Effect of ecdysone agonists on vitellogenesis and the expression of EcR and USP in codling moth (Cydia pomonella)

The effects of tebufenozide and methoxyfenozide on vitellogenin (Vg) synthesis/release in the fat body, translocation in hemolymph, uptake by the ovary, and the expression of the ecdysone receptor (EcR) and its heterodimer partner, ultraspiracle protein (USP) in fat body, were investigated in Cydia pomonella. The results indicated that both ecdysone agonists significantly increased the Vg level in the adult hemolymph when the moths were exposed to agonist-treated surfaces. However, these agonists did not affect Vg release from the fat body nor Vg deposition in the first batch oocytes. Western blot analysis revealed that the expression of EcR and USP was significantly increased in tebufenozide- and methoxyfenozide-treated samples compared to the control, suggesting that ecdysone agonists regulated the Vg synthesis via the EcR and USP proteins complex.     

Toxicity and residual activity of methoxyfenozide and tebufenozide to codling moth (Lepidoptera: Tortricidae) and oriental fruit moth (Lepidoptera: Tortricidae)

A series of studies were conducted to examine the residual activity and toxicity of the ecdysone agonists tebufenozide and methoxyfenozide to codling moth, Cydia pomonella (L.), and oriental fruit moth, Grapholita molesta (Busck), in North Carolina apple systems. Methoxyfenozide exhibited greater activity than tebufenozide against codling moth eggs in dose-response bioassays, with a 4.5- and 5.3-fold lower LC50 value to eggs laid on fruit treated before or after oviposition, respectively. Oriental fruit moth eggs were 57- and 12-fold less sensitive to methoxyfenozide than were codling moth eggs on fruit treated before and after oviposition, respectively. Methoxyfenozide was effective in reducing larval entries of both codling moth and oriental fruit moth in field residual activity bioassays, exhibiting activity for at least 28 d after application. Residue breakdown on fruit was approximately 80% at 28 d after treatment for both methoxyfenozide and tebufenozide, with the most rapid residue decline (60%) occurring during the first 14 d after application. Two applications of methoxyfenozide applied at 14-d intervals provided better canopy coverage and higher residue levels than one application. Spray volume (683 versus 2,057 liters/ha) did not affect the efficacy of methoxyfenozide. Leaf and fruit expansion during the season was measured to determine potential plant-growth dilution effects on residual activity. There was very little increase in leaf area after mid May, but increase in fruit surface area over the season was described by a second order polynomial regression. Implications for codling moth and oriental fruit moth management programs are discussed.     

Towards Coleoptera-specific high-throughput screening systems for compounds with ecdysone activity: development of EcR reporter assays using weevil (Anthonomus grandis)-derived cell lines and in silico analysis of ligand binding to A. grandis EcR ligand-binding pocket

Molting in insects is regulated by ecdysteroids and juvenile hormones. Several synthetic non-steroidal ecdysone agonists are on the market as insecticides. These ecdysone agonists are dibenzoylhydrazine (DBH) analogue compounds that manifest their toxicity via interaction with the ecdysone receptor (EcR). Of the four commercial available ecdysone agonists, three (tebufenozide, methoxyfenozide and chromafenozide) are highly lepidopteran specific, one (halofenozide) is used to control coleopteran and lepidopteran insects in turf and ornamentals. However, compared to the very high binding affinity of these DBH analogues to lepidopteran EcRs, halofenozide has a low binding affinity for coleopteran EcRs. For the discovery of ecdysone agonists that target non-lepidopteran insect groups, efficient screening systems that are based on the activation of the EcR are needed. We report here the development and evaluation of two coleopteran-specific reporter-based screening systems to discover and evaluate ecdysone agonists. The screening systems are based on the cell lines BRL-AG-3A and BRL-AG-3C that are derived from the weevil Anthonomus grandis, which can be efficiently transduced with an EcR reporter cassette for evaluation of induction of reporter activity by ecdysone agonists. We also cloned the almost full length coding sequence of EcR expressed in the cell line BRL-AG-3C and used it to make an initial in silico 3D-model of its ligand-binding pocket docked with ponasterone A and tebufenozide.     

