Ecdysone receptor hyperactivation leading to mortality via mis-timed activation of nuclear receptor cascade

You Song & co-authors

Norwegian Institute for Water Research (NIVA), Økernveien 94, NO-0579 Oslo, Norway

This AOP describes how hyperactivation of the ecdysone receptor (EcR) initiates a cascade of mis-timed nuclear receptor signaling, disrupting the tightly regulated endocrine control of insect molting. Dysregulation of key neuropeptides (ETH, CCAP) and subsequent impairment of motor programs and abdominal muscle contractions lead to incomplete ecdysis. Failure to properly shed the cuticle results in increased mortality.

This AOP highlights a critical endocrine mechanism relevant to insect growth and survival, providing a biologically plausible framework for linking molecular-level perturbations to population-relevant outcomes. It has direct regulatory relevance for evaluating endocrine-disrupting chemicals that target EcR signaling, such as insect growth regulators (IGRs), which are widely used in pest control but may pose risks to non-target insect populations, including pollinators and beneficial species.

By capturing key mechanistic events from receptor hyperactivation through behavioral and physiological impairment to organismal death, this AOP can support ecological risk assessment, guide the development of mechanistic testing strategies, and inform regulatory decision-making for chemicals acting on the EcR pathway.

The development of this AOP was motivated by the regulatory and ecological importance of chemicals that target the ecdysone receptor (EcR) in insects. EcR is the molecular target of several classes of insect growth regulators (IGRs), which are deliberately designed to disrupt molting and cause lethality in pest species. However, these same mechanisms raise concern for potential adverse effects on non-target insects, including pollinators and beneficial arthropods. Because ecdysis is a highly conserved and hormonally regulated process essential for insect survival, disruptions of EcR signaling provide a biologically plausible and ecologically relevant pathway to mortality.

This AOP was therefore prioritized to support risk assessment and decision-making for EcR-active substances. It provides a mechanistic framework that integrates a large body of invertebrate endocrinology and neuroethology, allowing regulators to trace the cascade from molecular receptor activation to survival outcomes. The background understanding of ecdysis neuroendocrinology—developed over decades of model insect research—offers strong biological plausibility and cross-species relevance, making it a valuable case study for mechanistic regulatory science.

The identification and evaluation of the key events (KEs) and key event relationships (KERs) were based on a structured strategy combining expert input, preliminary scoping, and systematic literature surveys:

1. Preliminary scoping and expert input

   • The starting point was established entomological literature on EcR signaling, molting neuroendocrine cascades, and motor program control.

   • Expert consultations with insect endocrinologists and toxicologists helped define the initial scope: perturbations to EcR signaling leading to lethal molting failure.

   • The AOP framework was cross-checked against existing knowledge of other insect hormone pathways (juvenile hormone, PTTH) to ensure specificity and avoid redundancy.

2. Literature identification and screening strategy

   • Databases searched: PubMed, Web of Science

   • Search terms: “ecdysone receptor agonist,” “EcR hyperactivation,” “ecdysis neuroendocrine cascade,” “ETH CCAP insect molting,” “ecdysis motor program,” “ecdysis mortality,” “insect growth regulator EcR,” “ecdysteroid signaling timing,” and combinations thereof.

   • Timeframe: Literature spanning ~1970 to 2023 was considered, as foundational insect endocrinology was established in the late 20th century and more recent studies provide mechanistic and toxicological data.

   • Searches emphasized both mechanistic biology (basic insect physiology and endocrinology) and applied ecotoxicology (chemical testing, IGR exposure studies).

3. Screening and selection

   • Abstracts were screened for relevance to EcR activation and downstream events leading to molting outcomes.

   • Inclusion criteria: mechanistic experimental data linking EcR signaling to endocrine output (ETH/CCAP), neural/motor activity, or apical ecdysis outcomes.

   • Exclusion criteria: studies limited to non-EcR endocrine pathways (unless providing comparative insights), purely descriptive morphology without mechanistic data, or vertebrate-focused studies.

4. Quality assessment and evidence integration

   • Evidence for each KE and KER was evaluated using OECD AOP WoE guidance (biological plausibility, essentiality, and empirical support).

   • Comparative studies across insect taxa were considered for domain of applicability.

   • Particular attention was paid to temporal concordance (e.g., timing of EcR activation vs. hormone release vs. motor output), dose–response information, and reproducibility across stressors (genetic manipulation vs. chemical activation).

This structured strategy ensured that the AOP is grounded in well-established insect physiology while incorporating toxicological evidence relevant to chemical risk assessment. The documentation of the search scope, terms, and expert scoping facilitates transparency, reproducibility, and re-use of the AOP and its components in other networks addressing arthropod endocrine disruption.

The overall weight of evidence (WoE) for this AOP is high, supported by strong biological plausibility, extensive empirical evidence, and moderate but growing quantitative understanding. The mechanistic underpinnings are grounded in well-established endocrine and neurophysiological processes that regulate molting in arthropods.

• Biological plausibility: The causal linkages between EcR activity, transcriptional cascades, neuropeptide release (ETH, CCAP), motor program activation, and abdominal muscle contraction are strongly supported by fundamental developmental biology. Each KE corresponds to a critical control point in the molting process, and their functional interdependencies are highly conserved across arthropods. The plausibility of the AOP is reinforced by decades of work in insect endocrinology and neurobiology, making the overall pathway mechanistically coherent.

