Plant essential oils are intricate blends comprising predominantly of monoterpenes and some sesquiterpenes. These oils display diverse bioactivities against targeted organisms, often arising from complex interactions among their constituents, which may demonstrate synergistic or antagonistic effects. Despite their wide use as botanical insecticides, the mechanisms behind these interactions and their effects on bioactivity are poorly understood. This study investigated the synergistic interaction of thymol and p-cymene, two major constituents of Thymus vulgaris essential oil, on the larvae and adults of the housefly, Musca domestica. The results showed that p-cymene synergized the insecticidal activity of thymol in adult houseflies, but not in larvae. GC-MS analyses and bioassays indicated the increased cuticular penetration of thymol by p-cymene was the mechanism of synergy, which was observed only in the adults. Two potential routes were proposed: the expansion of the wetting area, or the disruption of cuticular integrity through dissolving the wax layer. The sequential application and large-volume treatment bioassay results suggested that the former was the more likely mechanism. Also, the hydrophobicity of the cuticle seemed critical for this stage-specific synergy. Wax-devoid adults failed to show synergistic toxicity, whereas artificially wax-coated larvae gained a synergistic effect. Overall, the findings provide insights into the synergistic mechanism of insecticidal activity of plant essential oils and suggest potential applications in developing effective strategies using penetration-enhancing synergists.
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