Exposure to methoxyfenozide-treated surfaces reduces the responsiveness of adult male codling moth (Lepidoptera: Tortricidae) to codlemone and pear ester lures in a wind tunnel

The responsiveness of male codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), exposed to surfaces treated with the ecdysteroid agonist methoxyfenozide, toward lures loaded with the synthetic sex pheromone codlemone and/or the pear ester kairomone were investigated in wind tunnel experiments. Five different kinds of commercially available codling moth monitoring lures (obtained from Tr6c6 Inc., Adair, OK) were used in the bioassay: Pherocon CM Standard lure (loaded with 1 mg of codlemone), Pherocon CM Long-Life L2 (loaded with 3.5 mg of codlemone), Pherocon CM 10X (loaded with 10 mg of codlemone), Pherocon CM-DA Combo (loaded with 3.0 mg of codlemone and 3.0 mg of pear ester), and Pherocon DA (loaded with 3.0 mg of pear ester). Results from the study revealed that male codling moth exposed to surfaces treated with methoxyfenozide and the surfactant exhibited a significant decline in responsiveness toward lures loaded with either codlemone or pear ester. The full impact of how this negative effect might alter current moth monitoring procedures in orchards receiving ecdysone agonist sprays requires further investigation.     

Baseline monitoring of codling moth (Lepidoptera: Tortricidae) larval response to benzoylhydrazine insecticides

A diet-incorporation larval bioassay was developed to measure the response of codling moth, Cydia pomonella (L.), to the benzoylhydrazine insecticides tebufenozide and methoxyfenozide. The bioassay tested neonates and third, fourth, and fifth instars from a laboratory colony and neonates and fourth instars from a pooled population collected from five certified-organic apple orchards. Bioassays were scored after 6 and 14 d. No differences between the laboratory and field population were found for either insecticide. Significant differences were found in the response of third and fifth instars between the 6 and 14 d bioassays, primarily due to a high proportion of moribund larvae in the shorter assay. Larval age had a significant effect in bioassays and was more pronounced in 6- versus 14-d tests. Fifth instars were significantly less susceptible to both insecticides than other stages, while responses of third and fourth instars were similar. The response of neonates was significantly different from third and fourth instars to tebufenozide but not with methoxyfenozide in the 14-d test. Field bioassays excluded the use of fifth instars and were scored after 14 d. LC50s estimated for 18 field-collected populations varied five- and ninefold for tebufenozide and methoxyfenozide, respectively. The responses of all but six field-collected populations were significantly different from the laboratory strain. Five of these six populations were collected from orchards with no history of organophosphate insecticide use. The LC50 for methoxyfenozide of one field-collected population reared in the laboratory for three generations declined fourfold, but was still significantly different from the laboratory population. These data suggest that transforming current codling moth management programs in Washington from a reliance on organophosphate insecticides to benzoylhydrazines may be difficult.     

Effects of methoxyfenozide-treated surfaces on the attractiveness and responsiveness of adult codling moth (Lepidoptera: Tortricidae)

The attractiveness and responsiveness of adult codling moth, Cydia pomonella (L.), exposed to surfaces treated with the ecdysteroid agonist methoxyfenozide was investigated in wind tunnel and orientation tube assays. When males were exposed to either water- or surfactant-treated surfaces for 48 h, and regardless of what treatment surfaces the females had been exposed to, the mean percent recaptures of such treated males in the wind tunnel assay were sometimes significantly greater than the recaptures of males that had been exposed to methoxyfenozide. Similarly, in the orientation tube assay, males exposed to methoxyfenozide-treated surfaces almost always had significantly lower mean levels of individuals exhibiting sexual excitability and the mean distances traveled upwind, regardless of female exposures. The two assays demonstrated that male codling moths exposed to methoxyfenozide-treated surfaces were not as responsive to calling females (treated and nontreated) as were the nontreated males. Conversely, females exposed to methoxyfenozide-treated surfaces were just as attractive to nontreated males as were nontreated females. It appears that a male's ability to respond to a calling female is more negatively affected by the ecdysone agonist than a female's ability to call and attract males.     

