RNA silencing of hormonal biosynthetic genes impairs larval growth and development in cotton bollworm, Helicoverpa armigera.

The cotton bollworm, Helicoverpa armigera, is a highly polyphagous pest, causing enormous losses to various economically important crops. The identification and in vitro functional validation of target genes of a pest is a prerequisite to combat pest via host-mediated RNA interference (RNAi). In the present study, six hormonal biosynthesis genes of H. armigera were chosen and evaluated by feeding insect larvae with dsRNAs corresponding to each target gene, viz., juvenile hormone acid methyltransferase (HaJHAMT), prothoracicotropic hormone (HaPTTH), pheromone biosynthesis-activating peptide (HaPBAP), molt regulating transcription factor (HaHR3), activated protein 4 (HaAP-4) and eclosion hormone precursor (HaEHP). The loss of function phenotypes for these hormonal genes were observed by releasing second instar larvae on to artificial diet containing target gene-specific dsRNAs. Ingestion of dsRNAs resulted in mortality ranging from 60% to 90%, reduced larval weight, phenotypic deformities and delayed pupation. The quantitative real-time PCR (qRT-PCR) analysis showed that the target gene transcript levels were decreased drastically (31% to 77%) as compared to control or unrelated control (GFP-dsRNA), and correlated well with the mortality and developmental defects of larvae. Also, a comparison of the silencing efficacy of un-diced long HaPTTH -dsRNAwith RNase III diced HaPTTH-dsRNA (siRNAs) revealed that long dsRNAs were more efficient in silencing the target gene. These results indicated that the hormonal biosynthesis genes have varied sensitivity towards RNAi and could be the vital targets for insect resistance in crop plants like cotton which are infested by H. armigera.

Ecdysone-dependent feedback regulation of prothoracicotropic hormone controls the timing of developmental maturation.

The activation of a neuroendocrine system that induces a surge in steroid production is a conserved initiator of the juvenile-to-adult transition in many animals. The trigger for maturation is the secretion of brain-derived neuropeptides, yet the mechanisms controlling the timely onset of this event remain ill-defined. Here, we show that a regulatory feedback circuit controlling the Drosophila neuropeptide Prothoracicotropic hormone (PTTH) triggers maturation onset. We identify the Ecdysone Receptor (EcR) in the PTTH-expressing neurons (PTTHn) as a regulator of developmental maturation onset. Loss of EcR in these PTTHn impairs PTTH signaling, which delays maturation. We find that the steroid ecdysone dose-dependently affects Ptth transcription, promoting its expression at lower concentrations and inhibiting it at higher concentrations. Our findings indicate the existence of a feedback circuit in which rising ecdysone levels trigger, via EcR activity in the PTTHn, the PTTH surge that generates the maturation-inducing ecdysone peak toward the end of larval development. Because steroid feedback is also known to control the vertebrate maturation-inducing hypothalamic-pituitary-gonadal axis, our findings suggest an overall conservation of the feedback-regulatory neuroendocrine circuitry that controls the timing of maturation initiation.

Discovery of a Manduca sexta Allatotropin Antagonist from a Manduca sexta Allatotropin Receptor Homology Model.

Insect G protein coupled receptors (GPCRs) have important roles in modulating biology, physiology and behavior. They have been identified as candidate targets for next-generation insecticides, yet these targets have been relatively poorly exploited for insect control. In this study, we present a pipeline of novel Manduca sexta allatotropin (Manse-AT) antagonist discovery with homology modeling, docking, molecular dynamics simulation and structure-activity relationship. A series of truncated and alanine-replacement analogs of Manse-AT were assayed for the stimulation of juvenile hormone biosynthesis. The minimum sequence required to retain potent biological activity is the C-terminal amidated octapeptide Manse-AT (6-13). We identified three residues essential for bioactivity (Thr4, Arg6 and Phe8) by assaying alanine-replacement analogs of Manse-AT (6-13). Alanine replacement of other residues resulted in reduced potency but bioactivity was retained. The 3D structure of the receptor (Manse-ATR) was built and the binding pocket was identified. The binding affinities of all the analogs were estimated by calculating the free energy of binding. The calculated binding affinities corresponded to the biological activities of the analogs, which supporting our localization of the binding pocket. Then, based on the docking and molecular dynamics studies of Manse-AT (10-13), we described it can act as a potent Manse-AT antagonist. The antagonistic effect on JH biosynthesis of Manse-AT (10-13) validated our hypothesis. The IC50 value of antagonist Manse-AT (10-13) is 0.9 nM. The structure-activity relationship of antagonist Manse-AT (10-13) was also studied for the further purpose of investigating theoretically the structure factors influencing activity. These data will be useful for the design of new Manse-AT agonist and antagonist as potential pest control agents.

