Insecticidal toxicities of carvacrol and thymol derived from Thymus vulgaris Lin. against Pochazia shantungensis Chou & Lu., newly recorded pest

The insecticidal toxicities of five essential oils against Pochazia shantungensis adults and nymphs, newly recorded pests, were evaluated. The LC50 values of Thymus vulgaris, Ruta graveolens, Citrus aurantium, Leptospermum petersonii and Achillea millefolium oils were recorded as 57.48, 84.44, 92.58, 113.26 and 125.78 mg/L, respectively, against P. shantungensis nymphs using the leaf dipping bioassay, and 75.80, 109.86, 113.26, 145.06 and 153.74 mg/L, respectively, against P. shantungensis adults using the spray bioassay method. Regarding volatile components identified in T. vulgaris oil, the LC50 values of carvacrol and thymol using the leaf dipping bioassay against P. shantungensis nymphs were 56.74 and 28.52 mg/L, respectively. The insecticidal action of T. vulgaris oil against P. shantungensis could be attributed to carvacrol and thymol. Based on the structure-toxicity relationship between thymol analogs and insecticidal toxicities against P. shantungensis nymphs similar to the LC50 values against P. shantungensis adults, the LC50 values of thymol, carvacrol, citral, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol were 28.52, 56.74 and 89.12, 71.41, 82.49, and 111.28 mg/L, respectively. These results indicate that the insecticidal mode of action of thymol analogs may be largely attributed to the methyl functional group. Thymol analogues have promising potential as first-choice insecticides against P. shantungensis adults and nymphs.

Scientific RepoRts | 7:40902 | DOI: 10.1038/srep40902 So for no report has been received about the insecticidal toxicities of Thymus vulgaris oil-derived constituents against P. shantungensis. Therefore, the aims of the present study were first to investigate the insecticidal properties of T. vulgaris oil-derived components against P. shantungensis adults and nymphs, and then to determine the structure-activity relationship between thymol analogs and insecticidal toxicities.