Fecundity and fertility reductions in adult leafrollers exposed to surfaces treated with the ecdysteroid agonists tebufenozide and methoxyfenozide

The effects on the fecundity and fertility of redbanded leafroller, Argyrotaenia velutinana (Walker), and obliquebanded leafroller,Choristoneura rosaceana (Harris), exposed as adults to surfaces treated with the ecdysone agonists tebufenozide (RH-5992) and methoxyfenozide (RH-2485) were examined. The first part of the study consisted of recently emerged moths being exposed to treated surfaces continuously throughout their lives (including mating and oviposition). Continuous exposure to tebufenozide- or methoxyfenozide-treated surfaces significantly reduced the mean number of eggs laid and the percent of eggs that hatched in both species. The second part of the study involved exposure of recently emerged virgin moths (by sex) to treated surfaces for 24 h, after which, the exposed moths were paired with a nontreated partner to mate and oviposit on nontreated surfaces. In this experiment, for A. velutinana, significant reductions in fecundity occurred only when the female was exposed to methoxyfenozide-treated surfaces. Significant reductions in A. velutinana egg fertility occurred with both male and female exposure in the methoxyfenozide treatments and only female exposure in the tebufenozide treatments. For C. rosaceana, significant reductions in fecundity occurred with both male and female exposure in the tebufenozide and methoxyfenozide treatments. Significant reductions in C. rosaceana egg fertility occurred with both male and female exposure in the tebufenozide treatments and only with female exposure in the methoxyfenozide treatments.     

Resistance of the codling moth Cydia pomonella (L.) (Lep., Tortricidae) to pesticides in Israel

Abstract:  Resistance of the codling moth Cydia pomonella (L.) (Lep., Tortricidae) to the organophosphorus compound (OP) azinphosmethyl was observed in apple orchards in Israel. The level of resistance varied with the pest control strategy. Compared with a sensitive laboratory population, the resistance level was highest in insects from the preventative pest control strategy, intermediate in integrated pest management (IPM) orchards, and relatively low in the organic orchards. The level of azinphosmethyl resistance in larvae (but not in adults) exposed for 17 generations in the laboratory to a pesticide-free diet was reduced by 50%. Codling moth larvae resistant to azinphosmethyl were also resistant to various insect growth regulators (IGRs). The IGRs include three chitin synthesis inhibitors (diflubenzuron, novaluron and teflubenzuron), two juvenile hormone mimics (pyriproxyfen and fenoxycarb) and one ecdysone agonist (methoxyfenozide). Codling moth resistant to azinphosmethyl was tolerant to methoxyfenozide and novaluron without previous history of application in apple orchards, indicating the possibility of cross-resistance. According to this study, managing resistance programs in apple orchards should be based on IPM principles with minimum use of conventional neuroactive pesticides.     

Diel patterns of locomotor activity in Cydia pomonella: age and sex related differences and effects of insect hormone mimics

Abstract. The diel pattern of locomotor activity in the codling moth Cydia pomonella L. (Lepidoptera: Tortricidae) was investigated in the laboratory, using a computer-based infra-red actograph. The level of locomotor activity varied with sex and age. On the day of emergence, females were more active than males, but on days 2, 4, 6 and 12 males were approximately twice as active as females. Males reached their highest activity on day 4 and females on day 12. Both sexes were most active at dusk. Their activity patterns were fitted to a set of polynomial regression models. Treatment with 400 p.p.m. of Juvenile Hormone mimic, fenoxycarb, stimulated locomotor activity and provoked a marked activity peak at dawn in both virgin and mated females. A similar treatment with the ecdysone agonist tebufenozide showed a neutral effect on the locomotor activity of females. Possible implications of these findings are discussed in relation to the physiology of the moth and to its dispersal behaviour.     