Characterization of a set of abdominal neuroendocrine cells that regulate stress physiology using colocalized diuretic peptides in Drosophila.

Multiple neuropeptides are known to regulate water and ion balance in Drosophila melanogaster. Several of these peptides also have other functions in physiology and behavior. Examples are corticotropin-releasing factor-like diuretic hormone (diuretic hormone 44; DH44) and leucokinin (LK), both of which induce fluid secretion by Malpighian tubules (MTs), but also regulate stress responses, feeding, circadian activity and other behaviors. Here, we investigated the functional relations between the LK and DH44 signaling systems. DH44 and LK peptides are only colocalized in a set of abdominal neurosecretory cells (ABLKs). Targeted knockdown of each of these peptides in ABLKs leads to increased resistance to desiccation, starvation and ionic stress. Food ingestion is diminished by knockdown of DH44, but not LK, and water retention is increased by LK knockdown only. Thus, the two colocalized peptides display similar systemic actions, but differ with respect to regulation of feeding and body water retention. We also demonstrated that DH44 and LK have additive effects on fluid secretion by MTs. It is likely that the colocalized peptides are coreleased from ABLKs into the circulation and act on the tubules where they target different cell types and signaling systems to regulate diuresis and stress tolerance. Additional targets seem to be specific for each of the two peptides and subserve regulation of feeding and water retention. Our data suggest that the ABLKs and hormonal actions are sufficient for many of the known DH44 and LK functions, and that the remaining neurons in the CNS play other functional roles.

Allatostatin-C antagonizes the synergistic myostimulatory effect of allatotropin and serotonin in Rhodnius prolixus (Stal).

Haematophagous insects can ingest large quantities of blood in a single meal producing a large quantity of urine in the following hours to eliminate the excess of water and mineral ions incorporated. The excretory activity of the Malpighian tubules is facilitated by an increase in haemolymph circulation as a result of the intensification of aorta contractions, combined with an increase of anterior midgut peristaltic waves. We have recently shown that haemolymph circulation during post-prandial diuresis is modulated by the synergistic activity of allatotropin (AT) and serotonin, resulting in an increase in aorta and crop contraction rates. In the present study we describe the antagonistic effect of allatostatin-C (AST-C) on the increase of aorta frequency of contractions induced by serotonin/AT in Rhodnius prolixus. The administration of AST-C counteracted the increase in the frequency induced by the treatment with serotonin/AT, but did not affect the increase in frequency induced by the administration of serotonin alone, suggesting that AST-C is altering the synergism between serotonin and AT. Furthermore, the administration of AST-C during post-prandial diuresis decreases the number of peristaltic waves of the anterior midgut. The AST-C putative receptor is expressed in the hindgut, midgut and dorsal vessel, three critical organs involved in post-prandial diuresis. All together these findings provide evidence that AST-C plays a key role as a myoregulatory and cardioregulatory peptide in R. prolixus.

Molecular cloning and structural characterization of Ecdysis Triggering Hormone from Choristoneura fumiferana.

