Identification of a juvenile-hormone signaling inhibitor via high-throughput screening of a chemical library.

Insecticide resistance has recently become a serious problem in the agricultural field. Development of insecticides with new mechanisms of action is essential to overcome this limitation. Juvenile hormone (JH) is an insect-specific hormone that plays key roles in maintaining the larval stage of insects. Hence, JH signaling pathway is considered a suitable target in the development of novel insecticides; however, only a few JH signaling inhibitors (JHSIs) have been reported, and no practical JHSIs have been developed. Here, we established a high-throughput screening (HTS) system for exploration of novel JHSIs using a Bombyx mori cell line (BmN_JF&AR cells) and carried out a large-scale screening in this cell line using a chemical library. The four-step HTS yielded 69 compounds as candidate JHSIs. Topical application of JHSI48 to B. mori larvae caused precocious metamorphosis. In ex vivo culture of the epidermis, JHSI48 suppressed the expression of the Krüppel homolog 1 gene, which is directly activated by JH-liganded receptor. Moreover, JHSI48 caused a parallel rightward shift in the JH response curve, suggesting that JHSI48 possesses a competitive antagonist-like activity. Thus, large-scale HTS using chemical libraries may have applications in development of future insecticides targeting the JH signaling pathway.

3D-QSAR based optimization of insect neuropeptide allatostatin analogs.

Allatostatins (AST) are neuropeptides originally described as inhibitors of juvenile hormone (JH) synthesis in insects. Consequently, they have been considered as potential lead compounds for the discovery of new insect growth regulators (IGRs). In the present work, receptor-based three-dimensional quantitative structure-activity relationship (3D-QSAR) was studied with 48 AST analogs, and a general approach for novel potent bioactive AST analogs is proposed. Hence, six novel AST analogs were designed and synthesized. Bioassays indicated that the majority novel analogs exhibited potent JH inhibitory activity, especially analog A6 (IC50: 3.79 nmol/L), which can be used as lead compound to develop new IGRs.

Plant-derived compounds regulate formation of the insect juvenile hormone receptor complex.

Insect growth regulators (IGRs) are attractive pest control agents due to their high target specificity and relative safety to the environment. Recently, plants have been shown to synthesize IGRs that affect the insect juvenile hormone (JH) as a part of their defense mechanisms. Using a yeast two-hybrid system transformed with the Aedes aegypti JH receptor as a reporter system, we identified several JH agonists (JHAs) and antagonists (JHANs) causing retardation in the ovarian development of female Asian tiger mosquito, Aedes albopictus, from plant essential oil compounds. While the JHAs increased the expression of a JH-induced gene, the JHANs caused a reduction in the expression of the same gene. The compounds identified in this study could provide insights into plant-insect interactions and may be useful for the development of novel IGR insecticides.

A plant diterpene counteracts juvenile hormone-mediated gene regulation during Drosophila melanogaster larval development.

Many plant species possess compounds with juvenile hormone disruptor (JHD) activity. In some plant species, such activity has been attributed to diterpene secondary metabolites. Plant JHD diterpenes disrupt insect development by interfering with the juvenile hormone (JH)-mediated formation of JH receptor complexes. Here, we demonstrate that a plant extract and a diterpene from Lindera erythrocarpa (methyl lucidone) interfere with the formation of both methoprene-tolerant (Met)/Taiman and Germ cell-expressed (GCE)/Taiman heterodimer complexes in yeast two-hybrid assays in vitro. In addition to the in vitro JHD activity, the diterpene and the plant extract from L. erythrocarpa also disrupt the development of larvae and pupae in Drosophila melanogaster. Comparing the transcriptomes of juvenile hormone analog (JHA, methoprene)- and JHD (methyl lucidone)-fed wandering third-instar larvae revealed a large number of genes that were coregulated by JHA and JHD. Moreover, most (83%) of the genes that were repressed by methyl lucidone were significantly activated by methoprene, indicating that JHDs and JHAs have opposing effects on the transcriptional regulation of many JH-dependent genes. Gene ontology analysis also suggested that some of the genes activated-by-JHA/repressed-by-JHD play roles in spermatogenesis. Affymetrix microarray-based analysis indicated that the expression of genes activated-by-JHA/repressed-by-JHD was testis-specific. Together, these results suggest that JH is involved in testis-specific gene expression and that plant JHD diterpenes function as JH antagonists in such JHA-mediated gene regulation.