The Drosophila ecdysone receptor (EcR) gene is required maternally for normal oogenesis

Oogenesis in Drosophila is regulated by the steroid hormone ecdysone and the sesquiterpenoid juvenile hormone. Response to ecdysone is mediated by a heteromeric receptor composed of the EcR and USP proteins. We have identified a temperature-sensitive EcR mutation, EcR(A483T), from a previously isolated collection of EcR mutations. EcR(A483T) is predicted to affect all EcR protein products (EcR-A, EcR-B1, and EcR-B2) since it maps to a common exon encoding the ligand-binding domain. In wild-type females, we find that both EcR-A and EcR-B1 are expressed in nurse cells and follicle cells throughout oogenesis. EcR mutant females raised at permissive temperature and then shifted to restrictive temperature exhibit severe reductions in fecundity. Oogenesis in EcR mutant females is defective, and the spectrum of oogenic defects includes the presence of abnormal egg chambers and loss of vitellogenic egg stages. Our results demonstrate a requirement for EcR during female reproduction and suggest that EcR is required for normal oogenesis.     

Heterodimerization of ecdysone receptor and ultraspiracle on symmetric and asymmetric response elements

Heterodimerization of nuclear receptors is facilitated by the interaction of two dimerization interfaces: one spanning the DNA-binding (C domain) region and the adjacent hinge (D domain) region, and the other in the ligand-binding (E domain) region. Ultraspiracle (USP) heterodimerizes with ecdysone receptor (EcR) and this complex participates in ecdysone signal transduction. The natural ecdysone response elements (EcREs) discovered so far are asymmetric elements composed of either imperfect palindromes or direct repeats. However, gel mobility shift assays have shown that both symmetric (perfect palindromes) and asymmetric (imperfect palindromes and direct repeats) elements can bind to the EcR/USP complex. Therefore, we analyzed EcR/USP domains involved in heterodimerization on different types of response elements (RE). Gel shift assays using full-length and truncated EcR and USP proteins showed that heterodimerization of these two proteins in the presence of asymmetric RE (DR4 and the natural EcRE hsp27) requires both dimerization interfaces present in CD and E domains of both proteins. In contrast, the dimerization interface present in the E domain of either EcR or USP was not essential for heterodimerization on symmetric RE such as PAL1 or IR1. We conclude that the use of heterodimerization interfaces present in CD and E domains of EcR/USP depends on the nature of response elements they bind to.     

Ecdysone receptor expression and activity in adult Drosophila melanogaster

Disrupting components of the ecdysone/EcR/USP signaling pathway in insects leads to morphological defects and developmental arrest. In adult Drosophila melanogaster decreased EcR function affects fertility, lifespan, behavior, learning, and memory; however we lack a clear understanding of how EcR/USP expression and activity impacts these phenotypes. To shed light on this issue, we characterized the wild-type expression patterns and activity of EcR/USP in individual tissues during early adult life. EcR and usp were expressed in numerous adult tissues, but receptor activity varied depending on tissue type and adult age. Receptor activity did not detectably change in response to mating status, environmental stress, ecdysone treatment or gender but is reduced when a constitutively inactive ecdysone receptor is present. Since only a subset of adult tissues expressing EcR and usp contain active receptors, it appears that an important adult function of EcR/USP in some tissues may be repression of genes containing EcRE's.     

Interaction of proteins involved in ecdysone and juvenile hormone signal transduction

Ecdysteroids and juvenile hormones (JH) regulate a variety of developmental, physiological, behavioral, and metabolic processes. Ecdysteroids function through a heterodimeric complex of two nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP). An 85 kDa protein identified in Drosophila melanogaster methoprene-tolerant (Met) mutant binds to JH III with high affinity, and the mutant flies are resistant to juvenile hormone analog (JHA), methoprene. Reporter assays using the yeast two-hybrid system were performed in order to study the molecular interactions between EcR, USP and Met. As expected, EcR fused to the B42 activation domain and USP fused to the LexA DNA binding domain interacted with each other and supported induction of the reporter gene in the presence of stable ecdysteroid analog, RG-102240 or steroids, muristerone A and ponasterone A. The USP:USP homodimers supported expression of the reporter gene in the absence of ligand, and there was no significant increase in the reporter activity after addition of a JHA, methoprene. Similarly, Met:Met homodimers as well as Met:EcR and Met:USP heterodimers induced reporter activity in the absence of ligand and addition of ecdysteroid or JH analogs did not increase the reporter activity regulated by either homodimers or heterodimers of Met protein. Two-hybrid assays in insect cells and in vitro pull-down assays confirmed the interaction of Met with EcR and USP. These data suggest that the proteins that are involved in signal transduction of ecdysteroids (EcR and USP) and juvenile hormones (Met) interact to mediate cross-talk between these two important hormones. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.     