At the end of each stadium, insects undergo a precisely orchestrated process known as ecdysis which results in the replacement of the old cuticle with a new one. This physiological event is necessary to accommodate growth in arthropods since they have a rigid chitinous exoskeleton. Ecdysis is initiated by the direct action of Ecdysis Triggering Hormones on the central nervous system. Choristoneura fumiferana is a major defoliator of coniferous forests in Eastern North America. It is assumed that, studies on the ecdysis behavior of this pest might lead to the development of novel pest management strategies. Hence in this study, the cDNA of CfETH was cloned. The open reading frame of the cDNA sequence was found to encode three putative peptides viz., Pre-Ecdysis Triggering Hormone (PETH), Ecdysis Triggering Hormone (ETH), and Ecdysis Triggering Hormone Associated Peptide (ETH-AP). The CfETH transcript was detected in the epidermal tissue of larval and pupal stages, but not in eggs and adults. In order to explore the structural conformation of ETH, ab initio modelling and Molecular Dynamics (MD) Simulations were performed. Further, a library of insecticides was generated and virtual screening was performed to identify the compounds displaying high binding capacity to ETH.

Effect of exogenous hormones on transcription levels of pyridoxal 5'-phosphate biosynthetic enzymes in the silkworm (Bombyx mori).

Vitamin B6 includes 6 pyridine derivatives, among which pyridoxal 5'-phosphate is a coenzyme for over 140 enzymes. Animals acquire their vitamin B6 from food. Through a salvage pathway, pyridoxal 5'-phosphate is synthesized from pyridoxal, pyridoxine or pyridoxamine, in a series of reactions catalyzed by pyridoxal kinase and pyridoxine 5'-phosphate oxidase. The regulation of pyridoxal 5'-phospahte biosynthesis and pyridoxal 5'-phospahte homeostasis are at the center of study for vitamin B6 nutrition. How pyridoxal 5'-phosphate biosynthesis is regulated by hormones has not been reported so far. Our previous studies have shown that pyridoxal 5'-phosphate level in silkworm larva displays cyclic developmental changes. In the current study, effects of exogenous juvenile hormone and molting hormone on the transcription level of genes coding for the enzymes involved in the biosynthesis of pyridoxal 5'-phospahte were examined. Results show that pyridoxal kinase and pyridoxine 5'-phosphate oxidase are regulated at the transcription level by development and are responsive to hormones. Molting hormone stimulates the expression of genes coding for pyridoxal kinase and pyridoxine 5'-phosphate oxidase, and juvenile hormone appears to work against molting hormone. Whether pyridoxal 5'-phosphate biosynthesis is regulated by hormones in general is an important issue for further studies.

The Neuropeptide Allatostatin A Regulates Metabolism and Feeding Decisions in Drosophila.

Coordinating metabolism and feeding is important to avoid obesity and metabolic diseases, yet the underlying mechanisms, balancing nutrient intake and metabolic expenditure, are poorly understood. Several mechanisms controlling these processes are conserved in Drosophila, where homeostasis and energy mobilization are regulated by the glucagon-related adipokinetic hormone (AKH) and the Drosophila insulin-like peptides (DILPs). Here, we provide evidence that the Drosophila neuropeptide Allatostatin A (AstA) regulates AKH and DILP signaling. The AstA receptor gene, Dar-2, is expressed in both the insulin and AKH producing cells. Silencing of Dar-2 in these cells results in changes in gene expression and physiology associated with reduced DILP and AKH signaling and animals lacking AstA accumulate high lipid levels. This suggests that AstA is regulating the balance between DILP and AKH, believed to be important for the maintenance of nutrient homeostasis in response to changing ratios of dietary sugar and protein. Furthermore, AstA and Dar-2 are regulated differentially by dietary carbohydrates and protein and AstA-neuronal activity modulates feeding choices between these types of nutrients. Our results suggest that AstA is involved in assigning value to these nutrients to coordinate metabolic and feeding decisions, responses that are important to balance food intake according to metabolic needs.

Diapause hormone in the Helicoverpa/Heliothis complex: A review of gene expression, peptide structure and activity, analog and antagonist development, and the receptor.

