Identification of G Protein-Coupled Receptors Required for Vitellogenin Uptake into the Oocytes of the Red Flour Beetle, \u3cem\u3eTribolium castaneum\u3c/em\u3e

Previous studies suggested that a membrane receptor might be involved in mediating vitellogenin (Vg) uptake and juvenile hormone (JH)-regulated remodeling of follicular epithelium (also called ‘patency’). G protein-coupled receptor (GPCR) family is one of the largest membrane receptor protein families and controls many key physiological processes. To investigate the role of GPCRs in insect reproduction and juvenile hormone-regulated Vg uptake, we performed a comprehensive RNA interference (RNAi) screen targeting GPCRs in the red flour beetle, Tribolium castaneum. Out of 112 GPCRs tested, knockdown of 41 GPCRs resulted in a reduction in fecundity. Interestingly, RNAi against two GPCRs (a Rhodopsin-like receptor and a Dopamine D2-like receptor) led to a significant reduction in Vg accumulation in developing oocytes. Functional assays of these two GPCRs showed that JH triggers a dose-dependent inhibition of intracellular cAMP levels in HEK293 cells expressing Tribolium Dopamine D2-like receptor. These data suggest that Dopamine D2-like receptor plays crucial roles in regulating Vg uptake and is a promising candidate membrane receptor mediating JH regulation of patency in the red flour beetle

Identification of G Protein-Coupled Receptors Required for Vitellogenin Uptake into the Oocytes of the Red Flour Beetle, \u3cem\u3eTribolium castaneum\u3c/em\u3e

Previous studies suggested that a membrane receptor might be involved in mediating vitellogenin (Vg) uptake and juvenile hormone (JH)-regulated remodeling of follicular epithelium (also called ‘patency’). G protein-coupled receptor (GPCR) family is one of the largest membrane receptor protein families and controls many key physiological processes. To investigate the role of GPCRs in insect reproduction and juvenile hormone-regulated Vg uptake, we performed a comprehensive RNA interference (RNAi) screen targeting GPCRs in the red flour beetle, Tribolium castaneum. Out of 112 GPCRs tested, knockdown of 41 GPCRs resulted in a reduction in fecundity. Interestingly, RNAi against two GPCRs (a Rhodopsin-like receptor and a Dopamine D2-like receptor) led to a significant reduction in Vg accumulation in developing oocytes. Functional assays of these two GPCRs showed that JH triggers a dose-dependent inhibition of intracellular cAMP levels in HEK293 cells expressing Tribolium Dopamine D2-like receptor. These data suggest that Dopamine D2-like receptor plays crucial roles in regulating Vg uptake and is a promising candidate membrane receptor mediating JH regulation of patency in the red flour beetle

Juvenile hormone and insulin regulate trehalose homeostasis in the red flour beetle, Tribolium castaneum

Insulin/IGF-1 signaling (IIS) has been well studied for its role in the control of life span extension and resistance to a variety of stresses. The Drosophila melanogaster insulin-like receptor (InR) mutant showed extended life span due to reduced juvenile hormone (JH) levels. However, little is known about the mechanism of cross talk between IIS and JH in regulation of life span extension and resistance to starvation. In the current study, we investigated the role of IIS and JH signaling in regulation of resistance to starvation. Reduction in JH biosynthesis, JH action, or insulin-like peptide 2 (ILP2) syntheses by RNA interference (RNAi)-aided knockdown in the expression of genes coding for juvenile hormone acid methyltransferase (JHAMT), methoprene-tolerant (Met), or ILP2 respectively decreased lipid and carbohydrate metabolism and extended the survival of starved beetles. Interestingly, the extension of life span could be restored by injection of bovine insulin into JHAMT RNAi beetles but not by application of JH III to ILP2 RNAi beetles. These data suggest that JH controls starvation resistance by regulating synthesis of ILP2. More importantly, JH regulates trehalose homeostasis, including trehalose transport and metabolism, and controls utilization of stored nutrients in starved adults

Nutrition Regulation of Male Accessory Gland Growth and Maturation in \u3cem\u3eTribolium castaneum\u3c/em\u3e