Improved strength of silk fibers in Bombyx mori trimolters induced by an anti-juvenile hormone compound.

BACKGROUND: Bombyx mori silk fibers with thin diameters have advantages of lightness and crease-resistance. Many studies have used anti-juvenile hormones to induce trimolters in order to generate thin silk; however, there has been comparatively little analysis of the morphology, structure and mechanical properties of trimolter silk. METHODS: This study induced two kinds of trimolters by appling topically anti-juvenile hormones and obtained thin diameter silk. Scanning electron microscope (SEM), FTIR analysis, tensile mechanical testing, chitin staining were used to reveal that the morphology, conformation and mechanical property of the trimolter silk. RESULTS: Cocoon of trimolters were highly densely packed by thinner fibers and thus had small apertures. We found that the conformation of trimolter silk fibroin changed and formed more ß-sheet structures. In addition, analysis of mechanical parameters yielded a higher Young's modulus and strength in trimolter silk than in the control. By chitin staining of silk gland, we postulated that the mechanical properties of trimolters' silk was enhanced greatly during to the structural changes of silk gland. CONCLUSION: We induced trimolters by anti-juvenile hormones and the resulting cocoons were more closely packed and had smaller silk fiber diameters. We found that the conformation of trimolters silk fibroin had a higher content of ß-sheet structures and better mechanical properties. GENERAL SIGNIFICANCE: Our study revealed the structures and mechanical properties of trimolter silk, and provided a valuable reference to improve silk quality by influencing molting in silkworms.

Synthesis and biological activity of FGLamide allatostatin analogs with Phe(3) residue modifications.

A FGLamide allatostatin neuropeptide mimic (H17) is a potential insect growth regulator which inhibits the production of juvenile hormone by the corpora allata. To find more evidence to reveal the structure-activity relationships of the Phe(3) residue in the C-terminal conserved pentapeptide and search for novel analogs with high activity, a series of Phe(3) residue-modified analogs were designed and synthesized using H17 as the lead compound. Bioassay using juvenile hormone (JH) production by corpora allata of the cockroach Diploptera punctata indicated that analogs 4, 11, and 13 showed strong ability to inhibit JH production in vitro, with IC50 of 38.5, 22.5, and 26 nM, respectively. As well, the activity of analog 2 (IC50 : 89.5 nM) proved roughly equivalent to that of H17. Based on the primary structure-activity relationships of Phe(3) residue, we suggest that for analogs containing six-membered aromatic rings, removing the methylene group of Phe(3) or an o-halogen or p-halogen-substituted benzene ring could increase the ability to inhibit biosynthesis of JH. This study will be useful for the design of new allatostatin analogs for insect management. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Effect of low doses of precocene on reproduction and gene expression in green peach aphid.

Insect reproduction can be stimulated by exposure to sublethal doses of insecticide that kill the same insects at high doses. This bi-phasic dose response to a stressor is known as hormesis and has been demonstrated with many different insect-insecticide models. The specific mechanisms of the increased reproduction in insects following sublethal pesticide exposure are unknown, but may be related to juvenile hormone (JH), which has a major role in regulation of metamorphosis and reproductive development in insects. We tested the hypothesis that exposure to sublethal concentrations of precocene, an antagonist of JH, would not result in stimulated reproductive outputs in the green peach aphid, Myzus persicae, as can be demonstrated with many neurotoxic insecticides. We also measured JH titers and the expression of various developmental (FPPS I), stress response (Hsp60), and dispersal (OSD, TOL and ANT) genes in aphids following exposure to the same precocene treatments. We found that when aphid nymphs were treated with certain sublethal concentrations of precocene, 1.5- to 2-fold increased reproductive stimulation occurred when they became adults, but this effect subsided in the following generation. Precocene treatments to nymphs resulted in no measurable effects on JH levels in subsequent reproducing adults. Although we detected major effects on gene expression following some precocene treatments (e.g. 100- to 300-fold increased expression of some genes), there were no clear relationships between gene expression and reproductive responses for a given treatment.

