dsSad-injected adult females within 15 days. These results suggest that ClSad plays a critical role in the reproduction of C. lividipennis. The present study provides insights into the molecular mechanism of the ClSad gene for the reproduction of C. lividipennis.

miR-34 modulates wing polyphenism in planthopper.

Polyphenism is a successful strategy adopted by organisms to adapt to environmental changes. Brown planthoppers (BPH, Nilaparvata lugens) develop two wing phenotypes, including long-winged (LW) and short-winged (SW) morphs. Though insulin receptor (InR) and juvenile hormone (JH) have been known to regulate wing polyphenism in BPH, the interaction between these regulators remains largely elusive. Here, we discovered that a conserved microRNA, miR-34, modulates a positive autoregulatory feedback loop of JH and insulin/IGF signaling (IIS) pathway to control wing polyphenism in BPH. Nlu-miR-34 is abundant in SW BPHs and suppresses NlInR1 by targeting at two binding sites in the 3'UTR of NlInR1. Overexpressing miR-34 in LW BPHs by injecting agomir-34 induces the development towards SW BPHs, whereas knocking down miR-34 in SW BPHs by injecting antagomir-34 induces more LW BPHs when another NlInR1 suppressor, NlInR2, is also suppressed simultaneously. A cis-response element of Broad Complex (Br-C) is found in the promoter region of Nlu-miR-34, suggesting that 20-hydroxyecdysone (20E) might be involved in wing polyphenism regulation. Topic application of 20E downregulates miR-34 expression but does not change wing morphs. On the other hand, JH application upregulates miR-34 expression and induces more SW BPHs. Moreover, knocking down genes in IIS pathway changes JH titers and miR-34 abundance. In all, we showed that miRNA mediates the cross talk between JH, 20E and IIS pathway by forming a positive feedback loop, uncovering a comprehensive regulation mechanism which integrates almost all known regulators controlling wing polyphenism in insects.

Expression of 20-hydroxyecdysone-related genes during gonadal development of Teleogryllus emma (Orthoptera: Gryllidae).

Insect gonads develop under endocrine signals. In this study, we assessed the characters of partial complementary DNAs encoding the Teleogryllus emma orthologs of 20-hydroxyecdysone (20E)-related genes (RXR, E75, HR3, Hsc70, and Hsp90) and analyzed their expression patterns in both nymph and adult crickets. 20E treatment suppressed expression of TeEcR, TeRXR, TeE75, TeHR3, TeHsc70, and TeHsp90. Temporal expression analysis demonstrated that TeERR and 20E-related genes were expressed in four stages of gonadal development from the fourth-instar nymph stage to the adult stage. The expression pattern of these genes differed in testicular and ovarian development. TeRXR, HR3, TeHsc70, and TeHsp90 were irregularly expressed in gonads of the same developmental stages, while mRNAs encoding TeERR, TeEcR, and TeE75 accumulated in higher levels in ovaries than in testes. RNA interference (RNAi) of TeEcR expression led to decrease of the expression levels of TeEcR, TeRXR, TeHR3, and TeHsc70, while it enhanced TeE75 and TeHsp90 expressions. These results demonstrate that the TeERR and 20E-related genes help regulate gonadal development, while TeEcR appears to inhibit TeE75 expression, TeE75 inhibits HR3 expression. Hsc70 indirectly regulated the expression of the primary and secondary response genes E74A, E75B, and HR3. Hsp90 regulated Usp expression with no direct regulatory relationship with EcR.

Post-transcriptional regulation of insect metamorphosis and oogenesis.

Metamorphic transformation from larvae to adults along with the high fecundity is key to insect success. Insect metamorphosis and reproduction are governed by two critical endocrines, juvenile hormone (JH), and 20-hydroxyecdysone (20E). Recent studies have established a crucial role of microRNA (miRNA) in insect metamorphosis and oogenesis. While miRNAs target genes involved in JH and 20E-signaling pathways, these two hormones reciprocally regulate miRNA expression, forming regulatory loops of miRNA with JH and 20E-signaling cascades. Insect metamorphosis and oogenesis rely on the coordination of hormones, cognate genes, and miRNAs for precise regulation. In addition, the alternative splicing of genes in JH and 20E-signaling pathways has distinct functions in insect metamorphosis and oogenesis. We, therefore, focus in this review on recent advances in post-transcriptional regulation, with the emphasis on the regulatory role of miRNA and alternative splicing, in insect metamorphosis and oogenesis. We will highlight important new findings of miRNA interactions with hormonal signaling and alternative splicing of JH receptor heterodimer gene Taiman.

