Increase, Ecdysone receptor hyperactivation

112410-23-8

QYPNKSZPJQQLRK-UHFFFAOYSA-N

Tebufenozide

DTXSID4034948

5289-74-7

NKDFYOWSKOHCCO-YPVLXUMRSA-N

20-Hydroxyecdysone

20E

DTXSID5040388

13408-56-5

PJYYBCXMCWDUAZ-JJJZTNILSA-N

Ponasterone A

DTXSID0040595

161050-58-4

QCAWEPFNJXQPAN-UHFFFAOYSA-N

Methoxyfenozide

Benzoic acid, 3-methoxy-2-methyl-.2-(3,5-dimethylbenzoyl)-2-(1,1-dimethylethyl)hydrazide

DTXSID3032628

112226-61-6

CNKHSLKYRMDDNQ-UHFFFAOYSA-N

Halofenozide

Benzoic acid, 4-chloro-, 2-benzoyl-2-(1,1-dimethylethyl)hydrazide RH-70345 Mach-2

DTXSID4032619

143807-66-3

HPNSNYBUADCFDR-UHFFFAOYSA-N

Chromafenozide

2H-1-Benzopyran-6-carboxylic acid, 3,4-dihydro-5-methyl-, 2-(3,5-dimethylbenzoyl)-2-(1,1-dimethylethyl)hydrazide

DTXSID4057976

15130-85-5

JQNVCUBPURTQPQ-GYVHUXHASA-N

Inokosterone

DTXSID40164756

59456-70-1

WWJFFVUVFNBJTN-UIBIZFFUSA-N

Nikkomycins

β-D-Allofuranuronic acid, 5-[[(2S,3S,4S)-2-amino-4-hydroxy-4-(5-hydroxy-2-pyridinyl)-3-methyl-1-oxobutyl]amino]-1,5-dideoxy-1-(3,4-dihydro-2,4-dioxo-1(2H)-pyrimidinyl)-

DTXSID5058436

GO:0008230

GO ecdysone receptor holocomplex

C061375

MESH Eip75B protein, Drosophila

PR:P33244

PR nuclear hormone receptor FTZ-F1 (fruit fly)

PR:Q9VCW0

PR cardioactive peptide (fruit fly)

