Ferrochelatase inhibition: The upstream molecular target of helioxanthin triggering mitochondrial collapse and larval death in Aedes aegypti.

The widespread use of chemical insecticides has led to resistance and environmental concerns, driving the need for botanical insecticides with novel modes of action. This study identified ferrochelatase as the upstream molecular target of helioxanthin. Inhibition of this enzyme triggered the compound's potent insecticidal activity against Aedes aegypti larvae, which exhibited an LC50 value of 2.79 mg/L (comparable to rotenone). Beyond mortality, helioxanthin exposure also induced behavioral and growth inhibition. Treatment induced severe mitochondrial ultrastructural damage and dysfunction, including ATP depletion, inhibition of respiratory complexes IV-V, and early loss of membrane potential. Multi-omics analyses revealed significant ferrochelatase downregulation and disrupted heme biosynthesis. Molecular docking and dynamics simulations demonstrated high-affinity binding between helioxanthin and ferrochelatase (ΔGbind = -66.3 kJ/mol), primarily driven by van der Waals interactions within a hydrophobic pocket involving key residues such as Ile-39 and Met-23. In vivo assays confirmed dose-dependent ferrochelatase inhibition (>78%, 24 h, at 11.2 mg/L), leading to protoporphyrin IX accumulation, a condition known to induce an intracellular ROS burst. Thus, ferrochelatase inhibition was identified as the initiating molecular event leading to mitochondrial collapse and larval death, revealing a novel mechanism of action and providing a promising target for developing next-generation green insecticides.