This review summarizes recent studies focusing on diapause hormone (DH) in the Helicoverpa/Heliothis complex of agricultural pests. Moths in this complex overwinter in pupal diapause, a form of developmental arrest used to circumvent unfavorable seasons. DH was originally reported in the silkmoth Bombyx mori, a species that relies on DH to induce an embryonic diapause. But, in the case of Helicoverpa/Heliothis, levels of dh transcripts and DH peptides are more abundant in nondiapausing pupae than in diapausing individuals, and DH effectively terminates diapause within a specific temperature range. A structure activity relationship study indicated that the active core of DH is the C-terminal hepta-peptide, LWFGPRLa. We designed and synthesized a first generation of DH agonists and identified two agonists (PK-2Abf and PK-Etz) that were nearly 50- and 13-fold more potent than the native hormone. These studies revealed two structural characteristics of DH and its agonists that are essential for interaction with the receptor: a trans-Pro configuration to form a type I ß-turn and a hydrophobic moiety involved in ligand binding. Modification of DH at the active core yielded a potent DH antagonist (DH-Jo, acetyl-GLWA[Jo]RLa) as well as an agonist (DH-2Abf-K). Three compounds (Decyl-1963, Dodecyl-1967, Heptyl-1965) were identified as agents capable of penetrating the cuticle of young pupae and thereby preventing the entry into diapause. DH receptor cDNA was cloned and an effective in vitro high throughput screen system was established for future use. This work sets the stage for further development of DH analogs and antagonists that have the potential to disrupt insect diapause as a tool for pest management.

Knockdown of adipokinetic hormone synthesis increases susceptibility to oxidative stress in Drosophila--a role for dFoxO?

Insect adipokinetic hormones (AKHs) are pleiotropic hormones known to play a protective role in response to oxidative stress (OS). However, the precise signaling pathways are unclear. We present evidence that AKH may primarily employ the Forkhead box class O transcription factor (FoxO) to exert this effect. The impact of knocking down AKH synthesis or its over-expression in its response to OS was studied in Drosophila melanogaster. AKH knockdown (AKH-RNAi) as well as AKH overexpression (AKH-oex) was achieved using the Gal-4/UAS system and controls were w(1118) (+/+), AKH-Gal4/+, UAS-AKH/+ and UAS-AKH-RNAi/+. Exposure to 80 µM hydrogen peroxide (HP) revealed that AKH-RNAi flies showed significantly higher mortality than AKH-oex or the respective control lines. This susceptibility was evidenced by significantly enhanced levels of protein carbonyls - a biomarker of OS, in AKH-RNAi flies compared to controls and AKH-oex flies. Interestingly, AKH-oex flies had the least amount of protein carbonyls. AKH-RNAi flies had significantly less dFoxO transcript and translated protein compared to control and AKH-oex flies in un-challenged condition as well as when challenged with HP. Sestrin - a major antioxidant defense protein and one of the targets of dFoxO - was also significantly down-regulated (both at mRNA and protein level) in AKH-RNAi flies (both unchallenged and challenged with HP) compared to control flies and flies with over-expressed AKH. These findings imply that dFoxO may act downstream of AKH as a transcription factor to mediate response to OS in D. melanogaster.

A neuronal pathway that controls sperm ejection and storage in female Drosophila.

In polyandrous females, sperm storage permits competition between sperm of different mates, and in some species females influence the relative fertilization success of competing sperm in favor of a preferred mate [1, 2]. In female Drosophila melanogaster, sperm competition is strongly influenced by the timing of sperm ejection from the uterus [3, 4]. Understanding how female behavior influences sperm competition requires knowledge of the neuronal mechanisms controlling sperm retention and storage, which is currently lacking. Here, we show that D. melanogaster females eject male ejaculates from the uterus 1-6 hr after mating with a stereotypic behavior regulated by a brain signaling pathway composed of diuretic hormone 44 (Dh44), a neuropeptide related to vertebrate corticotropin-releasing factor (CRF), and its receptor, Dh44R1. Suppression of Dh44 signals in the brain expedites sperm ejection from the uterus, resulting in marked reduction of sperm in the storage organs and decreased fecundity, whereas enhancement of Dh44 signals delays sperm expulsion. The Dh44 function was mapped to six neurons located in the pars intercerebralis of the brain together with a small subset of Dh44R1 neurons that express the sex-specific transcription factor doublesex. This study identifies a neuronal pathway by which females can control sperm retention and storage and provides new insight into how the female might exercise post-copulatory sexual selection.

