p-Cymene enhances toxicity and penetration of thymol in the larvae of Trichoplusia ni

We investigated the penetration-enhancing effect of the synergistic insecticidal combination of the major constituents of thyme oil in the cabbage looper, Trichoplusia ni. Several synergistic interactions were observed among the four major constituents of thyme oil via topical application in third instar larvae, including the thymol+p-cymene combination at a 1:1 ratio. In vivo GC-MS analyses of penetration through the cuticle of third instar larvae showed increased detections of both compounds when mixed compared to individual applications, with twice as much thymol detection at 8h post-treatment. In vitro analyses using the integument of fifth instar larvae confirmed the same tendency (increased penetration of thymol in mixture), indicating the role of p-cymene in cuticular penetration, whereas in an injection assay, p-cymene made no direct contribution to the toxicity of the thymol+p-cymene combination. Further bioassays with topical application of thymol alone produced the same LD₅₀ value as the thymol+p-cymene mixture (i.e., requiring only one-half as much thymol), but the application of the two compounds to different locations on the body required twice the LD₅₀ value, the same dose as that of thymol. Contact angle measurements on a beeswax layer suggest increased spreadability of thymol on the wax layer of the insect by lowering the surface tension. These in vitro results can be considered the first conclusive evidence confirming a penetration-enhancing effect of synergistic combinations of plant essential oil constituents, and this technique can be adapted by other research areas to monitor penetration of synergistic combinations, structure-activity relationships, or insecticide resistance.