Ecdysone receptor antagonism leading to mortality via mis-timed ecdysone receptor-responsive nuclear receptor cascade

• You Song

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The applicability of this AOP is determined by the conservation of ecdysone receptor (EcR) signaling, nuclear receptor cascades, and the endocrine–neuromuscular coordination of molting in arthropods. These processes are essential and broadly conserved, making the AOP relevant to a wide range of taxa, life stages, and ecological contexts.

• Taxonomic applicability:

  • Insects: Evidence is strongest in holometabolous insects (Drosophila melanogaster, Manduca sexta, Bombyx mori, Tribolium castaneum), where EcR signaling pathways and their role in regulating neuropeptide release and ecdysis are well studied. Hemimetabolous insects (e.g., locusts, cockroaches) also depend on EcR-mediated signaling for molting, though fewer direct data on antagonism are available.

  • Crustaceans: EcR signaling and chitin synthesis are conserved in aquatic crustaceans (Daphnia magna, Carcinus maenas, Penaeus spp.). Several studies demonstrate EcR involvement in molting control, although direct evidence for EcR antagonism is more limited compared to agonism.

  • Other arthropods: Arachnids and myriapods also undergo EcR-regulated molting and chitin synthesis, suggesting the AOP is broadly relevant, although direct empirical data are scarce.

• Sex applicability: The AOP is equally applicable to both sexes. EcR signaling and molting regulation are not sex-dependent, and antagonism of EcR disrupts development and survival irrespective of sex.

• Life-stage applicability: This AOP is most relevant to juvenile and larval stages that must undergo molting to grow and reach maturity. In insects, applicability is strongest during larval and pupal molts. In crustaceans, molting occurs throughout life, making the AOP relevant across both juvenile and adult stages. Adults in insects that have completed metamorphosis are generally not susceptible, as EcR no longer drives molting processes.

• Environmental context: The AOP is applicable in both terrestrial and aquatic environments where arthropods are exposed to EcR antagonists. Possible exposure scenarios include:

  • Agricultural systems: pesticide use or residues with unintended EcR antagonist activity.

  • Aquatic ecosystems: runoff or leaching introducing EcR-active contaminants that affect crustaceans and aquatic insects.

  • Laboratory/experimental systems: synthetic EcR antagonists or candidate insecticides tested in model organisms.

Summary: This AOP is broadly applicable to arthropods, with high confidence for holometabolous insects, moderate confidence for crustaceans, and lower but plausible confidence for other arthropod groups. It applies to both sexes and is particularly critical during juvenile/larval stages, though in crustaceans it may extend into adulthood due to lifelong molting.

The essentiality of the key events (KEs) in this AOP is considered high overall, as demonstrated by genetic, pharmacological, and physiological evidence. Manipulations at each point of the pathway show that blocking or altering a KE prevents the normal sequence of downstream events, culminating in incomplete ecdysis and mortality.

MIE: EcR antagonism (Event 2374)

• Evidence: EcR is the master regulator of steroid-induced molting responses. Competitive antagonists, dominant-negative EcR mutations, or EcR knockdowns prevent induction of downstream nuclear receptor genes.

• Support: In insects, EcR antagonism leads to suppression of HR3, Ftz-f1, and E75 expression, resulting in developmental arrest and failed molting.

• Essentiality rating: High – antagonism at this level blocks the entire cascade.

KE: Mis-timed EcR-responsive nuclear receptor cascade (Event 2375)

• Evidence: The cascade of nuclear receptors is essential for establishing endocrine and neuronal competence for molting. If the cascade is not properly activated, subsequent events such as ETH and CCAP release cannot occur at the correct time or magnitude.

• Support: Knockdown of HR3 or Ftz-f1 in Drosophila prevents ETH induction and ecdysis behavior.

• Essentiality rating: High – disruption here directly leads to failure of peptide release and downstream events.

KE: Decreased circulating ETH (Event 998)

• Evidence: ETH released from Inka cells is indispensable for triggering the ecdysis motor program.

• Support: ETH knockout mutants or RNAi suppression in Drosophila abolish ecdysis behavior. Administration of synthetic ETH rescues ecdysis in some cases, even under EcR disruption.

• Essentiality rating: High – ETH is the proximate trigger of the motor program.

KE: Decreased circulating CCAP (Event 1266)

• Evidence: CCAP neurons sustain rhythmic motor outputs during ecdysis. Their activation depends on proper endocrine priming.

• Support: Ablation or silencing of CCAP neurons abolishes ecdysis motor activity; exogenous CCAP partially restores abdominal contractions. Under EcR antagonism, CCAP release is suppressed.

• Essentiality rating: High – CCAP is required for maintenance of motor activity during ecdysis.

KE: Decreased ecdysis motor program activity (Event 1267)

• Evidence: The central pattern generator driving ecdysis behavior must be activated by ETH and maintained by CCAP.