[Effect of plants synthesizing biologically active substances-precocenes on insects]. / Vliianie rastenii, sinteziruiushchikh biologicheski aktivnye veshchestva-prekotseny, na nasekomykh.

Ntegrated data on the direct and indirect influence of plants synthesizing biologically active substances--precocenes--on phytophagous insects developing and feeding on these plants. The experimental data on individual organs and tissues as well as organ systems in various insect groups are discussed.

Inhibition of specific degradation of 57-kDa protein in royal jelly during storage by ethylenediaminetetraacetic acid.

We have previously shown that 57-kDa protein in royal jelly (RJ) was specifically degraded in proportion to both storage temperature and storage period, and we suggested that it could be useful as a marker of freshness of RJ (Kamakura, M., Fukuda, T., Fukushima, M. and Yonekura, M., Biosci. Biotechnol. Biochem., 65, 277-284 (2001).). Here, we investigated the effects of various proteinase inhibitors on proteinase activity in RJ and on the specific degradation of 57-kDa protein during storage. Ethylenediaminetetraacetic acid (EDTA), but not other inhibitors, inhibited the proteinase activity in RJ, and dose-dependently suppressed storage-dependent degradation of 57-kDa protein. These results suggest that EDTA inhibits a specific proteinase activity in RJ, thereby suppressing the degradation of 57-kDa protein during storage at high temperature.

Synthesis of lipids in the fat body of Gryllus bimaculatus: age-dependency and regulation by adipokinetic hormone.

The free abdominal fat body of adult female Mediterranean field crickets, Gryllus bimaculatus, synthesizes lipids from [1-(14)C]-acetate in vitro. Up to an age of 12h, the incorporation of acetate into lipids is very low and then rises to a maximum 24h after adult emergence. Thereafter, the incorporation of acetate decreases to moderate levels at day 2 and then slowly decreases until day 30. The adipokinetic hormone of G. bimaculatus (Grb-AKH) significantly inhibits the incorporation of acetate at a concentration of 10(-11) M; maximum inhibition (approximately 95%) is reached at 10(-8) M. The inhibiting effect of Grb-AKH is fast, dose-dependent, and reversible. The periovaric fat body shows a similar pattern of acetate incorporation, although rates of incorporation are lower; the incorporation can be inhibited by Grb-AKH as well. The segmental abdominal fat body and the fat body from the head both incorporate acetate into lipids at low rates that cannot be inhibited significantly by AKH. Prepurified brain extracts significantly inhibit acetate incorporation by free abdominal fat bodies at a concentration of 0.1 brain equivalent. Allatostatins and crustacean cardioactive peptide, which are both present in cricket brains, are not responsible for this inhibiting effect. Octopamine causes a dose-dependent inhibition of acetate incorporation whereas synephrine had no such effect. The inhibiting effect of Grb-AKH on the formation of lipid stores in the fat body and its consequences for reproductive processes are discussed.

Rapid degradation of the silkworm diapause hormone by trypsin and its suppression by VAP-map, a synthetic analog of the cuticular peptide of silkworm, Bm ACP-6.7 (VAP-peptide).

Very fast tryptic degradation of the silkworm diapause hormone was found and the degradation pathway was analyzed by moderating the reaction conditions. It proceeded via cleavage at Arg23 and finally at Arg15 of DH. As the C-terminal structure of DH was essential for exhibiting bioactivity, the first cleavage caused rapid inactivation of the hormone. This tryptic digestion was strongly suppressed by adding VAP-map, a synthetic analog of the cuticular peptide of silkmoths, Bm ACP-6.7 (VAP-peptide), which is a natural synergist of DH. VAP-map suppressed the enzymic reaction by interacting with the substrate, but not with the enzyme.

Insect neuropeptide antagonists.