Insulin/IGF-1 signaling (IIS) pathway is known to control growth, development and reproduction. Insulin-like peptide mediated body size plasticity in Drosophila melanogaster has been reported. Here, our studies showed that IIS pathway and nutrition regulate growth and maturation of the male accessory gland (MAG) in the red flour beetle, Tribolium castaneum. The size of MAG increased from day 1 to day 5 post-adult emergence (PAE). This increase in the size of MAG is contributed by an increase in cell size, but not cell number. The growth of MAG was impaired after double-stranded RNA (dsRNA)-mediated knockdown in the expression of genes coding for ILP3, InR, Chico, PI3k, AKT, and GATA1 involved in IIS pathway. Interestingly, starvation showed similar effects on the growth and maturation of MAG. The phenotypes observed in animals where IIS signaling pathway genes were knocked down are similar to the phenotypes observed after starving beetles for 5 days PAE. These data suggest that nutrition signals working through IIS pathway regulate maturation of MAG by promoting the growth of MAG cells

Comparative Analysis of Double-Stranded RNA Degradation and Processing in Insects

RNA interference (RNAi) based methods are being developed for pest management. A few products for control of coleopteran pests are expected to be commercialized soon. However, variability in RNAi efficiency among insects is preventing the widespread use of this technology. In this study, we conducted research to identify reasons for variability in RNAi efficiency among thirty-seven (37) insects belonging to five orders. Studies on double-stranded RNA (dsRNA) degradation by dsRNases and processing of labeled dsRNA to siRNA showed that both dsRNA degradation and processing are variable among insects belonging to different orders as well as among different insect species within the same order. We identified homologs of key RNAi genes in the genomes of some of these insects and studied their domain architecture. These data suggest that dsRNA digestion by dsRNases and its processing to siRNAs in the cells are among the major factors contributing to differential RNAi efficiency reported among insects

Double-Stranded RNA Binding Protein, Staufen, Is Required for the Initiation of RNAi in Coleopteran Insects

RNA interference (RNAi) is being used to develop methods to control pests and disease vectors. RNAi is robust and systemic in coleopteran insects but is quite variable in other insects. The determinants of efficient RNAi in coleopterans, as well as its potential mechanisms of resistance, are not known. RNAi screen identified a double-stranded RNA binding protein (StaufenC) as a major player in RNAi. StaufenC homologs have been identified in only coleopteran insects. Experiments in two coleopteran insects, Leptinotarsa decemlineata and Tribolium castaneum, showed the requirement of StaufenC for RNAi, especially for processing of double-stranded RNA (dsRNA) to small interfering RNA. RNAi-resistant cells were selected by exposing L. decemlineata, Lepd-SL1 cells to the inhibitor of apoptosis 1 dsRNA for multiple generations. The resistant cells showed lower levels of StaufenC expression compared with its expression in susceptible cells. These studies showed that coleopteran-specific StaufenC is required for RNAi and is a potential target for RNAi resistance. The data included in this article will help improve RNAi in noncoleopteran insects and manage RNAi resistance in coleopteran insects

Identification of Ecdysone Hormone Receptor Agonists as a Therapeutic Approach for Treating Filarial Infections

Background A homologue of the ecdysone receptor has previously been identified in human filarial parasites. As the ecdysone receptor is not found in vertebrates, it and the regulatory pathways it controls represent attractive potential chemotherapeutic targets. Methodology/ Principal Findings Administration of 20-hydroxyecdysone to gerbils infected with B. malayi infective larvae disrupted their development to adult stage parasites. A stable mammalian cell line was created incorporating the B. malayi ecdysone receptor ligand-binding domain, its heterodimer partner and a secreted luciferase reporter in HEK293 cells. This was employed to screen a series of ecdysone agonist, identifying seven agonists active at sub-micromolar concentrations. A B. malayi ecdysone receptor ligand-binding domain was developed and used to study the ligand-receptor interactions of these agonists. An excellent correlation between the virtual screening results and the screening assay was observed. Based on both of these approaches, steroidal ecdysone agonists and the diacylhydrazine family of compounds were identified as a fruitful source of potential receptor agonists. In further confirmation of the modeling and screening results, Ponasterone A and Muristerone A, two compounds predicted to be strong ecdysone agonists stimulated expulsion of microfilaria and immature stages from adult parasites. Conclusions The studies validate the potential of the B. malayi ecdysone receptor as a drug target and provide a means to rapidly evaluate compounds for development of a new class of drugs against the human filarial parasites