CL:0000100

CL motor neuron

CHEBI:17029

CHEBI chitin

GO:0035076

GO ecdysone receptor-mediated signaling pathway

GO:0008255

GO ecdysis-triggering hormone activity

GO:0010467

GO gene expression

GO:0002790

GO peptide secretion

GO:0019226

GO transmission of nerve impulse

GO:0006936

GO muscle contraction

GO:0006031

GO chitin biosynthetic process

D009026

MESH mortality

WIKI increased

WIKI decreased

Tebufenozide

2016-11-29T18:42:27

2017-02-09T03:06:52

20-hydroxyecdysone

2017-02-09T03:06:05

Ponasterone A

2017-02-09T03:06:22

Methoxyfenozide

2017-02-06T12:28:03

2017-02-09T03:42:07

Halofenozide

2017-02-06T12:28:45

Chromafenozide

2017-02-09T03:41:19

Cyasterone

2017-02-09T03:42:50

Makisterone A

2017-02-09T03:43:03

Inokosterone

2017-02-09T03:43:16

Ecdysone

2017-02-09T03:43:26

RH-5849

2017-02-09T03:43:40

Polyoxin D

2020-10-23T06:20:12

Nikkomycins

2018-05-24T15:54:09

7375

NCBI Lucilia cuprina

WCS_35525

common ecological species Daphnia magna

WikiUser_5

ApacheUser insects

WCS_35525

common ecological species crustaceans

6656

NCBI Arthropoda

Increase, Ecdysone receptor hyperactivation

Increase, EcR hyperactivation

Molecular

CL:0000255

CL eukaryotic cell

AOPWiki 2016-11-29T18:41:22

2025-09-29T04:47:29

Decrease, Circulating ecdysis triggering hormone

Decrease, Circulating ETH

Tissue

AOPWiki 2016-11-29T18:41:28

2018-05-24T16:34:43

Increase, Nuclear receptor E75b gene expression

Increase, E75b expression

Molecular

CL:0000255

CL eukaryotic cell

AOPWiki 2017-02-09T03:20:45

2018-05-24T16:32:20

Decrease, Fushi tarazu factor-1 gene expression

Decrease, Ftz-f1 expression

Molecular

CL:0000255

CL eukaryotic cell

AOPWiki 2017-02-09T03:21:25

2025-09-26T04:58:05

Decrease, Circulating crustacean cardioactive peptide

Decrease, Circulating CCAP

Tissue

UBERON:0000179

UBERON haemolymphatic fluid

AOPWiki 2017-02-09T03:23:06

2018-05-24T16:37:35

Decrease, Ecdysis motor program activity

Tissue

UBERON:0001016

UBERON nervous system

AOPWiki 2017-02-09T03:24:07

2025-09-29T04:18:57

Decrease, Abdominal muscle contraction

Tissue

UBERON:0014895

UBERON somatic muscle

AOPWiki 2016-11-29T18:41:28

2018-05-24T16:41:01

Increase, Incomplete ecdysis

Individual

AOPWiki 2016-11-29T18:41:28

2018-05-24T16:41:34

Increase, Mortality

Individual

This key event is observed at the biological level of the individual and describes the increase of mortality of individuals upon exposure to a stressor.

The AO can be detected by observation, for example by immobilization of the respective organisms. There exist guidelines for the characterization of this AO in arthropods. For example, the OECD 202 Daphnia sp. Acute immobilization test (OECD 2004) which can also be modified depending on the effect one expects.

Taxonomic: This AO is applicable to all living organisms. Life stage: This AO is applicable to all life stages. Sex: This AO is applicable to all sexes. Chemical: Substances known to increase mortality in arthropods are of the family of pyrimidine nucleosides (e.g. polyoxin D and nikkomycin Z) (Gijswijt et al. 1979; Tellam et al. 2000; Arakawa et al. 2008).

High Unspecific

High

All life stages

High High

Arakawa T, Yukuhiro F, Noda H. 2008. Insecticidal effect of a fungicide containing polyoxin B on the larvae of Bombyx mori (Lepidoptera: Bombycidae), Mamestra brassicae, Mythimna separata, and Spodoptera litura (Lepidoptera: Noctuidae). Appl Entomol Zool. 43(2):173–181. doi:10.1303/aez.2008.173. Gijswijt MJ, Deul DH, de Jong BJ. 1979. Inhibition of chitin synthesis by benzoyl-phenylurea insecticides, III. Similarity in action in Pieris brassicae (L.) with Polyoxin D. Pestic Biochem Physiol. 12(1):87–94. doi:10.1016/0048-3575(79)90098-1. OECD. 2004. Test No. 202: Daphnia sp. Acute Immobilisation Test. OECD OECD Guidelines for the Testing of Chemicals, Section 2. [accessed 2020 Mar 3]. https://www.oecd-ilibrary.org/environment/test-no-202-daphnia-sp-acute-immobilisation-test_9789264069947-en. Tellam RL, Vuocolo T, Johnson SE, Jarmey J, Pearson RD. 2000. Insect chitin synthase. cDNA sequence, gene organization and expression. Eur J Biochem. 267(19):6025–6043. doi:10.1046/j.1432-1327.2000.01679.x. AOPWiki 2016-11-29T18:41:24

2020-10-26T05:18:16

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AOPWiki 2017-02-09T03:33:12

2017-02-09T03:33:12

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AOPWiki 2017-02-09T03:33:44

2017-02-09T03:33:44

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AOPWiki 2017-02-09T03:34:32

2017-02-09T03:34:32

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AOPWiki 2017-02-09T03:34:57

2017-02-09T03:34:57

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AOPWiki 2017-02-09T03:35:25

2017-02-09T03:35:25

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AOPWiki 2025-09-29T04:17:51

2025-09-29T04:17:51

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AOPWiki 2016-11-29T18:41:36

2016-12-03T16:38:02

<upstream-id>0bc82cd8-c09f-4b6c-b6d7-c97ab36dbffe</upstream-id> <downstream-id>e3f4565b-2b96-4c29-a071-6634ae047ae0</downstream-id>

AOPWiki 2016-11-29T18:41:36

2016-12-03T16:38:02

Ecdysone receptor agonism leading to mortality via suppression of Ftz-f1

EcR agonism leading to mortality via suppression of Ftz-f1

Knut Erik Tollefsen

You Song and Knut Erik Tollefsen Norwegian Institute for Water Research (NIVA), Økernveien 94, N-0579 Oslo, Norway

