Essential oils of Cupressus funebris, Juniperus communis, and J. chinensis (Cupressaceae) as repellents against ticks (Acari: Ixodidae) and mosquitoes (Diptera: Culicidae) and as toxicants against mosquitoes

Abstract

Juniperus communis leaf oil, J. chinensis wood oil, and Cupressus funebris wood oil (Cupressaceae) from China were analyzed by gas chromatography and gas chromatography-mass spectrometry. We identified 104 compounds, representing 66.8–95.5% of the oils. The major components were: α-pinene (27.0%), α-terpinene (14.0%), and linalool (10.9%) for J. communis; cuparene (11.3%) and δ-cadinene (7.8%) for J. chinensis; and α-cedrene (16.9%), cedrol (7.6%), and β-cedrene (5.7%) for C. funebris. The essential oils of C. funebris, J. chinensis, and J. communis were evaluated for repellency against adult yellow fever mosquitoes, Aedes aegypti (L.), host-seeking nymphs of the lone star tick, Amblyomma americanum (L.), and the blacklegged tick, Ixodes scapularis Say, and for toxicity against Ae. aegypti larvae and adults, all in laboratory bioassays. All the oils were repellent to both species of ticks. The EC95 values of C. funebris, J. communis, and J. chinensis against A. americanum were 0.426, 0.508, and 0.917 mg oil/cm2 filter paper, respectively, compared to 0.683 mg deet/cm2 filter paper. All I. scapularis nymphs were repelled by 0.103 mg oil/cm2 filter paper of C. funebris oil. At 4 h after application, 0.827 mg oil/cm2 filter paper, C. funebris and J. chinensis oils repelled ≥80% of A. americanum nymphs. The oils of C. funebris and J. chinensis did not prevent female Ae. aegypti from biting at the highest dosage tested (1.500 mg/cm2). However, the oil of J. communis had a Minimum Effective Dosage (estimate of ED99) for repellency of 0.029 ± 0.018 mg/cm2; this oil was nearly as potent as deet. The oil of J. chinensis showed a mild ability to kill Ae. aegypti larvae, at 80 and 100% at 125 and 250 ppm, respectively. Mosquito and tick-borne illnesses exact a considerable toll in human misery and financial expenditure. Problems with ticks and tick-borne diseases have continued to grow during the past three decades (Parola and Raoult 2001). Lyme disease (caused by the spirochete Borrelia burgdorferi) and its principal vector, Ixodes scapularis Say (Spielman et al. 1985), have deservedly received significant attention in the United States. The lone star tick, Amblyomma americanum (L.), has acquired notoriety not only as a nuisance biter, but also as a vector of Ehrlichia chaffeensis, which causes human monocytic ehrlichiosis (Childs and Paddock 2003, Armstrong et al. 2001). Mosquitoes are vectors for many pathogens that cause serious diseases, such as malaria, dengue fever, yellow fever, Rift Valley fever, and Chikungunya, which can attain epidemic levels with high rates of human morbidity and mortality. Repellents applied to skin or clothing are recommended as a means of personal protection against biting arthropods (e.g., CDC 2002). Toxicants can reduce the densities of mosquito and tick populations and the risk of disease transmission. The synthetic repellent deet (N,N-diethyl-3-methyl benzamide) has been used widely since the 1950s to defend against mosquitoes and ticks. Newer synthetics, such as picaridin and IR3535, now provide more options for users (Debboun et al. 2007). Permethrin, a synthetic pyrethroid, is approved to be used as a repellent, but only when applied on clothing (Schreck et al. 1982). As toxicants, pyrethroids are used to control a wide range of arthropods that are agricultural and public health pests (Hoel et al. 2010). Repeated use of pyrethroids, or insecticides of any class, can lead to insecticide resistance. Consequently, there is an urgent need to develop alternatives to chemical control of a wide variety of arthropod vectors of human diseases (Pridgeon et al. 2008, 2009). Many naturally occurring repellents and insecticides have potential for development into useful products because they combine efficacy, biodegradability, and limited risk to mammals and the environment (Dayan et al. 2009, Hoel et al. 2010, Carter 1989, Bissinger et al. 2009, Bissinger and Roe 2010). Plant essential oils and seed pressed oils comprise a significant portion of the market share for natural product-based insecticides, and some have served as the basis of commercial repellent formulations (Dayan et al. 2009, Nerio et al. 2010). A great diversity of medicinal and aromatic plants occurs in China, where these natural products exert a strong impact on peoples’ lives and culture (Li et al. 2006). Prominent among the useful taxa is the cypress family, Cupressaceae. Commercial cedarwood oils have been obtained from three genera of Cupressaceae: Juniperus (Texas, Virginia, and Africa), Cupressus (China), and Cedrus (Morocco, India) (Adams and Li 2008). Cupressus funebris Endl. is generally regarded as the botanical source of Chinese cedarwood oil, a well known perfume material (Adams and Li 2008), and its derivatives (Duquesnoy et al. 2006). The studies of Panella et al. (1997, 2005), Dietrich et al. (2006), and Dolan et al. (2007, 2009) indicate that the Cupressaceae may be a rich source of anti-tick compounds and that further investigation of cypress species for repellent and insecticidal chemicals is warranted. We investigated Chinese weeping cedar, C. funebris, Chinese juniper, Juniperus chinensis L., and common juniper, J. communis L., essential oils from China. One purpose of this study was to characterize the chemical composition of the essential oils of C. funebris, J. chinensis, and J. communis by analysis using gas chromatography (GC) and GC-mass spectrometry (GC-MS). The second purpose was to evaluate these oils as repellents against the ticks A. americanum and I. scapularis and as repellents and adult and larval toxicants against the yellow fever mosquito, Aedes aegypti (L.). Larvae of I. scapularis from a colony at Oklahoma State University were fed as larvae on rats at the USDA, ARS, Beltsville Agricultural Research Center, Beltsville, MD in accordance with USDA, ARS, Beltsville Area Animal Care and Use Committee Protocol #08–013. The fed larvae and resultant nymphs were held at 23–24° C, ∼97% RH and a photoperiod of 16:8 h (L:D). The A. americanum nymphs were from a colony at the USDA, ARS, Knipling-Bushland U. S. Livestock Insects Research Laboratory, Kerrville, TX and a colony at Oklahoma State University, Stillwater, OK (used in duration bioassay) and held at 23–24° C, ∼97% RH and a photoperiod of 16:8 h (L:D). The I. scapularis and A. americanum nymphs were tested three to five, and three to six months after molting, respectively. Pupae of Ae. aegypti from the Gainesville (CMAVE) colony were maintained in the laboratory at 28 ±1° C and 30–60% RH, and the resulting adults aged five to nine days were used for repellent testing. The A. aegypti mosquitoes (also from the CMAVE colony) used in the insecticide bioassays were held at 22–30° C, 80% RH, and a photoperiod of 14:10 (L:D) including 2 h of simulated dusk and dawn. The mosquitoes were maintained on 10% sucrose ad libitum and provided twice weekly with bovine blood in 1% heparin placed in pig intestine and warmed to 37° C. Juniperus communis oil was purchased from Shanghai Qika Corporation, Shanghai, China, and C. funebris and J. chinensis oils were purchased from Peking University Zoteq Co., Ltd., Beijing, China. Deet was purchased from Aldrich, Sigma-Aldrich, Inc., St. Louis, MO, and permethrin from Chem Service, West Chester, PA. The oils were analyzed by capillary GC-FID and GC/MS using an Agilent 5975 GC-MSD system (SEM Ltd., Istanbul, Turkey). A Hewlett Packard-Innowax FSC column (60 m × 0.25 mm, 0.25 mm film thickness) was used with helium as the carrier gas (0.8 ml/min). The GC oven temperature was held at 60° C for 10 min and ramped to 220° C at a rate of 4° C/min, then held constant at 220° C for 10 min with a final programmed ramp to 240° C at a rate of 1° C/min, and held a second time at 240° C for 20 min. The split ratio was adjusted to 40:1. The injector temperature was at 250° C. The mass spectrometer was operated with an electron energy of 70 eV. The mass range was m/z 35 to 425 at a scan rate of 3.46 scans/s. The GC analysis was carried out using an Agilent 6890N GC system equipped with a FID detector operated at a temperature of 300° C. To obtain the same elution order of peaks detected by GC/MS, simultaneous injection on the GC was performed using the same column and appropriate chromatographic conditions as those described for the GC/MS system. Identification of the essential oil components was carried out by comparing their relative retention times with those of authentic samples or by comparing their relative retention index (RRI) to a series of n-alkanes. Computer matching identified compounds used as references (Wiley and MassFinder 3.1) (Koenig et al. 2004, McLafferty and Stauffer 1989). An in-house “Başer Library of Essential Oil Constituents” composed of genuine compounds and components of known oils, and MS literature data (Joulain