Results and Discussion
This study was undertaken within the framework of a more general study involving the natural products for insecticidal toxicities against P. shantungensis adults and nymphs. Essential oils of Achillea millefolium flowers, Citrus aurantium fruits, Leptospermum petersonii leaves, Ruta graveolens leaves and T. vulgaris leaves were analyzed ( Table 1). The yields of A. millefolium, C. aurantium, L. petersonii, R. graveolens and T. vulgaris oils were 0.658, 1.451, 0.984, 0.924, and 1.122%, respectively. The insecticidal toxicities of the five oils against P. shantungensis adults and nymphs were evaluated after 48 and 72 h exposure ( Table 2). From the leaf dipping and spray bioassays against P. shantungensis adults and nymphs, the insecticidal responses and the LC 50 values increased from 48 to 72 h exposure. The LC 50 values of T. vulgaris, R. graveolens, C. aurantium, L. petersonii and A. millefolium oils at 72 h exposure were 75.80, 109.86, 113.26, 145.06 and 153.74 mg/L, respectively, in the spray bioassay against P. shantungensis adults, and 57.48, 84.44, 92.58, 113.26 and 125.78 mg/L, respectively, in the leaf dipping bioassay against P. shantungensis nymphs. Based on the LC 50 values against P. shantungensis adults and nymphs, T. vulgaris oil had the highest insecticidal toxicity followed by R. graveolens, C. aurantium, L. petersonii and A. millefolium oils. The insecticidal toxicity of T. vulgaris oil against P. shantungensis nymphs was about 1.3-fold more than that against P. shantungensis adults. There was no insect mortality in the distilled water treatment (negative control) of P. shantungensis adults and nymphs. Differences in the insecticidal toxicities of plant-derived oils may be explained on the basis of species-specific responses to plant species, phytochemicals, and the weight and size of P. shantungensis adults and nymphs 11 .
C. aurantium, L. petersonii, R. graveolens, and T. vulgaris oils using the leaf dipping bioassay were 28.52, 56.74 and 89.12 mg/L respectively. Using the spray bioassay against P. shantungensis adults, the LC 50 values of thymol, carvacrol and citral were 42.12, 75.62, and 102.74 mg/L, respectively. The insecticidal toxicity of thymol against P. shantungensis nymphs and adults was approximately 1.8-3.3 times greater than that of carvacrol and citral. In contrast, the other components (β -caryophyllene, camphene, caryophyllene oxide, ρ -cymene, linalool, limonene, myrcene, α -phellandrene, α -pinene, terpinolene, γ -terpinene, sabinene, β -pinene, camphor, (− )-carveol, geraniol, and bornyl acetate) did not exhibit any insecticidal toxicity against P. shantungensis adults and nymphs (data not shown). The insecticidal toxicities of the essential oils appear to be connected to their chemical composition. The insecticidal toxicities of T. vulgaris and R. graveolens oils could be due to the existence of thymol and carvacrol, which exhibited the greatest insecticidal toxicities. The essential oils of C. aurantium and L. petersonii contain citral, which showed insecticidal toxicities against P. shantungensis adults and nymphs, however its toxicity was weaker than thymol and carvacrol. Furthermore, P. shantungensis nymphs were more susceptible to T. vulgaris oil, carvacrol and thymol, when compared to P. shantungensis adults ( Fig. 1). In a previous study, the differential susceptibility shown by P. shantungensis adults and nymphs to thymol and carvacrol was attributed to differences in the weights and sizes of P. shantungensis adults and nymphs, as well as the potential to detoxify glutathione S-transferase and hydrolase [7][8][9] . The synergetic effect of thymol combined with carvacrol has previously been reported for other insects, such as beetles 14 and lepidopterans 6 . Medeiros et al. 15 suggested that thymol and carvacrol to different species of insects are connected with the insecticidal effect of these monoterpenes on the cells of target insects, since they cause disorganization in the cell membrane, leading it to lose permeability 15 . In contrast, although the A. millefolium oil did not contain thymol, carvacrol and citral, the insecticidal properties of A. millefolium against P. shantungensis adults and nymphs could be due to internal synergy or blend effect of their constituents. Previous study reported internal synergy or a blend effect of the main constituents of plant oil for Ocimum kenyenst 16 , Zanthoxylum armatum 17 and Plectranthus marruboides 18 oils against the mosquito species, Aedes aegypti and Anopheles gambiae. Our results indicate that some terpenes containing the other tested components may correlate with the detoxification mechanisms of P. shantungensis adults and nymphs by several terpenes. Treatment with terpenes of Melia azedarach against Spodoptera littoralis can significantly increase the activities of α -esterase and β -esterase, which are important detoxifying enzymes 19 and significant decreased the acid phosphatases, alkaline phosphatases, adenosine triphosphatases and the lactate dehydrogenase of Cnaphalocrocis medinalis 20 .
In order to establish the structure-toxicity relationship between thymol analogs and insecticidal toxicities against P. shantungensis adults and nymphs, thymol, carvacrol, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol were selected as thymol analogs for testing (Fig. 1). The insecticidal toxicities of thymol structurally related analogs and how activity varies with structure were investigated using leaf dipping and spray bioassays against P. shantungensis adults and nymphs (   .90 times greater than that of carvacrol, citral, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol. While the functional group in thymol was necessary for insecticidal toxicity, the removal of the methyl functional group reduced in insecticidal toxicity. Furthermore, the position of the methyl and isopropyl functional group in the phenol ring altered insecticidal toxicity. These results indicate that the insecticidal mode of action of thymol analogs may be largely attributable to the methyl functional group. This observation contrasts to an earlier finding that the isopropyl functional group in thymol analogs is key in imparting insecticidal toxicity against stored-food pests 21 .
The present results implicate T. vulgaris oil, thymol and thymol structurally related analogs as promising natural products of insecticides against exotic insects. Others have found visual evidence of leaf phytotoxicity caused by T. vulgaris oil to the host plant of P. shantungensis, grape leaf 22 . The LD 50 values of carvacrol, thymol, and T. vulgaris oil against rat are 810, 980 and 2,840 mg/kg, respectively, by oral administration and the dermal LD 50 value of thymol and T. vulgaris oil exceeds 2,000 mg/kg against rat and 5,000 mg/kg against rabbit, respectively 23 . These results suggest that T. vulgaris oil, carvacrol, thymol and thymol analogs have a relatively low acute toxicity in mammals.
Our study is the first to investigate the insecticidal toxicities of T. vulgaris oil, thymol and thymol analogs against P. shantungensis adults and nymphs. Considering the fact that T. vulgaris is a very inexpensive plant to acquire and is easily cultivated, and are not barriers for the commercial development of carvacrol, thymol, and T. vulgaris oil isolated from T. vulgaris. Further study is required to decrease the human toxicity of the T. vulgaris oil, thymol and thymol analogs and establish the insecticidal mode of action of thymol analogs against P. shantungensis adults and nymphs.

Insects and bioassays. P. shantungensis adults and nymphs were collected from persimmon trees in
Wanjugun, Korea and classified the fourth instar stages of P. shantungensis nymphs and adults as detailed elsewhere 2,4 . The insecticidal toxicities of the five essential oils against P. shantungensis adults and nymphs were assessed (  Gas chromatography-mass spectrometry. The components of the essential oil extracted from T. vulgaris leaves were quantified using the Hewlett-Packard HP 6890 and H5973IV series (Agilent, Santa Clara, CA, USA) and were separated with HP-Innowax capillary column and DB-5 column (0.25 mm i.d. × 0.25 μ m thickness × 2,990 cm L.). The conditions of the column were as follows: Helium at 0.75 mL/min; column temperature (51 to 201 °C) at 2 °C/min; injector temperature (211 °C); split ration (48:1); ion source temperature (231 °C); ionization potential (70e V); and mass spectra range (50-800 amu). The components of T. vulgaris oils were evaluated according to retention times, retention indices, and mass spectra and were identified by comparison with a spectrum library ( Table 3). The relative composition of each T. vulgaris oil constituent (%) was measured by comparison with internal standards.
Statistical analysis. Data obtained for each dose response bioassay were subjected to probit analysis. The median lethal concentration (LC 50 ) value and the slope of the regression lines were calculated using the statistical package SPSS, version 12.0 for Windows.