BmILF and I-motif Structure Are Involved in Transcriptional Regulation of \u3cem\u3eBmPOUM2\u3c/em\u3e in \u3cem\u3eBombyx mori\u3c/em\u3e

Guanine-rich and cytosine-rich DNA can form four-stranded DNA secondary structures called G-quadruplex (G4) and i-motif, respectively. These structures widely exist in genomes and play important roles in transcription, replication, translation and protection of telomeres. In this study, G4 and i-motif structures were identified in the promoter of the transcription factor gene BmPOUM2, which regulates the expression of the wing disc cuticle protein gene (BmWCP4) during metamorphosis. Disruption of the i-motif structure by base mutation, anti-sense oligonucleotides (ASOs) or inhibitory ligands resulted in significant decrease in the activity of the BmPOUM2 promoter. A novel i-motif binding protein (BmILF) was identified by pull-down experiment. BmILF specifically bound to the i-motif and activated the transcription of BmPOUM2. The promoter activity of BmPOUM2 was enhanced when BmILF was over-expressed and decreased when BmILF was knocked-down by RNA interference. This study for the first time demonstrated that BmILF and the i-motif structure participated in the regulation of gene transcription in insect metamorphosis and provides new insights into the molecular mechanism of the secondary structures in epigenetic regulation of gene transcription

Hormonal regulation of the humoral innate immune response in Drosophila melanogaster

Juvenile hormone (JH) and 20-hydroxy-ecdysone (20E) are highly versatile hormones, coordinating development, growth, reproduction and aging in insects. Pulses of 20E provide key signals for initiating developmental and physiological transitions, while JH promotes or inhibits these signals in a stage-specific manner. Previous evidence suggests that JH and 20E might modulate innate immunity, but whether and how these hormones interact to regulate the immune response remains unclear. Here we show that JH and 20E have antagonistic effects on the induction of antimicrobial peptide (AMP) genes in Drosophila melanogaster. 20E pretreatment of Schneider S2* cells promoted the robust induction of AMP genes, following immune stimulation. On the other hand, JH III, and its synthetic analogs (JHa) methoprene and pyriproxyfen, strongly interfered with this 20E-dependent immune potentiation, although these hormones did not inhibit other 20E-induced cellular changes. Similarly, in vivo analyses in adult flies confirmed that JH is a hormonal immuno-suppressor. RNA silencing of either partner of the ecdysone receptor heterodimer (EcR or Usp) in S2* cells prevented the 20E-induced immune potentiation. In contrast, silencing methoprene-tolerant (Met), a candidate JH receptor, did not impair immuno- suppression by JH III and JHa, indicating that in this context MET is not a necessary JH receptor. Our results suggest that 20E and JH play major roles in the regulation of gene expression in response to immune challenge

Methyl Farnesoate Plays a Dual Role in Regulating \u3cem\u3eDrosophila\u3c/em\u3e Metamorphosis

Corpus allatum (CA) ablation results in juvenile hormone (JH) deficiency and pupal lethality in Drosophila. The fly CA produces and releases three sesquiterpenoid hormones: JH III bisepoxide (JHB3), JH III, and methyl farnesoate (MF). In the whole body extracts, MF is the most abundant sesquiterpenoid, followed by JHB3 and JH III. Knockout of JH acid methyl transferase (jhamt) did not result in lethality; it decreased biosynthesis of JHB3, but MF biosynthesis was not affected. RNAi-mediated reduction of 3-hydroxy-3-methylglutaryl CoA reductase (hmgcr) expression in the CA decreased biosynthesis and titers of the three sesquiterpenoids, resulting in partial lethality. Reducing hmgcr expression in the CA of the jhamt mutant further decreased MF titer to a very low level, and caused complete lethality. JH III, JHB3, and MF function through Met and Gce, the two JH receptors, and induce expression of Kr-h1, a JH primary-response gene. As well, a portion of MF is converted to JHB3 in the hemolymph or peripheral tissues. Topical application of JHB3, JH III, or MF precluded lethality in JH-deficient animals, but not in the Met gce double mutant. Taken together, these experiments show that MF is produced by the larval CA and released into the hemolymph, from where it exerts its anti-metamorphic effects indirectly after conversion to JHB3, as well as acting as a hormone itself through the two JH receptors, Met and Gce