and Koenig 1998, ESO 2000, Jennings and Shibamoto 1980) were also used for the identification. The relative concentrations of the separated compounds based on percentage were computed from chromatograms obtained with the GC/FID system. Host-seeking ticks of many species tend to climb vertical surfaces, particularly in the presence of host-produced stimuli. This behavior was used to expose A. americanum nymphs to repellent treatments in an in vitro bioassay described in detail by Carroll et al. (2004). Briefly, a 4 × 7-cm rectangle of Whatman No. 4 filter paper was marked with a pencil into two 1 × 4-cm zones at the far ends of the paper strip and a central 4 × 5-cm zone. Using a pipettor, 165 μl of test solution was evenly applied to both sides of the central 4 × 5 cm of the filter paper. After drying for 10–15 min, the paper strip was suspended from a bulldog clip hung from a slender horizontal dowel held by an Aptex No. 10 double clip work holder (Aptex, Bethel, CT). A Petri dish (9 cm diameter) glued in the center of a 15 cm Petri dish created a moat when water was added between their walls (1.5 cm high). The moated Petri dishes were placed directly beneath the suspended filter paper. When A. americanum nymphs climbed to the rim of a storage vial opened in the center of the moated Petri dishes (5.5 and 9 cm diameters), the paper strip was removed from the dowel and held so that ten ticks crawled onto the lower untreated zone. Because nymphs of I. scapularis tended to be slower and more apt to drop from untreated filter paper, they were screened for readiness to crawl while the test solution dried on the filter paper (Schreck et al. 1995). As the treated filter paper dried, I. scapularis were transferred with forceps to an untreated vertical filter paper. Ten ticks that climbed >5 mm were selected for testing and placed in a moated petri dish until 10–15 min post-application had elapsed. Using forceps, the I. scapularis nymphs were then transferred individually from the moated Petri dish to the lower untreated zone of the filter paper. The locations of the ticks were recorded at 1, 3, 5, 10, and 15 min after the tenth A. americanum or I. scapularis nymph grasped the lower untreated zone of the filter paper. Ticks were considered repelled if they were in the lower untreated zone at 15 min or if they fell from the filter paper without having crossed the upper boundary of the treated zone. Test solutions and ethanol controls were tested in random order. A control was run each day that the oils and deet were tested. Ticks were tested in replicates of ten ticks per combination of concentration and oil or deet. Essential oils from C. funebris and J. chinensis were tested against three replicates of A. americanum at 0.827, 0.413, 0.206, and 0.103 mg/cm2filter paper, and J. communis at 0.827, 0.413, and 0.206 mg/cm2 filter paper. A single concentration (0.103 mg oil/cm2 filter paper) of each of C. funebris, J. communis, and J. chinensis essential oil was tested against three replicates (ten ticks per replicate) of I. scapularis nymphs. To determine the duration of the repellency of C. funebris and J. chinensis, we tested their essential oils (0.827 mg oil/cm2 filter paper) against A. americanum nymphs at 2, 4, and 6 h after application. Deet, at 0.827 mg oil/cm2 filter paper, was included in the duration tests for comparison. Four replicates (ten ticks per replicate) were tested at 2 and 4 h post-application and three replicates (ten ticks per replicate) at 6 h. Female mosquitoes were selected from the stock cages by a hand-draw box (Posey and Schreck 1981). Approximately 500 (± 10%) mosquitoes, primarily females, were transferred into a test cage (capacity 59,000 cm3, dimensions 45 cm × 37.5 cm × 35 cm) and held therein for 25 (± 2.5) min before initiating repellency assays (Barnard et al. 2007). Extracts were weighed and placed in a 2-dram vial to which 2 ml acetone was added. The initial weight of extract was measured so that when one half (1 ml of solution) was removed and a 50 cm2 muslin cloth was added to the vial, the remaining 1 ml solution would produce an initial concentration on cloth of 1.500 mg/cm2. were then such that the concentrations on cloth for the remaining 1 ml solution were: and mg/cm2. were and at C in a until testing test of cloth from the vial and onto two of stock cm × of cm the cloth onto the The and cloth was then placed on a drying for min before testing. A single test of the of a with a by a Corporation, A was then placed the and A of was the final