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

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

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 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.

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.

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.