BY-SA

1.0

This Adverse Outcome Pathway (AOP) describes how hyperactivation of the ecdysone receptor (EcR) in arthropods can lead to lethal molting disruption and mortality. Binding of natural ligands (ecdysteroids) to EcR is critical for regulating molting and metamorphosis in insects and crustaceans. However, inappropriate or prolonged activation of EcR by exogenous chemicals disrupts the tightly regulated temporal gene expression cascade required for successful ecdysis. Key events (KEs) include altered expression of early transcription factors (E75B, Ftz-f1), reduction of circulating neuropeptides (CCAP, ETH), impaired motor program activity, and suppression of abdominal muscle contraction, ultimately resulting in incomplete ecdysis and death. This AOP provides mechanistic understanding relevant for environmental chemical safety assessment, particularly regarding pesticides and other compounds targeting insect endocrine systems. The development of this AOP is motivated by regulatory needs to understand and predict the impacts of insect growth regulators and other endocrine-active chemicals that target EcR. Such compounds are widely used in pest control but pose risks to non-target arthropods, including pollinators and aquatic invertebrates. The AOP formalizes knowledge of conserved molting endocrine pathways to support hazard identification and potential regulatory screening frameworks.

The AOP was developed based on structured literature reviews and expert knowledge. Key sources included primary research on EcR signaling, molting neuropeptides (ETH, CCAP), transcriptional cascades in Drosophila melanogaster and other model insects, as well as crustacean endocrinology. Literature searches were conducted in PubMed, Web of Science, and Scopus using terms such as ecdysone receptor agonists, ecdysis motor program, insect molting disruption, 20-hydroxyecdysone, and ecdysteroid signaling. Priority was given to studies demonstrating experimental perturbation of EcR or downstream KEs and their effects on molting success and survival. Reviews and AOP frameworks (e.g., OECD guidance) were used to ensure structured evaluation and alignment with regulatory relevance.

The Adverse Outcome is highly significant from a regulatory point of view. It is employed as regulatory endpoint in most studies assessing the toxicity of stressors.

adjacent

Not Specified

High adjacent

Not Specified

High adjacent

Not Specified

Moderate adjacent

Not Specified

Moderate adjacent

Not Specified

Moderate adjacent

Not Specified

Moderate adjacent

Not Specified

Moderate adjacent

Not Specified

High

Moderate Unspecific

High

Juvenile

High

Adult

High Moderate High

The overall weight of evidence supporting this AOP is strong, with high biological plausibility and multiple lines of empirical support linking EcR hyperactivation to lethal molting disruption. The key endocrine and neuropeptide signaling pathways involved are highly conserved across arthropods, increasing confidence in broad taxonomic applicability, particularly to insects and crustaceans undergoing ecdysis. Essentiality of key events is well demonstrated through genetic and pharmacological manipulations, and direct causal linkages between upstream molecular initiating events and downstream organism-level outcomes are supported by both in vitro and in vivo studies. While quantitative understanding remains incomplete, especially regarding cross-species dose-response relationships, the evidence base is sufficient to support application in chemical screening, prioritization, and risk assessment. The AOP is considered reliable for use in evaluating the hazards of EcR agonists and related endocrine-active chemicals, with clear regulatory relevance to the assessment of insect growth regulators and protection of non-target arthropods. <ul> <li> Taxa: Arthropods, primarily insects (Diptera, Lepidoptera, Coleoptera) and crustaceans. </li> <li> Life stage: Juvenile and larval stages undergoing molting. </li> <li> Sex: Both sexes are equally affected, as molting regulation is not sex-specific. </li> <li> Other considerations: The pathway is most relevant in holometabolous insects but is applicable across arthropods where molting is controlled by EcR signaling. </li> </ul>

<ul> <li> EcR hyperactivation (MIE): Genetic or pharmacological overactivation prevents correct timing of molting cascades, leading to lethality. </li> <li> E75B and Ftz-f1 expression changes: Knockout or overexpression experiments demonstrate disruption of subsequent endocrine signals and molting success. </li> <li> Circulating ETH/CCAP: Blocking or reducing peptide release suppresses ecdysis behavior. Rescue experiments with exogenous peptides restore motor program activity. </li> <li> Motor program activity and abdominal contractions: Neurophysiological studies show that impaired muscle activity directly prevents exuviation. </li> <li> Incomplete ecdysis: Universally essential for linking endocrine dysfunction to mortality. </li> </ul> Overall, essentiality of KEs is supported by direct experimental evidence in multiple model arthropods.