Identification of plant compounds that disrupt the insect juvenile hormone receptor complex.

Insects impact human health through vector-borne diseases and cause major economic losses by damaging crops and stored agricultural products. Insect-specific growth regulators represent attractive control agents because of their safety to the environment and humans. We identified plant compounds that serve as juvenile hormone antagonists (PJHANs). Using the yeast two-hybrid system transformed with the mosquito JH receptor as a reporter system, we demonstrate that PJHANs affect the JH receptor, methoprene-tolerant (Met), by disrupting its complex with CYCLE or FISC, formation of which is required for mediating JH action. We isolated five diterpene secondary metabolites with JH antagonist activity from two plants: Lindera erythrocarpa and Solidago serotina. They are effective in causing mortality of mosquito larvae at relatively low LD50 values. Topical application of two diterpenes caused reduction in the expression of Met target genes and retardation of follicle development in mosquito ovaries. Hence, the newly discovered PJHANs may lead to development of a new class of safe and effective pesticides.

Control of larval and egg development in Aedes aegypti with RNA interference against juvenile hormone acid methyl transferase.

RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including mosquitoes and many other insects. Little has been done, however, to harness this approach in order to control adult and larval mosquitoes. Juvenile hormone (JH) plays a pivotal role in the control of reproduction in adults and metamorphism in larval mosquitoes. This report describes an approach to control Aedes aegypti using RNAi against JH acid methyl transferase (AeaJHAMT), the ultimate enzyme in the biosynthetic pathway of JH III that converts JH acid III (JHA III) into JH III. In female A. aegypti that were injected or fed jmtA dsRNA targeting the AeaJHAMT gene (jmtA) transcript, egg development was inhibited in 50% of the treated females. In mosquito larvae that were fed transgenic Pichia pastoris cells expressing long hair pin (LHP) RNA, adult eclosion was delayed by 3 weeks causing high mortality. Northern blot analyses and qPCR studies show that jmtA dsRNA causes inhibition of jmtA transcript in adults and larvae, which is consistent with the observed inhibition of egg maturation and larval development. Taken together, these results suggest that jmtA LHP RNA expressed in heat inactivated genetically modified P. pastoris cells could be used to control mosquito populations in the marsh.

Ecdysone differentially regulates metamorphic timing relative to 20-hydroxyecdysone by antagonizing juvenile hormone in Drosophila melanogaster.

In insects, a steroid hormone, 20-hydroxyecdysone (20E), plays important roles in the regulation of developmental transitions by initiating signaling cascades via the ecdysone receptor (EcR). Although 20E has been well characterized as the molting hormone, its precursor ecdysone (E) has been considered to be a relatively inactive compound because it has little or no effect on classic EcR mediated responses. I found that feeding E to wild-type third instar larvae of Drosophila melanogaster accelerates the metamorphic timing, which results in elevation of lethality during metamorphosis and reduced body size, while 20E has only a minor effect. The addition of a juvenile hormone analog (JHA) to E impeded their precocious pupariation and thereby rescued the reduced body size. The ability of JHA impeding the effect of E was not observed in the Methoprene-tolerant (Met) and germ-cell expressed (gce) double mutant animals lacking JH signaling, indicating that antagonistic action of JH against E is transduced via a primary JH receptor, Met, or a product of its homolog, Gce. I also found that L3 larvae are susceptible to E around the time when they reach their minimum viable weight. These results indicate that E, and not just 20E, is also essential for proper regulation of developmental timing and body size. Furthermore, the precocious pupariation triggered by E is impeded by the action of JH to ensure that animals attain body size to survive metamorphosis.

