Ecdysteroid Agonist Research Articles

Combined Transcriptomic Analysis and RNA Interference Reveal the Effects of Methoxyfenozide on Ecdysone Signaling Pathway of Spodoptera exigua.

Spodoptera exigua is a worldwide pest afflicting edible vegetables and has developed varying levels of resistance to insecticides. Methoxyfenozide (MET), an ecdysteroid agonist, is effective against lepidopteran pests such as S. exigua. However, the mechanism of MET to S. exigua remains unclear. In this study, we analyzed the expression patterns of genes related to the ecdysone signaling pathway in transcriptome data treated with sublethal doses of MET and analyzed how expression levels of key genes affect the toxicity of MET on S. exigua. Our results demonstrated that 2639 genes were up-regulated and 2512 genes were down-regulated in S. exigua treated with LC30 of MET. Of these, 15 genes were involved in the ecdysone signaling pathway. qPCR results demonstrated that ecdysone receptor A (EcRA) expression levels significantly increased in S. exigua when treated with different doses of MET, and that the RNAi-mediated silencing of EcRA significantly increased mortality to 55.43% at 72 h when L3 S. exigua larvae were exposed to MET at the LC30 dose. Additionally, knocking down EcRA suppressed the most genes expressed in the ecdysone signaling pathway. The combination of MET and dsEcRA affected the expression of E74 and enhanced the expression of TREA. These results demonstrate that the adverse effects of sublethal MET disturb the ecdysone signaling pathway in S. exigua, and EcRA is closely related to MET toxic effect. This study increases our collective understanding of the mechanisms of MET in insect pests.

Ecdysis triggering hormone is essential for larva-pupa-adult transformation in Leptinotarsa decemlineata.

In Drosophila melanogaster, ecdysis triggering hormone (ETH) is the key factor triggering ecdysis behaviour and promoting trachea clearance. However, whether ETH plays the dual roles in non-dipteran insects is unknown. In this survey, we found that Ldeth mRNA levels were positively correlated with circulating 20-hydroxyecdysone (20E) titers in Leptinotarsa decemlineata. Ingestion of an ecdysteroid agonist halofenozide or 20E stimulated the transcription of Ldeth, whereas RNA interference (RNAi) of ecdysteroidogenesis (LdPTTH or LdSHD) or 20E signalling (LdEcR, LdUSP or LdFTZ-F1) genes inhibited the expression, indicating ETH acts downstream of 20E. RNAi of Ldeth at the final instar stage impaired pupation. More than 80% of the Ldeth-depleted beetles remained as prepupae, completely wrapped in the old larval cuticles. These prepupae became withered, dried and darkened gradually, and finally died in soil. The remaining Ldeth hypomorphs pupated and emerged as abnormal adults, bearing smaller and wrinkle elytrum and hindwing. Moreover, the tracheae in the Ldeth hypomorphs were full of liquid. We accordingly proposed that the failure of trachea clearance disenabled air-swallowing after pupa-adult ecdysis and impacted wing expansion. Our results suggest that ETH plays the dual roles, initiation of ecdysis and motivation of trachea clearance, in a coleopteran.

Dissecting the roles of FTZ-F1 in larval molting and pupation, and the sublethal effects of methoxyfenozide on Helicoverpa armigera.

In holometabolous insects, the major developmental transitions - larval molting and pupation - are triggered by a pulse of 20-hydroxyecdysone (20E) and coordinated by juvenile hormone. Methoxyfenozide (MF), an ecdysteroid agonist, represents a new class of insect growth regulators and is effective against lepidopteran pests. Fushi-tarazu factor 1 (FTZ-F1) is an ecdysone-inducible transcription factor. To date, the effect of MF on 20E-response genes remains unclear, and we speculate the involvement of FTZ-F1 in MF's growth regulating effect. MF at LC25 and LC10 caused severe ecdysis failure in Helicoverpa armigera, extended their larval duration, lowered their pupal weight, and reduced the respiratory, pupation and emergence rates. Furthermore, sublethal doses of MF inhibited ecdysteroidogenesis and lowered the intrinsic 20E titer, but showed an inductive effect on 20E-response genes including HaFTZ-F1. HaFTZ-F1, predominantly expressed in larval epidermis, was markedly upregulated before or right after larval ecdysis, and maintained a high level in prepupal stage. Knockdown of HaFTZ-F1 in 4th-instar larvae severely impaired larval ecdysis, whereas its knockdown in final-instar larvae caused abnormal pupation. Moreover, knocking down HaFTZ-F1 downregulated three critical ecdysteroidogenesis genes, lowered 20E titer, and suppressed the expression of 20E receptors and 20E-response genes. The introduction of 20E into HaFTZ-F1-RNAi larvae partly relieved the negative effects on the 20E-induced signaling cascade. Our findings reveal the adverse effects of sublethal doses of MF on the development of H. armigera and elucidate the resulting perturbations on the 20E-induced signaling cascade; we propose that HaFTZ-F1 regulates ecdysis and pupation by mediating 20E titer and its signaling pathway. © 2020 Society of Chemical Industry.