Juvenile hormone-regulated alternative splicing of the taiman gene primes the ecdysteroid response in adult mosquitoes.

Juvenile hormone (JH) regulates many aspects of insect development and reproduction. In some processes, JH plays a critical role in defining the action of the steroid hormone 20-hydroxyecdysone (20E). In Aedes aegypti mosquitoes, JH prepares newly emerged female adults to become competent to synthesize vitellogenin in response to 20E after blood ingestion. The molecular basis of this competence is still not well understood. Here, we report that JH regulates pre-mRNA splicing of the taiman gene, which encodes a key transcriptional regulator required for both JH- and 20E-controlled gene expression. JH stimulated the production of the Taiman isoforms A/B, while reducing the levels of the isoforms C/D, in the fat body after adult eclosion. The appearance of the A/B isoforms in maturing mosquitoes was accompanied by acquisition of the competence to respond to 20E. Depletion of the A/B isoforms, by inhibiting the alternative splicing or by isoform-specific RNA interference, considerably diminished the 20E-induced gene expression after a blood meal and substantially impaired oocyte development. In accordance with this observation, further studies indicated that in the presence of 20E, the Taiman A/B isoforms showed much stronger interactions with the 20E receptor complex than the Taiman C/D isoforms. In contrast, all four isoforms displayed similar capabilities of forming active JH receptor complexes with the methoprene-tolerant protein (Met). This study suggested that JH confers the competence to newly emerged female mosquitoes by regulating mRNA splicing to generate the Taiman isoforms that are essential for the vitellogenic 20E response.

Steroid Hormone Signaling Is Essential for Pheromone Production and Oenocyte Survival.

Many of the lipids found on the cuticles of insects function as pheromones and communicate information about age, sex, and reproductive status. In Drosophila, the composition of the information-rich lipid profile is dynamic and changes over the lifetime of an individual. However, the molecular basis of this change is not well understood. To identify genes that control cuticular lipid production in Drosophila, we performed a RNA interference screen and used Direct Analysis in Real Time and gas chromatography mass spectrometry to quantify changes in the chemical profiles. Twelve putative genes were identified whereby transcriptional silencing led to significant differences in cuticular lipid production. Amongst them, we characterized a gene which we name spidey, and which encodes a putative steroid dehydrogenase that has sex- and age-dependent effects on viability, pheromone production, and oenocyte survival. Transcriptional silencing or overexpression of spidey during embryonic development results in pupal lethality and significant changes in levels of the ecdysone metabolite 20-hydroxyecdysonic acid and 20-hydroxyecdysone. In contrast, inhibiting gene expression only during adulthood resulted in a striking loss of oenocyte cells and a concomitant reduction of cuticular hydrocarbons, desiccation resistance, and lifespan. Oenocyte loss and cuticular lipid levels were partially rescued by 20-hydroxyecdysone supplementation. Taken together, these results identify a novel regulator of pheromone synthesis and reveal that ecdysteroid signaling is essential for the maintenance of cuticular lipids and oenocytes throughout adulthood.

Host miRNAs are involved in hormonal regulation of HaSNPV-triggered climbing behaviour in Helicoverpa armigera.

Baculoviruses manipulate host climbing behaviour to ensure that the hosts die at elevated positions on host plants to facilitate virus proliferation and transmission, which is a process referred to as tree-top disease. However, the detailed molecular mechanism underlying tree-top disease has not been elucidated. Using transcriptome analysis, we showed that two hormone signals, juvenile hormone (JH) and 20-hydroxyecdysone (20E), are key components involved in HaSNPV-induced tree-top disease in Helicoverpa armigera larvae. RNAi-mediated knockdown and exogenous hormone treatment assays demonstrated that 20E inhibits virus-induced tree-top disease, while JH mediates tree-top disease behaviour. Knockdown of BrZ2, a downstream signal of JH and 20E, promoted HaSNPV-induced tree-top disease. We also found that two miRNAs target BrZ2 and are involved in the cross-talk regulation between 20E and JH manipulating HaSNPV replication, time to death and HaSNPV-induced tree-top disease.