KER 103 → 1264 (EcR hyperactivation → Increased E75B expression) <ul> <li> Biological plausibility: Strong. E75B is a primary-response gene in the ecdysone signaling cascade. Overactivation of EcR drives prolonged or elevated expression of E75B. </li> <li> Empirical support: Multiple in vitro and in vivo studies (e.g., Drosophila, lepidopterans) show dose-dependent induction of E75B following EcR agonist exposure. </li> <li> Uncertainties: Quantitative thresholds vary across taxa. </li> </ul> KER 103 → 1265 (EcR hyperactivation → Decreased Ftz-f1 expression) <ul> <li> Biological plausibility: Strong. Ftz-f1 is normally induced after a decline in ecdysone signaling, serving as a competence factor for subsequent developmental transitions. Sustained EcR activity suppresses Ftz-f1 expression. </li> <li> Empirical support: Genetic experiments in Drosophila demonstrate that EcR hyperactivation prevents Ftz-f1 induction, leading to molting defects. </li> <li> Uncertainties: The precise timing of downregulation differs between insect species. </li> </ul> KER 1265 → 998 (Decreased Ftz-f1 expression → Decreased circulating ETH) <ul> <li> Biological plausibility: Moderate to strong. ETH release from Inka cells requires proper transcriptional programming, in which Ftz-f1 plays a permissive role. </li> <li> Empirical support: ETH levels are reduced in Ftz-f1 mutant or RNAi knockdown insects, with corresponding ecdysis failure. </li> <li> Uncertainties: Direct mechanistic links in crustaceans are less studied. </li> </ul> KER 1264 → 1266 (Increased E75B expression → Decreased circulating CCAP) <ul> <li> Biological plausibility: Moderate. E75B dysregulation affects downstream neural peptide release patterns, including crustacean cardioactive peptide (CCAP). </li> <li> Empirical support: Studies in Manduca sexta and Drosophila show disrupted CCAP neuron activation in response to EcR agonists. </li> <li> Uncertainties: Empirical dose-response data linking E75B overexpression directly to CCAP suppression are limited. </li> </ul> KER 1266 → 1267 (Decreased circulating CCAP → Decreased ecdysis motor program activity) <ul> <li> Biological plausibility: Strong. CCAP is required to initiate and maintain the motor patterns driving ecdysis behavior. </li> <li> Empirical support: Ablation or silencing of CCAP neurons abolishes normal ecdysis behavior in insects. Exogenous CCAP restores motor program activity in some experimental systems. </li> <li> Uncertainties: Quantitative thresholds for peptide levels triggering full motor program are not well established. </li> </ul> KER 998 → 993 (Decreased circulating ETH → Decreased abdominal muscle contraction) <ul> <li> Biological plausibility: Strong. ETH directly triggers ecdysis motor output by activating central nervous system circuits. </li> <li> Empirical support: ETH knockout or peptide inhibition prevents abdominal contractions in Drosophila larvae. ETH injection can rescue the phenotype. </li> <li> Uncertainties: Effects may be stage-dependent. </li> </ul> KER 1267 → 990 (Decreased ecdysis motor program activity → Incomplete ecdysis) <ul> <li> Biological plausibility: Strong. Motor program activity is essential for successful shedding of the old cuticle. </li> <li> Empirical support: Neurophysiological and behavioral studies show that impaired motor activity directly correlates with failed or incomplete ecdysis. </li> <li> Uncertainties: Variation in motor outputs among species may influence severity. </li> </ul> KER 993 → 990 (Decreased abdominal muscle contraction → Incomplete ecdysis) <ul> <li> Biological plausibility: Strong. Abdominal contractions generate the mechanical force required for cuticle shedding. </li> <li> Empirical support: Pharmacological or genetic inhibition of abdominal muscle contraction prevents complete ecdysis in Drosophila and other insects. </li> <li> Uncertainties: Contribution of other body muscles (e.g., thoracic) not fully quantified. </li> </ul> KER 990 → 350 (Incomplete ecdysis → Increased mortality) <ul> <li> Biological plausibility: Strong. Inability to shed the old cuticle is incompatible with survival. </li> <li> Empirical support: High mortality rates are consistently observed in laboratory and field studies where molting is disrupted by EcR agonists or neuropeptide blockers. </li> <li> Uncertainties: Mortality timing (immediate vs delayed) may vary with species and stage. </li> </ul>