Sublethal Effects of Insect Growth Regulators on Boll Weevil (Coleoptera: Curculionidae).

Immature stages of the boll weevil, Anthonomus grandis grandis Boh. (Coleoptera: Curculionidae), develop protected inside cotton fruiting structures. Therefore, the adult beetles have become the main target of insecticide applications. The use of insect growth regulators (IGRs) is recommended against immatures, even though they may also affect the survival and reproductive traits of adult insects. The present study evaluated the impact of a juvenile hormone analog (pyriproxyfen), an ecdysteroid agonist (methoxyfenozide), and a chitin biosynthesis inhibitor (lufenuron) on adult cotton boll weevils, a key cotton pest. Mated and virgin beetles were treated by feeding them contaminated squares and cotton leaf discs that were previously immersed into pyriproxyfen, methoxyfenozide, and lufenuron solutions at field-rate concentrations. After exposure, treated couples were caged onto cotton plants, and survival, fecundity, and egg viability were evaluated. The IGRs neither affected the survival nor fecundity of adult boll weevils. On the other hand, egg viability was significantly reduced by lufenuron, regardless of whether the females were treated premating or postmating or whether their pairs were either treated or untreated. However, egg viability increased as the females aged since the initial exposure date to lufenuron, indicating a potential transovarial effect of this insecticide. Our results indicate that pyriproxyfen and methoxyfenozide do not affect adult boll weevils, whereas lufenuron temporarily reduces the egg viability of this key cotton pest.

Transcriptional response of Methoprene-tolerant (Met) gene to three insect growth disruptors in Leptinotarsa decemlineata (Say)

Abstract Some insect growth disruptors (IGDs), such as pyriproxyfen and halofenozide, may be used to control Leptinotarsa decemlineata. However, their mechanism of action remains elusive. Methoprene-tolerant (Met) mediates juvenile hormone (JH) signal to control numerous essential physiological processes. In the present paper, we identified a Met gene (LdMet). LdMet protein was a typical basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) transcription factor with a bHLH domain, two PAS domains (PAS-A and PAS-B) and a region called PAS associated C terminal (PAC). Eight conserved amino acids critical for JH binding were located in PAS-B and PAC domains. The temporal expression pattern of LdMet was in accordance with the variation of circulating JH titers. Feeding of juvenoid methoprene or pyriproxyfen, or provide for JH dose-dependently stimulated the expression of LdMet. RNA interference-mediated knockdown of two JH degradation genes increased the transcription of LdMet, while silencing of a JH biosynthesis gene repressed the transcription. Conversely, ingestion of an ecdysteroid agonist halofenozide or 20-hydroxyecdysone (20E) reduced the mRNA levels of LdMet, in a dose-dependent manner, whereas knockdown of either ecdysteroidogenesis or 20E signaling genes increased the mRNA accumulation. Providing that the expression of LdMet can be disturbed by methoprene, pyriproxyfen and halofenozide, LdMet may be a potential target of these IGDs in L. decemlineata larvae.

Methoxyfenozide, an Ecdysteroid Agonist Insecticide, Alters Oocyte Growth During Metamorphosis ofEphestia kuehniellaZeller

Methoxyfenozide is a lepidopteran-specific insecticide belonging to non-steroidal ecdysteroid agonists. Its effects were evaluated under laboratory conditions on the morphometry and histology of ovaries in the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Treatment at LD50 (0.01 µg/pupa) and LD90 (0.37 µg/pupa) was made by topical applications on newly ecdysed female pupae. The compound was found to increase the size of the ovaries and to reduce the thickness of the follicular epithelium compared to controls. Moreover, the histological study of ovaries showed alterations including vacuolation, the absence of nurse cells, and shrinkage, leaving a space around the oocyte tissue.