Differential Expression of Ecdysone Receptor Leads to Variation in Phenotypic Plasticity across Serial Homologs.

Bodies are often made of repeated units, or serial homologs, that develop using the same core gene regulatory network. Local inputs and modifications to this network allow serial homologs to evolve different morphologies, but currently we do not understand which modifications allow these repeated traits to evolve different levels of phenotypic plasticity. Here we describe variation in phenotypic plasticity across serial homologous eyespots of the butterfly Bicyclus anynana, hypothesized to be under selection for similar or different functions in the wet and dry seasonal forms. Specifically, we document the presence of eyespot size and scale brightness plasticity in hindwing eyespots hypothesized to vary in function across seasons, and reduced size plasticity and absence of brightness plasticity in forewing eyespots hypothesized to have the same function across seasons. By exploring the molecular and physiological causes of this variation in plasticity across fore and hindwing serial homologs we discover that: 1) temperature experienced during the wandering stages of larval development alters titers of an ecdysteroid hormone, 20-hydroxyecdysone (20E), in the hemolymph of wet and dry seasonal forms at that stage; 2) the 20E receptor (EcR) is differentially expressed in the forewing and hindwing eyespot centers of both seasonal forms during this critical developmental stage; and 3) manipulations of EcR signaling disproportionately affected hindwing eyespots relative to forewing eyespots. We propose that differential EcR expression across forewing and hindwing eyespots at a critical stage of development explains the variation in levels of phenotypic plasticity across these serial homologues. This finding provides a novel signaling pathway, 20E, and a novel molecular candidate, EcR, for the regulation of levels of phenotypic plasticity across body parts or serial homologs.

20-Hydroxyecdysone (20E) Primary Response Gene E75 Isoforms Mediate Steroidogenesis Autoregulation and Regulate Developmental Timing in Bombyx.

The temporal control mechanisms that precisely control animal development remain largely elusive. The timing of major developmental transitions in insects, including molting and metamorphosis, is coordinated by the steroid hormone 20-hydroxyecdysone (20E). 20E involves feedback loops to maintain pulses of ecdysteroid biosynthesis leading to its upsurge, whereas the underpinning molecular mechanisms are not well understood. Using the silkworm Bombyx mori as a model, we demonstrated that E75, the 20E primary response gene, mediates a regulatory loop between ecdysteroid biosynthesis and 20E signaling. E75 isoforms A and C directly bind to retinoic acid receptor-related response elements in Halloween gene promoter regions to induce gene expression thus promoting ecdysteroid biosynthesis and developmental transition, whereas isoform B antagonizes the transcriptional activity of isoform A/C through physical interaction. As the expression of E75 isoforms is differentially induced by 20E, the E75-mediated regulatory loop represents a fine autoregulation of steroidogenesis, which contributes to the precise control of developmental timing.

The genome of Tetranychus urticae reveals herbivorous pest adaptations.

The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and an extreme record of pesticide resistance. Here we present the completely sequenced and annotated spider mite genome, representing the first complete chelicerate genome. At 90 megabases T. urticae has the smallest sequenced arthropod genome. Compared with other arthropods, the spider mite genome shows unique changes in the hormonal environment and organization of the Hox complex, and also reveals evolutionary innovation of silk production. We find strong signatures of polyphagy and detoxification in gene families associated with feeding on different hosts and in new gene families acquired by lateral gene transfer. Deep transcriptome analysis of mites feeding on different plants shows how this pest responds to a changing host environment. The T. urticae genome thus offers new insights into arthropod evolution and plant-herbivore interactions, and provides unique opportunities for developing novel plant protection strategies.

Steroid signaling promotes stem cell maintenance in the Drosophila testis.