• Essentiality of key events: Essentiality is considered high. Genetic, pharmacological, and RNAi manipulations at multiple nodes (EcR activity, nuclear receptor genes, ETH/CCAP neurons, motor programs) consistently demonstrate that disruption prevents progression to downstream events and results in incomplete ecdysis. Rescue experiments (e.g., exogenous ETH or CCAP) further confirm causal roles of upstream events in controlling downstream outcomes.

• Empirical support: Empirical evidence is strong, with consistent observations across multiple model systems, particularly holometabolous insects (Drosophila melanogaster, Manduca sexta, Bombyx mori, Tribolium castaneum). Hyperactivation of EcR by diacylhydrazine insecticides reliably produces predictable phenotypes: altered gene expression, reduced peptide release, suppression of motor activity, incomplete ecdysis, and mortality. Similar outcomes have been observed in crustaceans, though fewer studies exist. The relationships are reproducible and concordant across experimental systems.

• Quantitative understanding: Current quantitative data are moderate. Some dose–response and temporal dynamics have been characterized for EcR agonists, transcriptional markers, and ETH release, showing concordance between exposure intensity and severity of downstream disruption. However, quantitative thresholds vary by species, and detailed response–response models remain limited. Cross-species extrapolation requires further development.

• Domain of applicability: The AOP is broadly applicable to arthropods. Evidence is strongest for holometabolous insects, with moderate confidence for crustaceans. Applicability is equal in both sexes and most relevant for juvenile and larval life stages undergoing ecdysis.

Regulatory relevance: This AOP is highly relevant for the assessment of insect growth regulators and other EcR-active chemicals. The robustness of the evidence supports its application in screening, prioritization, test guideline refinement, and ecological risk assessment of non-target arthropods. While quantitative relationships require strengthening, the AOP already provides a reliable mechanistic framework linking receptor-level perturbations to population-relevant adverse outcomes.

Overall conclusion: The AOP is supported by strong mechanistic plausibility, consistent empirical evidence, and partial quantitative understanding. Confidence in the AOP is high for insects and moderate but growing for crustaceans. This AOP is sufficiently developed for regulatory consideration and provides a valuable tool for integrating mechanistic data into chemical hazard and risk assessment.

This AOP provides a mechanistic framework that can be applied across several domains of chemical hazard assessment and regulatory decision-making. Because EcR is a critical developmental regulator in arthropods, perturbations at this receptor level are highly predictive of adverse outcomes that impact survival and population sustainability. The structured evidence in this AOP allows for multiple scientific and regulatory applications:

1. Screening and Prioritization of Chemicals

• The molecular initiating event (EcR hyperactivation) can be evaluated using in vitro receptor-binding assays, reporter gene assays, or in silico modeling (e.g., docking and QSAR approaches).

• This allows early identification of chemicals with EcR agonist activity, facilitating prioritization for further testing.

2. Development and Refinement of Test Guidelines

• Intermediate KEs (mis-timed transcriptional cascades, altered neuropeptide release, suppression of motor activity) provide mechanistic biomarkers that could be incorporated into OECD guideline refinements.

• For example, ETH and CCAP measurements or transcriptomic markers (E75, HR3, Ftz-f1) could be developed into endpoints for short-term assays, reducing reliance on labor-intensive whole-organism tests.

3. Integrated Approaches to Testing and Assessment (IATA)

• The AOP provides the scientific foundation for integrating multiple data streams (in vitro assays, omics biomarkers, neuropeptide quantification, behavioral endpoints) into tiered testing strategies.

• Such IATAs can reduce animal use and provide mechanistic clarity for regulatory submissions.

4. Grouping and Read-Across

• The AOP supports chemical grouping strategies by identifying shared modes of action (EcR agonism).

• Structural alerts (e.g., diacylhydrazine scaffolds) and activity profiling can be used to apply read-across approaches to new or untested compounds.

5. Ecological Risk Assessment

• By mechanistically linking molecular initiating events to population-level mortality, this AOP provides a foundation for extrapolating laboratory mechanistic data to ecological risk scenarios.

• Particularly relevant for non-target insects (e.g., pollinators, natural enemies) and aquatic crustaceans that may be exposed to EcR-active pesticides in agricultural runoff.

• Population models could incorporate incomplete ecdysis as a measurable endpoint to predict ecological consequences.

6. Regulatory Decision-Making

• Regulators may use this AOP in weight-of-evidence evaluations for substances with suspected endocrine-disrupting activity in arthropods.

• It provides a transparent mechanistic rationale that strengthens the scientific basis for restrictions, mitigation measures, or prioritization of monitoring for non-target exposure.

7. Research and Method Development

• The AOP highlights knowledge gaps in quantitative relationships between KEs, encouraging targeted research.

• Development of quantitative models (e.g., dose-response functions for ETH and CCAP suppression) could improve predictive utility.

Summary: This AOP has strong potential applications in screening, test guideline refinement, grouping/read-across, and ecological risk assessment. It provides a mechanistic bridge from molecular initiating events to organismal mortality, supporting regulatory decisions concerning the use and approval of EcR-active insecticides and other endocrine-active substances.

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