BDE-47 causes developmental retardation with down-regulated expression profiles of ecdysteroid signaling pathway-involved nuclear receptor (NR) genes in the copepod Tigriopus japonicus

2,2′,4,4′-Tetrabromodiphenyl ether (BDE-47) is a persistent organic pollutant (POP) in marine environments. Despite its adverse effects (e.g. developmental retardation) in ecdysozoa, the effects of BDE-47 on transcription of ecdysteroid signaling pathway-involved-nuclear receptor (NR) genes and metamorphosis-related genes have not been examined in copepods. To examine the deleterious effect of BDE-47 on copepod molting and metamorphosis, BDE-47 was exposed to the harpacticoid copepod Tigriopus japonicus, followed by monitoring developmental retardation and transcriptional alteration of NR genes. The developmental rate was significantly inhibited (P<0.05) in response to BDE-47 and the agricultural insecticide gamma-hexachlorocyclohexane. Conversely, the ecdysteroid agonist ponasterone A (PoA) led to decreased molting and metamorphosis time (P<0.05) from the nauplius stage to the adult stage. In particular, expression profiles of all NR genes were the highest at naupliar stages 5–6 except for SVP, FTZ-F1, and HR96 genes. Nuclear receptor USP, HR96, and FTZ-F1 genes also showed significant sex differences (P<0.05) in gene expression levels over different developmental stages, indicating that these genes may be involved in vitellogenesis. NR gene expression patterns showed significant decreases (P<0.05) in response to BDE-47 exposure, implying that molting and metamorphosis retardation is likely associated with NR gene expression. In summary, BDE-47 leads to molting and metamorphosis retardation and suppresses transcription of NR genes. This information will be helpful in understanding the molting and metamorphosis delay mechanism in response to BDE-47 exposure.

IDENTIFICATION AND HORMONE INDUCTION OF PUTATIVE CHITIN SYNTHASE GENES AND SPLICE VARIANTS IN Leptinotarsa decemlineata (SAY).

Chitin synthase (ChS) plays a critical role in chitin synthesis and excretion. In this study, two ChS genes (LdChSA and LdChSB) were identified in Leptinotarsa decemlineata. LdChSA contains two splicing variants, LdChSAa and LdChSAb. Within the first, second, and third larval instars, the mRNA levels of LdChSAa, LdChSAb, and LdChSB coincide with the peaks of circulating 20-hydroxyecdysone (20E) and juvenile hormone (JH). In vitro culture of midguts and an in vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide stimulated the expression of the three LdChSs. Conversely, a reduction of 20E by RNA interference (RNAi) of an ecdysteroidogenesis gene LdSHD repressed the expression of these LdChSs, and ingestion of halofenozide by LdSHD RNAi larvae rescued the repression. Moreover, disruption of 20E signaling by RNAi of LdEcR, LdE75, LdHR3, and LdFTZ-F1 reduced the expression levels of these genes. Similarly, in vitro culture and an in vivo bioassay showed that exogenous JH and a JH analog methoprene activated the expression of the three LdChSs, whereas a decrease in JH by RNAi of a JH biosynthesis gene LdJHAMT downregulated these LdChSs. It seems that JH upregulates LdChSs at the early stage of each instar, whereas a 20E pulse triggers the transcription of LdChSs during molting in L. decemlineata.

Synthesis and Larvicidal Activity of Novel Thenoylhydrazide Derivatives.

A pair of chemical isomeric structures of novel N-tert-butylphenyl thenoylhydrazide compounds I and II were designed and synthesized. Their structures were characterized by MS, IR, 1H NMR, elemental analysis and X-ray single crystal diffraction. The regioselectivity of the Meerwein arylation reaction and the electrophilic substitution reaction of N-tert-butyl hydrazine were studied by density functional theory (DFT) quantum chemical method. The larvicidal tests revealed that some compounds I had excellent larvicidal activity against Culex pipiens pallens. As the candidates of insect growth regulators (IGRs), the larval growth inhibition and regulation against Culex pipiens pallens were examined for some compounds, especially I1 and I7. Compounds I1 and I7 were further indicated as an ecdysteroid agonist by reporter gene assay on the Spodoptera frugiperda cell line (Sf9 cells). Finally, a molecular docking study of compound I7 was conducted, which was not only beneficial to understand the structure-activity relationship, but also useful for development of new IGRs for the control of mosquitos.