• Support: Neural recordings show that suppression of ETH/CCAP leads to reduced or absent motor programs. Without this motor program, ecdysis cannot proceed.

• Essentiality rating: High – the motor program is indispensable for shedding the cuticle.

KE: Decreased abdominal muscle contraction (Event 993)

• Evidence: Abdominal contractions generate the mechanical forces required for exuviation.

• Support: Pharmacological suppression of muscle activity or targeted neuromuscular inhibition prevents molting. EcR antagonism reduces contraction frequency and intensity through ETH/CCAP suppression.

• Essentiality rating: High – abdominal contractions are necessary for successful ecdysis.

KE: Incomplete ecdysis (Event 990)

• Evidence: Incomplete shedding of the old cuticle results in entrapment and lethality.

• Support: Consistently observed in EcR antagonist treatments and in peptide or motor program knockdowns.

• Essentiality rating: High – incomplete ecdysis is the direct cause of mortality.

AO: Mortality (Event 350)

• Evidence: Mortality is the inevitable outcome of incomplete ecdysis. Individuals unable to shed their old cuticle cannot survive to the next developmental stage.

• Support: Observed across all insect species tested with EcR antagonists and confirmed in crustaceans under molting disruption conditions.

Overall conclusion on essentiality:

Essentiality of the KEs is high across the entire pathway. Direct manipulations demonstrate that EcR antagonism initiates a cascade of failures in neuropeptide signaling and neuromuscular function, leading inevitably to incomplete ecdysis and death. Rescue studies (e.g., peptide supplementation, gene overexpression) provide further causal evidence that intermediate KEs (ETH, CCAP, motor activity) are indispensable. Confidence in essentiality is highest in insects and moderate but increasing in crustaceans.

The overall evidence for this AOP is supported by strong biological plausibility and increasing empirical support from insect and crustacean models. Each KER is assessed below in terms of biological plausibility, empirical evidence, and quantitative understanding.

KER 2374 → 2375 (EcR antagonism → Mis-timed nuclear receptor cascade)

• Biological plausibility: Strong. EcR is the central nuclear receptor mediating steroid hormone signaling during molting. Antagonism prevents proper activation of downstream nuclear receptors (e.g., HR3, Ftz-f1, E75).

• Empirical support: In Drosophila melanogaster and Manduca sexta, EcR knockdowns or exposure to EcR inhibitors suppress nuclear receptor expression, leading to blocked molting.

• Quantitative understanding: Limited. Qualitative suppression of nuclear receptor targets is clear, but dose–response thresholds for EcR antagonists remain incompletely defined.

KER 2375 → 998 (Mis-timed nuclear receptor cascade → Decreased ETH release)

• Biological plausibility: Strong. Nuclear receptor signaling is required to establish Inka cell competence for ETH secretion.

• Empirical support: HR3 and Ftz-f1 knockdown in insects prevents ETH release, resulting in molting failure. In EcR antagonist exposures, ETH levels are reduced or absent.

• Quantitative understanding: Limited. ETH titer measurements exist for insects exposed to antagonists, but quantitative relationships to receptor inhibition are not fully characterized.

KER 2375 → 1266 (Mis-timed nuclear receptor cascade → Decreased CCAP release)

• Biological plausibility: Moderate to strong. CCAP neuron activation is downstream of transcriptional and endocrine priming. Antagonism of EcR prevents the cascade needed for their proper activation.

• Empirical support: Reduced CCAP immunoreactivity and neuronal activity observed in antagonist-treated insects. CCAP knockdown studies independently confirm that reduced CCAP leads to impaired motor programs.

• Quantitative understanding: Sparse. While CCAP suppression is qualitatively observed, detailed dose–response data are lacking.

KER 998 → 1267 (Decreased ETH → Decreased motor program activity)

• Biological plausibility: Strong. ETH directly activates central pattern generators in the insect CNS to initiate ecdysis behavior.

• Empirical support: ETH knockouts in Drosophila abolish motor program activity. Rescue experiments with exogenous ETH restore normal contractions. In EcR antagonist studies, suppression of ETH correlates with reduced or absent motor patterns.

• Quantitative understanding: Moderate. Threshold ETH concentrations needed for CNS activation have been estimated, but species-specific differences exist.

KER 1266 → 1267 (Decreased CCAP → Decreased motor program activity)

• Biological plausibility: Strong. CCAP sustains motor activity during ecdysis.

• Empirical support: Silencing CCAP neurons abolishes rhythmic motor outputs. Exogenous CCAP restores abdominal contractions in insects exposed to EcR modulators.

• Quantitative understanding: Limited. Data demonstrate binary presence/absence of motor activity but lack dose–response characterization.

KER 1267 → 990 (Decreased motor program activity → Incomplete ecdysis)

• Biological plausibility: Strong. Coordinated motor programs are required for shedding the old cuticle. Without motor output, the exuvia cannot be shed.