Stem cell regulation by local signals is intensely studied, but less is known about the effects of hormonal signals on stem cells. In Drosophila, the primary steroid twenty-hydroxyecdysone (20E) regulates ovarian germline stem cells (GSCs) but was considered dispensable for testis GSC maintenance. Male GSCs reside in a microenvironment (niche) generated by somatic hub cells and adjacent cyst stem cells (CySCs). Here, we show that depletion of 20E from adult males by overexpressing a dominant negative form of the Ecdysone receptor (EcR) or its heterodimeric partner ultraspiracle (usp) causes GSC and CySC loss that is rescued by 20E feeding, uncovering a requirement for 20E in stem cell maintenance. EcR and USP are expressed, activated and autonomously required in the CySC lineage to promote CySC maintenance, as are downstream genes ftz-f1 and E75. In contrast, GSCs non-autonomously require ecdysone signaling. Global inactivation of EcR increases cell death in the testis that is rescued by expression of EcR-B2 in the CySC lineage, indicating that ecdysone signaling supports stem cell viability primarily through a specific receptor isoform. Finally, EcR genetically interacts with the NURF chromatin-remodeling complex, which we previously showed maintains CySCs. Thus, although 20E levels are lower in males than females, ecdysone signaling acts through distinct cell types and effectors to ensure both ovarian and testis stem cell maintenance.

Phospholipase Cγ1 connects the cell membrane pathway to the nuclear receptor pathway in insect steroid hormone signaling.

In addition to the classical nuclear receptor pathway, there is a nongenomic pathway in the cell membrane that regulates gene expression in animal steroid hormone signaling; however, this mechanism is unclear. Here, we report that the insect steroid hormone 20-hydroxyecdysone (20E) regulates calcium influx via phospholipase Cγ1 (PLCG1) to modulate the protein kinase C phosphorylation of the transcription factor ultraspiracle (USP1) in the lepidopteran insect Helicoverpa armigera. The PLCG1 mRNA levels are increased during the molting and metamorphic stages. The depletion of PLCG1 by RNA interference can block 20E-enhanced pupation, cause larvae death and pupation defects, and repress 20E-induced gene expression. 20E may induce the tyrosine phosphorylation of PLCG1 at the cytosolic tyrosine kinase (Src) homology 2 domains and then determine the migration of PLCG1 toward the plasma membrane. The G-protein-coupled receptor (GPCR) inhibitor suramin, Src family kinase inhibitor PP2, and the depletions of ecdysone-responsible GPCR (ErGPCR) and Gαq restrain the 20E-induced tyrosine phosphorylation of PLCG1. PLCG1 participates in the 20E-induced Ca(2+) influx. The inhibition of GPCR, PLC, inositol 1,4,5-trisphosphate receptor, and calcium channels represses the 20E-induced Ca(2+) influx. Through calcium signaling, PLCG1 mediates the transcriptional activation driven by the ecdysone-response element. Through PLCG1 and calcium signaling, 20E regulates PKC phosphorylation of USP1 at Ser-21 to determine its ecdysone-response element binding activity. These results suggest that 20E activates PLCG1 via the ErGPCR and Src family kinases to regulate Ca(2+) influx and PKC phosphorylation of USP1 to subsequently modulate gene transcription for metamorphosis.

Characterization of a novel RXR receptor in the salmon louse (Lepeophtheirus salmonis, Copepoda) regulating growth and female reproduction.

BACKGROUND: Nuclear receptors have crucial roles in all metazoan animals as regulators of gene transcription. A wide range of studies have elucidated molecular and biological significance of nuclear receptors but there are still a large number of animals where the knowledge is very limited. In the present study we have identified an RXR type of nuclear receptor in the salmon louse (Lepeophtheirus salmonis) (i.e. LsRXR). RXR is one of the two partners of the Ecdysteroid receptor in arthropods, the receptor for the main molting hormone 20-hydroxyecdysone (E20) with a wide array of effects in arthropods. RESULTS: Five different LsRXR transcripts were identified by RACE showing large differences in domain structure. The largest isoforms contained complete DNA binding domain (DBD) and ligand binding domain (LBD), whereas some variants had incomplete or no DBD. LsRXR is transcribed in several tissues in the salmon louse including ovary, subcuticular tissue, intestine and glands. By using Q-PCR it is evident that the LsRXR mRNA levels vary throughout the L. salmonis life cycle. We also show that the truncated LsRXR transcript comprise about 50% in all examined samples. We used RNAi to knock-down the transcription in adult reproducing female lice. This resulted in close to zero viable offspring. We also assessed the LsRXR RNAi effects using a L. salmonis microarray and saw significant effects on transcription in the female lice. Transcription of the major yolk proteins was strongly reduced by knock-down of LsRXR. Genes involved in lipid metabolism and transport were also down regulated. Furthermore, different types of growth processes were up regulated and many cuticle proteins were present in this group. CONCLUSIONS: The present study demonstrates the significance of LsRXR in adult female L. salmonis and discusses the functional aspects in relation to other arthropods. LsRXR has a unique structure that should be elucidated in the future.