Knockdown of a nutrient amino acid transporter gene LdNAT1 reduces free neutral amino acid contents and impairs Leptinotarsa decemlineata pupation.

A Leptinotarsa decemlineata SLC6 NAT gene (LdNAT1) was cloned. LdNAT1 was highly expressed in the larval alimentary canal especially midgut. LdNAT1 mRNA levels were high right after the molt and low just before the molt. JH and a JH analog pyriproxyfen activated LdNAT1 expression. RNAi of an allatostatin gene LdAS-C increased JH and upregulated LdNAT1 transcription. Conversely, silencing of a JH biosynthesis gene LdJHAMT decreased JH and reduced LdNAT1 expression. Moreover, 20E and an ecdysteroid agonist halofenozide repressed LdNAT1 expression, whereas a decrease in 20E by RNAi of an ecdysteroidogenesis gene LdSHD and disruption of 20E signaling by knockdown of LdE75 and LdFTZ-F1 activated LdNAT1 expression. Thus, LdNAT1 responded to both 20E and JH. Moreover, knockdown of LdNAT1 reduced the contents of cysteine, histidine, isoleucine, leucine, methionine, phenylalanine and serine in the larval bodies and increased the contents of these amino acids in the larval feces. Furthermore, RNAi of LdNAT1 inhibited insulin/target of rapamycin pathway, lowered 20E and JH titers, reduced 20E and JH signaling, retarded larval growth and impaired pupation. These data showed that LdNAT1 was involved in the absorption of several neutral amino acids critical for larval growth and metamorphosis.

Two Leptinotarsa uridine diphosphate N-acetylglucosamine pyrophosphorylases are specialized for chitin synthesis in larval epidermal cuticle and midgut peritrophic matrix

Uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (UAP) is involved in the biosynthesis of chitin, an essential component of the epidermal cuticle and midgut peritrophic matrix (PM) in insects. In the present paper, two putative LdUAP genes were cloned in Leptinotarsa decemlineata. In vivo bioassay revealed that 20-hydroxyecdysone (20E) and an ecdysteroid agonist halofenozide activated the expression of the two LdUAPs, whereas a decrease in 20E by RNA interference (RNAi) of an ecdysteroidogenesis gene LdSHD and a 20E signaling gene LdFTZ-F1 repressed the expression. Juvenile hormone (JH), a JH analog pyriproxyfen and an increase in JH by RNAi of an allatostatin gene LdAS-C downregulated LdUAP1 but upregulated LdUAP2, whereas a decrease in JH by silencing of a JH biosynthesis gene LdJHAMT had converse effects. Thus, expression of LdUAPs responded to both 20E and JH. Moreover, knockdown of LdUAP1 reduced chitin contents in whole larvae and integument samples, thinned tracheal taenidia, impaired larval–larval molt, larval-pupal ecdysis and adult emergence. In contrast, silencing of LdUAP2 significantly reduced foliage consumption, decreased chitin content in midgut samples, damaged PM, and retarded larval growth. The resulting larvae had lighter fresh weights, smaller body sizes and depleted fat body. As a result, the development was arrested. Combined knockdown of LdUAP1 and LdUAP2 caused an additive negative effect. Our data suggest that LdUAP1 and LdUAP2 have specialized functions in biosynthesizing chitin in the epidermal cuticle and PM respectively in L. decemlineata.

Nuclear receptor ecdysone-induced protein 75 is required for larval-pupal metamorphosis in the Colorado potato beetle Leptinotarsa decemlineata (Say).