Expression and purification of the hormone binding domain of the Drosophila ecdysone and ultraspiracle receptors

Escherichia coli vectors were constructed for the production of a protein complex that mimics the native ecdysone receptor (EcR) isolated from Drosophila. The two steroid receptors, ultraspiracle (USP) and EcR, were expressed as truncations, retaining primarily the hormone binding domains. The recombinant receptor complex was able to mimic the pharmacology of the native receptor with respect to both synthetic and natural agonists. USP and EcR fusion proteins could be expressed in separate cell lines and then recombined following isolation to yield a ligand binding preparation with a dissociation constant (K(D)) for Ponasterone A of 1.5 nM and a total yield of 1.9 pmol ligand binding sites/mg protein. Alternatively, the simultaneous coexpression of both receptors increased yields by several orders of magnitude to 6 nmol ligand binding sites/mg protein with a K(D) of 0.6 nM. Chromatographic analysis under native conditions showed that EcR, when expressed alone, migrated as a variety of complexes, mostly coming out in the void volume as denatured, insoluble, aggregate. In contrast, purified extracts of coexpressed EcR and USP eluted as a single peak with a mobility indicating a heterodimer. The majority of the coexpressed fusion receptors, following purification, formed functional steroid binding sites. A detailed scheme is provided for the expression and isolation of milligram quantities of highly purified receptor dimer.     

Inhibition of [3H]ponasterone A binding by ecdysone agonists in the intact Kc cell line.

Inhibition of the binding of [3H]ponasterone A ([3H]PoA) by ecdysone agonists including diacylhydrazines such as RH-5849, tebufenozide (RH-5992) and methoxyfenozide (RH-2485) was examined in intact Drosophila Kc cells. The reciprocal logarithm of the concentration at which there is 50% inhibition of [3H]PoA binding, pIC(50) (M), was determined as the binding activity for all compounds from each concentration-response curve. The order of the activity was PoA>20-hydroxyecdysone>cyasterone>inokosterone>or=makisterone A>methoxyfenozide>or=tebufenozide>ecdysone>RH-5849. The ranking of steroidal ecdysone analogs is consistent with that obtained against Spodoptera Sf-9 cells. Furthermore, in terms of pIC(50), all binding activity for ecdysone analogs, except ecdysone, estimated in the Kc cell line system was significantly higher than that for the Sf-9 cell line system. However, the activity of ecdysone was comparable between Kc and Sf-9 cells. The activity of diacylhydrazine analogs against Kc cells was significantly low compared with that against Sf-9 cells. The potency of methoxyfenozide was 1/200 that of PoA, which showed the highest activity in the Kc cell line system among all compounds tested. The activity of tebufenozide analogs having an n-pentyl or n-hexyl group instead of a 4-ethylphenyl group was similar to that of RH-5849.     

Oriental fruit moth (Lepidoptera: Tortricidae) phenology and management with methoxyfenozide in North Carolina apples

The phenology of oriental fruit moth, Grapholita molesta (Busck), on apple (Malus spp.) in North Carolina was studied using pheromone traps and egg sampling in abandoned and commercial orchards in 2000 and 2001, with subsequent development of an oviposition degree-day model and management studies in relation to codling moth, Cydia pomonella (L.), phenology. Oriental fruit moth eggs were found in greater numbers on leaves early and on fruit later in the growing season, on the top versus the bottom of the leaf surface, and on the calyx area versus the side or stem end of the fruit. A degree-day (DD) model to predict oriental fruit moth oviposition was developed based on temperature accumulations from peak moth trap capture of the first (overwintering) generation, by using 7.2 and 32.2 degrees C as the temperature limits. The model predicted four ovipositing generations of oriental fruit moth with the second beginning 507 DD after peak moth catch. Using predictions of the oriental fruit moth and codling moth degree-day oviposition models, an experiment was conducted to determine the level of second generation oriental fruit moth control with methoxyfenozide applied under different scenarios for first generation codling moth. Methoxyfenozide was equally effective in managing codling moth and oriental fruit moth for all treatment timings.