• Empirical support: In multiple insect species, reduced or absent motor activity directly correlates with incomplete ecdysis. Behavioral recordings show that even partial motor impairment prevents full shedding.

• Quantitative understanding: Moderate. Time-course and behavioral data are available, but quantitative thresholds for activity loss leading to incomplete ecdysis remain undefined.

KER 993 → 990 (Decreased abdominal muscle contraction → Incomplete ecdysis)

• Biological plausibility: Strong. Abdominal contractions are the physical mechanism for exuviation.

• Empirical support: Pharmacological or neural inhibition of muscle contractions leads to incomplete molting in Manduca sexta and Drosophila. In EcR antagonist treatments, contraction amplitude and frequency are suppressed.

• Quantitative understanding: Limited. Data are mostly descriptive, with few quantitative models linking contraction deficits to molting failure.

KER 990 → 350 (Incomplete ecdysis → Mortality)

• Biological plausibility: Strong. Failure to shed the cuticle is universally lethal, as the animal remains trapped in the old exoskeleton.

• Empirical support: In both insects and crustaceans, incomplete molting caused by EcR disruption results in nearly 100% mortality.

• Quantitative understanding: High at the AO level; survival is binary (ecdysis complete = survival, incomplete = death).

Overall WoE conclusion:

• Biological plausibility: High, with strong support across all KERs.

• Empirical support: Moderate to strong. Evidence is strongest in insects, with emerging but more limited support in crustaceans.

• Quantitative understanding: Moderate. While qualitative evidence is robust, quantitative dose–response data are incomplete, and cross-species extrapolation remains uncertain.

Confidence in the overall AOP: High for insects (Diptera, Lepidoptera, Coleoptera); moderate for crustaceans; plausible for other arthropods but requiring additional data.

Quantitative understanding of this AOP is moderate, with several well-described dose–response relationships for EcR antagonists in insects, but limited cross-species data and few predictive models that cover the entire cascade. The available evidence supports the directionality and causal links between KEs, but precise thresholds, dynamic ranges, and interspecies extrapolation remain uncertain.

• Molecular initiating event (EcR antagonism): In vitro receptor-binding and reporter assays have been used to determine antagonist potency (IC₅₀, Ki values) for specific compounds. Competitive inhibition assays demonstrate dose-dependent suppression of EcR activation, but interspecies differences in receptor isoforms complicate extrapolation.

• EcR-responsive nuclear receptor cascade: Transcriptomic studies in Drosophila melanogaster and Manduca sexta have shown that antagonists reduce or delay expression of nuclear receptor targets (e.g., HR3, Ftz-f1, E75) in a concentration-dependent manner. While these data establish concordance, quantitative thresholds vary widely by chemical and species.

• Neuropeptide levels (ETH, CCAP): Dose-dependent decreases in ETH and CCAP release have been observed under EcR antagonism. ETH titers in hemolymph decline significantly at antagonist concentrations above established IC₅₀ values. However, quantitative models linking peptide suppression to motor program failure are incomplete, and available data are primarily descriptive.

• Motor program activity and abdominal contractions: Electrophysiological and behavioral assays reveal that reductions in ETH/CCAP correspond to impaired or absent motor activity. Some dose–response data exist, showing stepwise decreases in contraction frequency and amplitude with antagonist exposure. However, thresholds at which contractions become insufficient for complete molting are not fully defined.

• Incomplete ecdysis and mortality: Laboratory exposures to EcR antagonists show a clear dose-dependent increase in incomplete molting, with mortality approaching 100% at high concentrations. Survival curves for insects exposed to different antagonist doses show steep responses, indicating limited tolerance margins. These data provide the strongest quantitative linkage in the pathway.

• Cross-species extrapolation: Data are most developed for holometabolous insects. Crustacean studies (e.g., Daphnia magna) report delayed or failed molts under antagonist exposure, but quantitative ETH/CCAP measurements are lacking. Differences in life-history traits (e.g., molting frequency, hormonal dynamics) complicate extrapolation between taxa.

• Uncertainties and data gaps:

  • Lack of standardized assays for quantifying ETH and CCAP suppression across species.

  • Few dynamic or mathematical models describing response–response relationships between KEs.

  • Limited quantitative data for crustaceans, arachnids, and other non-insect arthropods.

  • Species-specific EcR isoforms may show variable sensitivity to antagonists, complicating generalization.

Summary: Quantitative relationships are best defined at the extremes of the pathway—at the MIE (receptor binding/activation assays) and the AO (dose-dependent mortality). Intermediate KEs (nuclear receptor expression, peptide release, motor activity) are supported by concentration-dependent observations but lack robust dose–response models. The overall confidence in quantitative understanding is moderate for insects, low to moderate for crustaceans, and uncertain for other arthropods. Developing standardized biomarkers and quantitative models would significantly improve predictive application of this AOP.