20-hydroxyecdysone (20E) and juvenile hormone (JH) are key regulators of insect development. In this study, three Leptinotarsa decemlineata Ecdysone-induced protein 75 (LdE75) cDNAs (LdE75A, B and C) were cloned from L. decemlineata. The three LdE75 isoforms were highly expressed just before or right after each moult. Within the fourth larval instar, they showed a small rise and a big peak 40 and 80 h after ecdysis. The expression peaks of the three LdE75s coincided with the peaks of circulating 20E levels. In vitro midgut culture and in vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide (Hal) enhanced LdE75 expression in the day 1 final larval instars. Conversely, a decrease in 20E by feeding a double-stranded RNA (dsRNA) against an ecdysteroidogenesis gene, Shade (LdSHD), repressed the expression of LdE75. Moreover, Hal upregulated the expression of the three LdE75s in LdSHD-silenced larvae. Thus, 20E pulses activate the transcription of LdE75s. Furthermore, ingesting dsE75-1 and dsE75-2 from a common fragment of the three isoforms successfully knocked down these LdE75s, and caused developmental arrest. Finally, knocking down LdE75s significantly repressed the transcription of three ecdysteroidogenesis genes, lowered the 20E titre and affected the expression of two 20E-response genes. Silencing LdE75s also induced the expression of a JH biosynthesis gene, increased JH titre and activated the transcription of a JH early-inducible gene. Thus, Ld E75s are required for larval-pupal metamorphosis and act mainly by modulating 20E and JH titres and mediating their signalling pathways.

Functions of nuclear receptor HR3 during larval-pupal molting in Leptinotarsa decemlineata (Say) revealed by in vivo RNA interference

Our previous results revealed that RNA interference-aided knockdown of Leptinotarsa decemlineata FTZ-F1 (LdFTZ-F1) reduced 20E titer, and impaired pupation. In this study, we characterized a putative LdHR3 gene, an early-late 20E-response gene upstream of LdFTZ-F1. Within the first, second and third larval instars, three expression peaks of LdHR3 occurred just before the molt. In the fourth (final) larval instar 80 h after ecdysis and prepupal stage 3 days after burying into soil, two LdHR3 peaks occurred. The LdHR3 expression peaks coincide with the peaks of circulating 20E level. In vitro midgut culture and in vivo bioassay revealed that 20E and an ecdysteroid agonist halofenozide (Hal) enhanced LdHR3 expression in the final larval instars. Conversely, a decrease in 20E by feeding a double-stranded RNA (dsRNA) against an ecdysteroidogenesis gene Ldshd repressed the expression. Moreover, Hal rescued the transcript levels in the Ldshd-silenced larvae. Thus, 20E peaks activate the expression of LdHR3. Furthermore, ingesting dsRNA against LdHR3 successfully knocked down the target gene, and impaired pupation. Finally, knockdown of LdHR3 upregulated the transcription of three ecdysteroidogenesis genes (Ldphm, Lddib and Ldshd), increased 20E titer, and activated the expression of two 20E-response genes (LdEcR and LdFTZ-F1). Thus, LdHR3 functions in regulation of pupation in the Colorado potato beetle.

Involvement of FTZ-F1 in the regulation of pupation in Leptinotarsa decemlineata (Say)

During the final instar larvae of holometabolous insects, a pulse of 20-hydroxyecdysone (20E) and a drop in juvenile hormone (JH) trigger larval-pupal metamorphosis. In this study, two LdFTZ-F1 cDNAs (LdFTZ-F1-1 and LdFTZ-F1-2) were cloned in Leptinotarsa decemlineata. Both LdFTZ-F1-1 and LdFTZ-F1-2 were highly expressed just before or right after each molt, similar to the expression pattern of an ecdysteroidogenesis gene LdSHD. Ingestion of an ecdysteroid agonist halofenozide (Hal) enhanced LdFTZ-F1-1 and LdFTZ-F1-2 expression in the final larval instar. Conversely, a decrease in 20E by feeding a double-stranded RNA (dsRNA) against LdSHD repressed the expression. Moreover, Hal rescued the expression levels in LdSHD-silenced larvae. Thus, 20E peaks seem to induce the transcription of LdFTZ-F1s. Furthermore, ingesting dsLdFTZ-F1 from a common fragment of LdFTZ-F1-1 and LdFTZ-F1-2 successfully knocked down both LdFTZ-F1s, and impaired pupation. Finally, knocking down LdFTZ-F1s significantly repressed the transcription of three ecdysteroidogenesis genes, lowered 20E titer, and reduced the expression of two 20E receptor genes. Silencing LdFTZ-F1s also induced the expression of a JH biosynthesis gene, increased JH titer, but decreased the mRNA level of a JH early-inducible gene. Thus, LdFTZ-F1s are involved in the regulation of pupation by modulating 20E and JH titers and mediating their signaling pathways.