Starvation increases insulin sensitivity and reduces juvenile hormone synthesis in mosquitoes.

BACKGROUND: The interactions between the insulin signaling pathway (ISP) and juvenile hormone (JH) controlling reproductive trade-offs are well documented in insects. JH and insulin regulate reproductive output in mosquitoes; both hormones are involved in a complex regulatory network, in which they influence each other and in which the mosquito's nutritional status is a crucial determinant of the network's output. Previous studies reported that the insulin-TOR (target of rapamacyn) signaling pathway is involved in the nutritional regulation of JH synthesis in female mosquitoes. The present studies further investigate the regulatory circuitry that controls both JH synthesis and reproductive output in response to nutrient availability. METHODS: We used a combination of diet restriction, RNA interference (RNAi) and insulin treatments to modify insulin signaling and study the cross-talk between insulin and JH in response to starvation. JH synthesis was analyzed using a newly developed assay utilizing fluorescent tags. CONCLUSIONS: Our results reveal that starvation decreased JH synthesis via a decrease in insulin signaling in the corpora allata (CA). Paradoxically, starvation-induced up regulation of insulin receptor transcripts and therefore "primed" the gland to respond rapidly to increases in insulin levels. During this response to starvation the synthetic potential of the CA remained unaffected, and the gland rapidly and efficiently responded to insulin stimulation by increasing JH synthesis to rates similar to those of CA from non-starved females.

[Antijuvenile influence of the precocene on the development of adult antennae in the apple surface eating tortricid Archips podana Scop. (Lepidoptera: Tortricidae)].

The influence ofprecocene II, an antijuvenile agent, on the development of adult antennae in the apple surface eating tortricid A. podana Scop was demonstrated. Treatment of the fifth instar larvae and prepupae with different doses of precocene proved to cause different sensitivity of the specimens to the juvenile hormone deficit. Treatment with 450 and 600 microg precocene per specimen during the first days after ecdysis to the fifth instar caused the death of larvae. Treatment with 300, 450, and 600 microg per specimen on the third day of the fifth instar larvae and prepupae caused a delay in the development of adult antennae. The results are discussed with respect to the role of the juvenile hormone in the development of imaginal structures during metamorphosis.

An anti-juvenile hormone agent, ethyl 4-(2-benzylhexyloxy)benzoate, inhibits juvenile hormone synthesis through the suppression of the transcription of juvenile hormone biosynthetic enzymes in the corpora allata in Bombyx mori.

Ethyl 4-[(S)-2-benzylhexyloxy)]benzoate (KF-13S), derived from ethyl 4-[2-(tert-butylcarbonyloxy)butoxy]benzoate (ETB), has strong anti-juvenile hormone (JH) activity which causes precocious metamorphosis in Bombyx mori, and the mode of action of this compound was studied. Application of KF-13S inhibited JH biosynthesis by the corpora allata (CA) in a reversible manner, and in vitro culture experiments showed that this inhibition was due to the direct action of this compound on the CA. When mRNA expression of the JH biosynthetic enzymes were studied, KF-13S strongly suppressed those of HMG Co-A synthase and HMG Co-A reductase. mRNA levels of other mevalonate enzymes and JH acid O-methyltransferase were also suppressed but were less sensitive to the compound. These studies showed that KF-13S prevents the transcription of many of the JH biosynthetic enzymes so that JH synthesis is suppressed.

Assessment of the pesticides atrazine, endosulfan sulphate and chlorpyrifos for juvenoid-related endocrine activity using Daphnia magna.

