Repellency and insecticidal activity of Zanthoxylum oil constituents against Stomoxys calcitrans L. (Diptera: Muscidae)

Abstract

The stable fly, Stomoxys calcitrans L., is one of the important insect pests as a blood-sucking ectoparasite that mainly bites cattle, horses, pigs, and dogs, but also bites humans. In this study, an assessment is made of the repellency of 22 plant essential oils (EOs), Zanthoxylum oil constituents, and the mixtures with tamanu, Calophyllum inophyllum, nut oil (CI-NO) and the olfactory responses of the fly antennae to Zanthoxylum-derived constituents. The fumigant toxicity and possible insecticide mode of action of the constituents also were evaluated. In a repellency test using the EOs at a dose of 0.5 mg/cm2 with six human male volunteers based on protection time (PT) to first bite, patchouli, clove bud, lovage root, and clove leaf EOs were the most effective (PT, 3.20–3.62 h) but were less active than N,N-diethyl-m-toluamide (DEET) (PT, 4.35 h). Strong repellency was also produced by thyme white EO (PT, 2.07 h), while moderate repellency was obtained from Z. piperitum pericarp steam distillate (ZP-SD), geranium, thyme red, and oregano EOs (PT, 1.01–1.12 h). Z. armatum seed oil (ZA-SO) and bergamot EO exhibited weak repellency (PT, 0.60 and 0.57 h). At 0.25 mg/cm2, protection time of patchouli, savory, thyme white and ZP-SD (PT, ~0.6 h) and clove bud, lovage root and clove leaf EOs (PT, ~1.0 h) were also shorter than that of DEET (PT, ~2.0 h). Binary mixtures of the active EOs and CI-NO produced a significant increase in the protection time, although the repellency of each EO was weaker than that of DEET. PT of binary mixtures (PT, 2.17–2.64 h) of five EOs (lovage root, patchouli, clove bud, thyme white, and clove leaf) and CI-NO (0.25 + 2.0 mg/cm2) was similar to that of DEET + CI-NO mixture (PT, 2.69 h) at the same doses and showed longer protection time than that of either DEET alone (PT, 2.03 h), the constituted EOs (PT, 0.54–1.07 h), or CI-NO alone (PT, 0.54 h). With the exception of savory, the other EOs, CI-NO, and binary mixtures did not induce any adverse effects on the human volunteers at 0.5 mg/cm2. ZP-SD and ZA-SO volatile constituents, cuminaldehyde, cuminyl alcohol, geraniol, limonene, linalool, methyl cinnamate, neral, peperitone, and α-phelandrene alone gave short protection time (PT, ~0.5 h) and were significantly less effective than DEET alone (PT, ~2.0 h) at 0.25 mg/cm2. However, the binary mixtures of the four compounds (limonene, α-phellandrene, cuminyl alcohol, and cuminaldehyde) and CI-NO (0.25 + 1.0 mg/cm2) provided strong protection (PT, 2.36 – 2.57 h), similar to that of the DEET + CI-NO mixture (PT, 2.52 h), and gave significantly stronger effects than that of DEET alone. Two major fatty acids of CI-NO such as oleic acid and linoleic acid, and the fatty acid methyl esters such as methyl oleate and methyl linoleate were found to provide longer protection than CI-NO. In a repellency test using eight aerosol formulations containing ZP-SD or ZA-SO and CI-NO as active ingredients, the repellency of the formulations against S. calcitrans females was comparable with that of DEET + CI-NO mixtures and DEET alone. The behavioral and olfactory response patterns to 1,8-cineole, citronellal, cuminaldehyde, linalool oxide, linalool, neral, piperitone, and terpinen-4-ol from Zanthoxylum plants in combination with the attractants such as 1-octen-3-ol and butyric acid were investigated on the fly antenna. The patterns of behavioral and olfactory responses were significantly altered depending on the ratios of 1-octen-3-ol or butyric acid to the EOs or compounds in the air mixtures. The present study demonstrated that Zanthoxylum EOs lowered the levels of response of flight behaviors of the stable fly toward the test attractants. The results indicate that an olfactory receptor on the fly antenna detects directly the repellent chemicals. In the vapor-phase mortality assay with ZP-SD and ZA-SO, their 28 constituents, and eight structurally related compounds against female stable fly, cuminaldehyde, thymol, (1S)-(–)-verbenone, (–)-myrtenal, carvacrol, (S)-(Z)-verbenol, ZP-SD, cuminyl alcohol, ZA-SO, piperitone, (–)-(Z)-myrtanol, and citronellal (LC50, 0.075–0.456 µg/cm3) showed strong activity, although their magnitude was less toxic than either chlorpyrifos or dichlorvos. Structure–activity relationship investigation indicated that structural characteristics such as carbon skeleton, degrees of saturation and types of functional groups, and vapor pressure parameter, appear to play a role in determining toxicities of the test compounds. In the acetylcholinesterase (AChE) inhibition assay, citronellyl acetate, alpha-pinene, thymol, carvacrol, and alpha-terpineol (IC50, 1.20–2.73 mM) inhibited the fly head AChE but their activities were weaker than that of chlorpyrifos and dichlorvos. However, octopamine and dopamine remarkably stimulate adenylate cyclase in brain of stable flies. At 100 nmol/ml, octopamine increased cAMP level 2.7-fold compared with control. The octopamine-stimulated cAMP level significantly reduced 13.7-fold and 3.9-fold by citronellal and thymol at 100 nmol/ml, respectively, and 2.0–2.9-fold by cuminaldehyde, cuminyl alcohol, (–)-myrtenal, and (1S)-(–)-verbenone. These results indicate that the possible target site for neurotoxicity of EO fumigants is the octopamine-sensitive adenylate cyclase in the nervous system. In conclusion, global efforts to reduce the level of highly toxic synthetic repellents or insecticides in the agricultural environment justify further study on plant EOs and Zanthoxylum-derived constituents described as potential biocides for the control of stable fly populations as repellents or insecticides with fumigant action.

Key words: Stomoxys calcitrans, plant essential oil, Zanthoxylum piperitum, natural repellent, natural fumigant, acetylcholinesterase inhibition, cAMP immunoassay, olfactory response

Student number: 2005 – 30877