The P450 enzyme Shade mediates the hydroxylation of ecdysone to 20-hydroxyecdysone in the Colorado potato beetle, Leptinotarsa decemlineata.

Ecdysone 20-monooxygenase (E20MO), a cytochrome P450 monooxygenase (CYP314A1), catalyses the conversion of ecdysone (E) to 20-hydroxyecdysone (20E). We report here the cloning and characterization of the Halloween gene Shade (Shd) encoding E20MO in the Colorado potato beetle, Leptinotarsa decemlineata. LdSHD has five conserved motifs typical of insect P450s, ie the Helix-C, Helix-I, Helix-K, PxxFxPE/DRF (PERF) and heme-binding motifs. LdShd was expressed in developing eggs, the first to fourth instars, wandering larvae, pupae and adults, with statistically significant fluctuations. Its mRNA was ubiquitously distributed in the head, thorax and abdomen. The recombinant LdSHD protein expressed in Spodoptera frugiperda 9 (Sf9) cells catalysed the conversion of E to 20E. Dietary introduction of double-stranded RNA (dsRNA) of LdShd into the second instar larvae successfully knocked down the LdShd expression level, decreased the mRNA level of the ecdysone receptor (LdEcR) gene, caused larval lethality, delayed development and affected pupation. Moreover, ingestion of LdShd-dsRNA by the fourth instars also down-regulated LdShd and LdEcR expression, reduced the 20E titre, and negatively influenced pupation. Introduction of 20E and a nonsteroidal ecdysteroid agonist halofenozide into the LdShd-dsRNA-ingested second instars, and of halofenozide into the LdShd-dsRNA-ingested fourth instars almost completely relieved the negative effects on larval performance. Thus, LdSHD functions to regulate metamorphotic processes by converting E to 20E in a coleopteran insect species Le. decemlineata.

Toxicity of Tebufenozide, an Ecdysteroid Agonist, toBlattella germanica(Blattodea: Blattellidae)

The toxicity of the non-steroidal ecdysteroid agonist tebufenozide was evaluated topically on adults of the cosmopolitan cockroach Blattella germanica (Blattodea: Blattellidae). The LD50 value was estimated at 402 μg/g of insect (range, 95% fiducial limits: 293–552) at 15 days after treatment. The effects on reproduction were observed in adult females of cockroaches that survived exposure to a LD25 (146 μg/g of insect) of tebufenozide. Dissection of treated females showed a clear reduction in both the number of oocytes per paired ovaries and the size of basal oocytes, as compared to controls. In addition, tebufenozide significantly reduced the mating success of pairs and altered the fertility as shown through the reduction of ootheca development and total absence of viable nymphs. Tebufenozide disrupted the Germancockroach reproduction by interferring with the homeostasis of the insect hormones.

Biological and Reproduction Activities of Mosquito Larvae of Culiseta morsitans (Theobald) after Treatment by Ecdysone Agonist Methoxyfenozide

Aims: Insect growth regulators (IGRs) offer alternatives to conventional chemical larvicides that pose problem of resistance and environmental safety. However, only a limited number of IGRs have been approved for use in mosquito control. The present investigation was to evaluate the insecticidal activity of a non -steroidal ecdysteroid agonist, methoxyfenozide (RH-2485), against last -instar larvae ofCuliseta morsitans (Culicidae: Diptera) under the laboratory conditions, and its effect on fecundity and fertility of females. Methodology: Based on preliminarybioassays, five concentrati ons ranging from 0.012 to 0.072mg/L were tested. The larvicidal test was made according to the World Health Organization(WHO) protocol standard by exposing newly ecdysed fourth -instar larvae for 24h. CL50value of methoxyfenozide value was determined by probit analysis. Results: The insecticidal activity of methoxyfenozide result in a dose - dependent reduction in adult emergence. In addition, larval mortality was relatively importan t compared with the mortality recorded for the other following developmental stages. The larval mortality varies betw een 16.84±3.89 % and 66.24±3.50 % for 0.012 mg/L and 0.072 OriginalResearch Article