The water flea Daphnia magna belongs to the cyclical parthenogenic species, which can reproduce by either parthenogenesis or sexual reproduction. Recent studies have reported the involvement of the methylfarnesoate hormone, in male sex determination of D. magna. The aim of this study was to evaluate the juvenoid and anti-juvenoid activity of atrazine, endosulfan sulphate, and chlorpyrifos. To assess the juvenoid activity we exposed maternal daphnids to several concentrations of the respective pesticides, using the percentage of male production as endpoint. Fenoxycarb (1microgL(-1)) was used as a positive control. The anti-juvenoid activity was assessed using a similar bioassay after the addition of fenoxycarb (1microgL(-1)) to all test solutions. Fenoxycarb is an insect growth regulator that mimics the action of methylfarnesoate, and promoted the production of 95% of male offspring at the given concentration. Weak juvenoid-agonist activity was detected for endosulfan sulphate, with a significant increase of the percentage of male daphnids detected through logistic regression. In addition, atrazine and endosulfan sulphate antagonized the juvenoid activity of fenoxycarb. These results also corroborate the hypothesis that weak juvenoid agonists can simultaneously act as juvenoid antagonists as it has been observed in other hormonal systems.

Pest toxicology: the primary mechanisms of pesticide action.

Pesticides are used to control pests before they harm us or our crops. They are selective toxicants in the form and manner used. Pesticides must be effective without human or crop injury. They must also be safe relative to human and environmental toxicology. The study of how the pesticide works on the pest is referred to here as pest toxicology. About 700 pesticides, including insecticides, herbicides, and fungicides, act on perhaps 95 biochemical targets in pest insects, weeds, and destructive fungi. Current insecticides act primarily on four nerve targets, i.e., acetylcholinesterase, the voltage-gated chloride channel, the acetylcholine receptor, and the gamma-aminobutyric acid receptor, systems which are present in animals but not plants. Herbicides act mostly on plant specific pathways by blocking photosynthesis, carotenoid synthesis, or aromatic and branched chain amino acid synthesis essential in plants but not mammals. Many fungicides block ergosterol (the fungal sterol) or tubulin biosynthesis or cytochrome c reductase, while others disrupt basic cellular functions. A major limiting factor in the continuing use of almost all pesticides is the selection of strains not only resistant to the selecting or pressuring compounds but also cross-resistant to other pesticides acting at the same target. One approach to reinstating control is to shift from compounds with the resistant target site or mode of action to another set which have a sensitive target. This type of pesticide management led to the formation of Resistance Action Committees for insecticides, herbicides, and fungicides with very knowledgable experts to define resistance groups, which are in fact listings of primary target sites in pest toxicology. Continued success in pest and pesticide management requires an understanding of comparative biochemistry and molecular toxicology considering pests, people, and crops. Defining and applying the principles of pest toxicology are critical to food production and human health.

Identification of Phe-Gly-Leu-amide type allatostatin-7 in Reticulitermes flavipes: its localization in tissues and relation to juvenile hormone synthesis.

The allatostatins (ASTs), with a Tyr/Phe-Xaa-Phe-Gly-Leu/Ile-amide C-terminus, are neuropeptides that occur in many orders of insects, but are known to inhibit juvenile hormone (JH) synthesis by corpora allata (CA) only in cockroaches, crickets, and termites. 5 AST peptides with similar sequences to those of 6 species of cockroaches have been isolated and sequenced from extract of brain tissue of the termite Reticulitermes flavipes. The amino acid sequence of a 6th peptide, R. flavipes AST-7, determined by LC-MS/MS following HPLC fractionation of brain extract, is S-P-S-S-G-N-Q-R-L-Y-G-F-G-L-NH(2). The 8 terminal amino acids are identical to AST-7 of the cockroach Diploptera punctata. R. flavipes and D. punctata AST-7s inhibited JH synthesis by CA of both species equally and their affinity for antibody against D. punctata AST-7 is similar. Immunoreactivity of termite tissue with this antibody indicates neuro- and myomodulatory activity of the peptide in addition to its demonstrated allatostatic function. The density of AST immunostaining in axons within the CA of R. flavipes and the rate of JH synthesis by similar glands were negatively correlated. This is evidence that when AST is abundant in the glands it is being released in vivo to limit JH production.

Isolation of cockroach Phe-Gly-Leu-amide allatostatins from the termite Reticulitermes flavipes and their effect on juvenile hormone synthesis.