<div> <ul> <li> Temperature: Affects hormone turnover and molting periodicity. </li> <li> Nutritional status: Influences steroid hormone synthesis and peptide release. </li> <li> Species-specific sensitivity: Different insects and crustaceans vary in susceptibility to EcR agonists. </li> <li> Developmental stage: Early versus late larval stages may have differing vulnerability. </li> </ul> <table class="table table-bordered table-fullwidth"> <thead> <tr> <th>Modulating Factor (MF)</th> <th>Influence or Outcome</th> <th>KER(s) involved</th> </tr> </thead> <tbody> <tr> <td> </td> <td> </td> <td> </td> </tr> </tbody> </table> </div>

Quantitative relationships between EcR activation and downstream transcriptional responses are partially characterized, especially in Drosophila. Dose-response data for diacylhydrazine insecticides provide empirical linkage between exposure and incomplete ecdysis. However, quantitative models are still limited and largely taxon-specific.

This AOP has clear utility for both scientific and regulatory applications related to the environmental assessment of endocrine-active substances targeting arthropod molting. Because EcR is the primary molecular target of many insect growth regulators (IGRs), this pathway provides a mechanistic framework for interpreting how chemical binding at the receptor level translates to population-level adverse outcomes such as mortality. From a regulatory perspective, the AOP can inform the development and refinement of OECD test guidelines addressing arthropod development and molting. It can also support the design of integrated approaches to testing and assessment (IATA), in which data from in vitro receptor-binding assays, transcriptomic biomarkers (e.g., E75B, Ftz-f1), and neuropeptide measurements can be combined with higher-tier organismal studies to streamline hazard characterization. Furthermore, the pathway may facilitate the identification of molecular biomarkers that can be incorporated into early screening assays, reducing reliance on animal-intensive in vivo tests. The AOP is also relevant for chemical grouping and read-across approaches, particularly for compounds within the diacylhydrazine class and other EcR agonists. Structure–activity relationship ((Q)SAR) models or chemical profilers trained on these endpoints could help predict EcR activity and prioritize substances for further testing. For ecological risk assessment, this AOP highlights the potential for population-level impacts on non-target arthropods, including beneficial insects (e.g., pollinators) and aquatic crustaceans. Given the essential role of molting for growth and reproduction, disruptions captured in this pathway provide a mechanistic basis to link molecular initiating events to ecologically relevant endpoints. Overall, this AOP offers opportunities to improve chemical safety decision-making by providing a structured framework to integrate mechanistic data into regulatory contexts, enabling screening, prioritization, and risk assessment of chemicals that act through EcR hyperactivation.

High

Song, Y.; Villeneuve, D. L.; Toyota, K.; Iguchi, T.; Tollefsen, K. E., 2017. Ecdysone receptor agonism leading to lethal molting disruption in arthropods: review and adverse outcome pathway development. Environ Sci Technol, 51, (8), 4142-4157. Song, Y., Evenseth, L.M., Iguchi, T., Tollefsen, K.E., 2017. Release of chitobiase as an indicator of potential molting disruption in juvenile Daphnia magna exposed to the ecdysone receptor agonist 20-hydroxyecdysone. J Toxicol Environ Health A, 1-9 Fay, K. A., Villeneuve, D. L., LaLone, C. A., Song, Y., Tollefsen, K. E. and Ankley, G. T., 2017. Practical approaches to adverse outcome pathway (AOP) development and weight of evidence evaluation as illustrated by ecotoxicological case studies. Environ. Toxicol. Chem. 36(6):1429-1449. Miyakawa, H., Sato, T., Song, Y., Tollefsen, K.E., Iguchi, T., 2017. Ecdysteroid and juvenile hormone biosynthesis, receptors and their signaling in the freshwater microcrustacean Daphnia. J Steroid Biochem Mol Biol. pii: S0960-0760(17), 30370-30379.   AOPWiki 2016-11-29T18:41:15

2025-10-02T04:18:29