The hormone-dependent function of Hsp90 in the crosstalk between 20-hydroxyecdysone and juvenile hormone signaling pathways in insects is determined by differential phosphorylation and protein interactions.

BACKGROUND: Heat shock protein 90 (Hsp90) interacts with steroid hormone receptors, signaling kinases, and various transcription factors. However, the mechanism by which Hsp90 interacts with different proteins in various pathways remains unclear. METHODS: Western blot was used to study Hsp90 expression profile in Helicoverpa armigera (Lepidoptera). RNA interference was performed to investigate the function of Hsp90 in 20-hydroxyecdysone (20E) and juvenile hormone (JH) signal pathways. The binding of Hsp90 to the transcription factor ultraspiracle protein (USP1) and JH candidate receptor methoprene-tolerant (Met1) was analyzed by co-immunoprecipitation. Phospho-(Ser) PKC substrate antibody was used to detect Hsp90 phosphorylation. RESULTS: Hsp90 participated in 20E- or JH-induced gene expression. 20E induced the interaction between Hsp90 and USP1, whereas JH III and methoprene induced the interaction between Hsp90 and Met1, respectively. 20E and JH counteracted each other for these protein interactions. Both JH III and methoprene induced protein kinase C (PKC) phosphorylation of Hsp90. This process could be inhibited by phospholipase C (PLC) and PKC inhibitors. 20E suppressed JH III- or methoprene-induced PKC phosphorylation of Hsp90. CONCLUSION: 20E maintained the non-PKC-phosphorylation status of Hsp90. Hsp90 interacted with USP1 to induce gene expression in the 20E pathway. JH regulated the PKC-phosphorylation status of Hsp90. Hsp90 also interacted with Met1 to induce gene expression in the JH pathway. GENERAL SIGNIFICANCE: Our study describes a novel mechanism of Hsp90 action by altering phosphorylation and protein interaction in various hormonal signaling pathways.

The Drosophila nuclear receptors DHR3 and betaFTZ-F1 control overlapping developmental responses in late embryos.

Studies of the onset of metamorphosis have identified an ecdysone-triggered transcriptional cascade that consists of the sequential expression of the transcription-factor-encoding genes DHR3, betaFTZ-F1, E74A and E75A. Although the regulatory interactions between these genes have been well characterized by genetic and molecular studies over the past 20 years, their developmental functions have remained more poorly understood. In addition, a transcriptional sequence similar to that observed in prepupae is repeated before each developmental transition in the life cycle, including mid-embryogenesis and the larval molts. Whether the regulatory interactions between DHR3, betaFTZ-F1, E74A and E75A at these earlier stages are similar to those defined at the onset of metamorphosis, however, is unknown. In this study, we turn to embryonic development to address these two issues. We show that mid-embryonic expression of DHR3 and betaFTZ-F1 is part of a 20-hydroxyecdysone (20E)-triggered transcriptional cascade similar to that seen in mid-prepupae, directing maximal expression of E74A and E75A during late embryogenesis. In addition, DHR3 and betaFTZ-F1 exert overlapping developmental functions at the end of embryogenesis. Both genes are required for tracheal air filling, whereas DHR3 is required for ventral nerve cord condensation and betaFTZ-F1 is required for proper maturation of the cuticular denticles. Rescue experiments support these observations, indicating that DHR3 has essential functions independent from those of betaFTZ-F1. DHR3 and betaFTZ-F1 also contribute to overlapping transcriptional responses during embryogenesis. Taken together, these studies define the lethal phenotypes of DHR3 and betaFTZ-F1 mutants, and provide evidence for functional bifurcation in the 20E-responsive transcriptional cascade.