Immunoreactivity to cockroach Diploptera punctata allatostatin-7 (Dippu AST-7) has been demonstrated previously in axons innervating the corpora allata of the termite Reticulitermes flavipes. This peptide and Dippu AST-11 inhibited juvenile hormone (JH) synthesis by corpora allata (CA) of brachypterous neotenic reproductives (secondary reproductives) of termites. The present study shows that R. flavipes CA are also inhibited by Dippu AST-2, AST-5, AST-8, and AST-9 at approximately the same rank order of potency as demonstrated in D. punctata. Another allatostatin from Periplaneta americana (Peram AST-12) also inhibits JH synthesis by R. flavipes CA. Sensitivity to the allatostatins is higher in glands with low rates of JH synthesis than in those with relatively high JH synthetic rates as has been demonstrated in CA from male and female secondary reproductives as well as in those from non-egg-laying and egg-laying females. The identical inhibitory effects of R. flavipes brain extract on CA from both D. punctata and R. flavipes and the isolation and identification of five cockroach allatostatins (Dippu AST-1, AST-2, AST-5, AST-8, and Peram AST-12) from termite brain extract reflect the close relationship between cockroaches and termites.

Structure-activity studies with endogenous allatostatins from Periplaneta americana: expressed receptor compared with functional bioassay.

The A-allatostatins (F/YXFGLamides) are insect neuropeptides with inhibitory actions on juvenile hormone (JH) synthesis, muscular contraction and vitellogenesis. They exist in multiple forms within each species. In the cockroach, Periplaneta americana, only one receptor for A-allatostatin has been identified thus far. Here, we have characterised the receptor response to all 15 of the endogenous A-allatostatins encoded by the P. americana allatostatin prohormone gene, together with some analogues, using an indirect heterologous system involving co-expression of the receptor and a potassium channel subunit in Xenopus laevis oocytes and electrophysiological measurements. We have also determined the relative potency of the same peptides to inhibit JH synthesis in corpora allata. Our data reveal that the heterologously expressed receptor responds to all of the endogenous allatostatins and, although differences in potency are recorded, this cannot readily be related to particular differences in the primary structure of the peptides. Similarly, all allatostatins act on the corpora allata to inhibit the synthesis of JH, again with varying potency not readily related to peptide structure. Interestingly, some of the peptides did not perform consistently across the two assays. We show that the receptor is widely expressed in adult P. americana tissues (head, retrocerebral glands, fat body, ovary, male accessory gland, gut, leg muscle, Malpighian tubule and nerve cord) as well as in early larval instars. The spatial expression supports the known pleiotropic activity of allatostatins and role as a paracrine effector. This is the first report of such a detailed characterisation of an invertebrate receptor for allatostatin.

Juvenile hormone analogs do not affect directly the activity of the ecdysteroid receptor complex in insect culture cell lines.

During insect development, ecdysteroids and juvenile hormones (JHs) interact to regulate larval growth, metamorphosis and reproduction but the molecular mechanisms by which both hormones influence each other's activity remain unknown. Because of their ease of use and straightforward genetic manipulation, insect cell lines often have been used to clarify the actions and interactions of hormones at the molecular level. Here we report on the use of two insect culture cell lines, Drosophila melanogaster S2 and Bombyx mori Bm5 cells, to investigate two molecular processes in which ecdysteroids and JH have been shown to interact: (1) direct modulation of the activity of the ecdysteroid receptor transcription complex and (2) interference at the level of induction of the primary gene E75. Our data do not support JH analogs (JHAs) acting through the above processes: 'antagonism' of ecdysteroid receptor activity by JHAs correlated with cytotoxicity and induction of E75 expression by JHAs was not demonstrated. However, we confirm previous studies in which it was observed that methoprene can partially reverse the growth inhibition by 20E in S2 cells (but not Bm5 cells). Therefore, the molecular mechanism by which both hormones influence each other's activity to regulate cell growth in S2 cells remains unknown.