Tebufenozide disrupts ovarian development and function in silkmoths

Adult development and production of up to 400 eggs within the pupal case of female silkmoths are both dependent on 20-hydroxyecdysone (20E), the steroid hormone of insects. When adult development was initiated with tebufenozide, the non-steroidal ecdysteroid agonist, instead of 20E, full development of all epidermal tissues like the wing was witnessed, but ovarian growth and egg formation was minimal. Administration of tebufenozide to female pharate adults caused disruption of the follicular epithelium, produced nurse cell damage, and inhibited oogenesis. Reduced ability to synthesize RNA and protein accompanied these tebufenozide induced morphological disturbances of the follicles. In vivo accumulation of vitellogenin (Vg) from the hemolymph was reduced in tebufenozide treated female ovaries as well as their ability to accumulate Vg in vitro. Determination of protein staining intensity and antibody reactivity of Vg pointed out that hemolymph Vg level remained fairly constant all through adult development whether induced by 20E or tebufenozide. Measurement of hemolymph volumes and hemolymph Vg levels of control and experimental animals allowed us to conclude that egg development involves the uptake of all the hemolymph proteins and not Vg alone. The loss of hemolymph that accompanies egg maturation was considerably reduced in tebufenozide initiated female pharate adults. 20E could not overcome ovarian growth inhibitory effects of tebufenozide. Dual mechanisms, one involving ecdysteroid antagonist action at the beginning of development, and the other unrelated to that function during heightened egg formation, are needed explain the biphasic inhibitory actions of tebufenozide on silkmoth ovaries.

Design and synthesis of N-alkyl-N′-substituted 2,4-dioxo-3,4-dihydropyrimidin-1-diacylhydrazine derivatives as ecdysone receptor agonist

Based on the discovery of thymine as an ecdysteroid agonist, a series of 1,4-disubstituted diacylhydrazine derivatives with a thymine moiety were designed and synthesized. The activities of these compounds against Spodoptera litura (Fabricius) were evaluated by the insect immersion method. Results showed that compound 2h with an N-cyclohexylmethyl substituent exhibits the most potent agonist activity with a median lethal concentration of 23.21μg/mL. This compound also caused malformation of molting larvae and adults. Compound 2h was further demonstrated as an ecdysteroid agonist by reporter gene assay on the Spodoptera frugiperda cell line (Sf9 cells). A molecular docking study indicated that hydrophobic interactions and the formation of hydrogen bonds between the compounds and the ecdysone receptor play critical roles in promoting the binding affinity of the compound. The structure of compound 2h may serve as a favorable template for the development of new ecdysteroid agonists with a pyrimidinedione moiety.

Antennal responses of Cydia pomonella (L.) exposed to surfaces treated with methoxyfenozide

Electroantennogram (EAG) measurements were recorded from the antennae of male and female codling moth, Cydia pomonella L., to determine whether adult moths exposed to surfaces treated with the ecdysteroid agonist methoxyfenozide experience a decline in their antennal reception and thus olfactory sensitivity. Such a phenomenon would offer a possible mechanism for the previously reported decreased responsiveness from moths treated with methoxyfenozide to pheromone- and plant volatile-based monitoring lures. Mean EAG data revealed that the antennae from methoxyfenozide-treated male moths appear to be just as sensitive to various doses of synthetic codlemone as the antennae from the control and surfactant-treated moths, but they appeared to be less sensitive to the pheromone component 12OH (collected from female effluvia) than the control male antennae. Mean male EAG responses to the pheromone components E8,E10-12Al and codlemone collected from methoxyfenozide-treated females were significantly less than the responses towards those two pheromone components collected from the control and surfactant-treated females. Female moth exposure to methoxyfenozide did not negatively impact the sensitivity of female antennae to the plant volatile pear ester, but it did towards the apple volatile butyl hexanoate. Data from this study show that adult C. pomonella exposure to methoxyfenozide-treated surfaces does appear to negatively impact, in a minor way, the (i) olfactory sensitivity (or detection) of male antennae towards some components of the female sex pheromone, (ii) the female antennal sensitivity towards a key apple volatile and (iii) the attractiveness of female pheromone effluvia.