The development of a new integrated approach to the generation of a novel type of insect neuropeptide (Np) antagonists and putative insect control agents based on backbone cyclic compounds is described. The approach, termed the backbone cyclic neuropeptide-based antagonist (BBC-NBA), was applied to the insect pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) family as a model, and led to the discovery of a potent linear lead antagonist and several highly potent, metabolically stable BBC antagonists, devoid of agonistic activity, which inhibited PBAN-mediated activities in moths in vivo. This review briefly summarizes our knowledge of insect Nps, describes the PK/PBAN Np family, presents the basic concepts behind the BBC-NBA approach, and introduces the advantages of this method for generation of Np agonists, antagonists and insecticide prototype molecules.

Alterations in ultraspiracle (USP) content and phosphorylation state accompany feedback regulation of ecdysone synthesis in the insect prothoracic gland.

Insect molting and metamorphosis are elicited by a class of ecdysteroids, mainly 20-hydroxyecdysone (20E), the precursor of which is synthesized in the prothoracic gland. 20E acts via the ecdysone receptor (EcR) and its heterodimer partner ultraspiracle (USP). Analysis of the prothoracic gland of Manduca sexta revealed that the developmental expression and phosphorylation of a specific USP form, p47, is positively correlated with ecdysteroidogenesis and that 20E, but not ecdysone, is responsible for initiating the translational expression and phosphorylation of p47. The latter forms a functional complex with EcR and the ligand-complex interaction results in the down regulation of ecdysteroidogenesis and the inhibition of prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis. The composite data suggest that USP plays a key role in modulating PTTH-stimulated ecdysteroid biosynthesis through the selective expression and phosphorylation of the p47 USP isoform.

Ecdysteroidostatin from the house fly, Musca domestica.

Ovaries from house flies maintained on sucrose secrete large amounts of ecdysteroid when they are cultured with ovarian ecdysteroidogenic hormone, OEH. However, ovarian ecdysteroid secretion is reduced by incubation with both OEH and the ovarian ecdysteroidostatin (OES). A partially purified OES fraction from a semi-preparative reverse phase HPLC C18 column caused a 98% inhibition of ovarian ecdysteroid secretion in vitro at a concentration of 0.8 equivalents per microliter. Ovaries can be activated to produce ecdysteroid in vivo by feeding diet containing protein to flies maintained on sucrose. Ecdysteroid secretion was inhibited when the in vivo stimulated ovaries were cultured with OES. This suggests that OES does not interfere with the OEH activation mechanism, but blocks ovarian ecdysteroid synthesis or release. Furthermore, OES inhibition is reversible and ecdysteroid secretion resumes when OES is removed. Musca OES could explain the decrease in ecdysteroid levels found in flies after mid-vitellogenesis. Both adult male and female abdomens contain OES, but OES was not transferred to females during mating. Evidence is presented that OES is not a trypsin modulating oostatic factor.

Effects of estrogenic xenobiotics on molting of the water flea, Daphnia magna.

The effects of five xenobiotics, 2,4,5-trichloribiphenyl (PCB29), the polychlorinated biphenyl (PCB) Aroclor 1242, diethyl phthalate, lindane, and 4-octylphenol, on molting of Daphnia magna were investigated. All except PCB29 are known to have unexpected estrogenicity in vertebrates. Daphnids exposed to PCB29, Aroclor 1242, and diethyl phthalate took significantly more time to complete four molts than did the controls. The inhibitory effects of these ortho-chlorinated PCBs suggest that certain structural features, most probably including ortho-chlorination, are related to the ability of a PCB to affect molting. Agents with multicyclic structures, such as PCBs, are more effective in inhibiting molting than are single-ringed xenobiotics, such as diethyl phthalate, which suggests that hydrophobicity may be a requirement for binding to the ecdysteroid receptor. These molt-inhibiting agents with multiple rings appear to bear more structural resemblance to the steroidal molting hormones of arthropods, the ecdysteroids, than do the single-ringed ones. While the possibility of alternative mechanisms, such as impairment of ecdysteroidogenesis exists, the results obtained herein support the hypothesis that some xenobiotics which disrupt endocrine processes in vertebrates can also interfere with the hormonally regulated molting process in arthropods through acting as antagonists of endogenous ecdysteroids by binding to and thereby blocking the ecdysteroid receptor.