the The was the by a between the and a 4 × cm in the to mosquito and biting from the skin and mosquitoes to the this cm2 was with muslin The order in which treated were tested was among and to any to A test when the and cloth were into the mosquito were received during the 1 min, the dosage of repellent on cloth was considered to have A in which five of 500 mosquitoes in the in 1 min was considered a an dosage (e.g., was tested on this concentration or or lower concentrations were evaluated with all until each had their concentration that the 1% the 1.500 mg/cm2 highest on cloth was not in 1 then the dosage was as at the highest concentration tested. Because the mosquitoes to repellent and from the a of ten tests were after which the mosquitoes were a 15 min tested and a female in the studies of of the oils a one or both a and tested each for 1 min were between the was evaluated 10 min after the drying to any that may from of the from the cloth during the duration of the The provided The was approved by the University of Use bioassays were performed as described in et al. Briefly, five Ae. aegypti larvae were placed into of a μl water and μl of a of pig to be tested were in for a stock concentration of 50 concentrations in a series in were tested for by 10 μl of each concentration into the After the of larvae was were continued until was for each All concentrations were tested in included carrier and chemicals from were into acetone for a final concentration of Ten adult Ae. aegypti female mosquitoes, three to five days were and placed on at 4° C. Using a onto a μl of the test chemical was applied to the of each with a final of per After mosquitoes were placed in with 10% sucrose and maintained at C and 80% relative All assays were performed in included carrier and The of ticks in the vertical filter paper bioassay that were repelled was in the using the with and EC95 values computed using the in the A between the repelled on the and concentration was obtained after a on concentration for some essential oils. When from high concentrations were the repelled was lower in the highest concentration at a lower concentrations this the The same was used to the mosquito was compared to the essential oils in the adult mosquito data (also in a Essential oils from of J. communis and wood oils from J. chinensis and C. funebris were and identified by gas chromatography and gas spectrometry. The relative of the are in and compounds were identified from J. communis, J. chinensis, and C. funebris essential oils, respectively. The major components of each species were: α-pinene (14.0%), linalool and for J. communis; cuparene δ-cadinene and α-cedrene for J. chinensis; α-cedrene (16.9%), cedrol (7.6%), β-cedrene and cuparene for C. funebris. All three essential oils were repellent to A. americanum and I. scapularis nymphs but were against I. scapularis nymphs at a lower concentration those to of A. 1 of A. americanum nymphs to the three oils and to deet. The EC95 values of C. funebris, J. chinensis, and J. communis are and mg oil/cm2 filter paper, respectively, the EC95 for deet is mg oil/cm2 filter paper the oils, the of J. communis mg oil/cm2 filter paper) was the but that of deet was Cupressus funebris oil, at 0.103 mg oil/cm2 filter paper, repelled all I. scapularis and the same concentration of J. chinensis and J. communis oils repelled and respectively. of A. americanum nymphs to the essential oils of C. funebris, J. chinensis, and J. communis, and an ethanol control of in vertical filter paper bioassays. and EC95 by The oils of C. funebris and J. chinensis, at 0.827 mg oil/cm2 filter paper, repelled americanum 4 h after repellent for both oils at 6 h showed the oil of C. funebris that of J. chinensis repelled female Ae. aegypti mosquitoes from biting cloth treated at the highest dosage tested (1.500 mg/cm2). However, the oil of J. communis had a of ± mg/cm2 of and at 250 and and at 125 in Ae. aegypti larvae were for J. chinensis, C. funebris, and J. communis oils, respectively. The concentrations to produce these levels of by these oils are the 0.25 of the used permethrin which (SEM mortality. The and of the oils and deet for Ae. aegypti larvae are in of adult Ae. aegypti was also at a of for all three oils. in of the leaf oils of J. communis from of the et al. 2006). major components of the oil of J. chinensis that we analyzed were cuparene δ-cadinene α-cedrene and cedrol The components of wood essential oils of J. chinensis at and from were by and Li to be cedrol α-cedrene and β-cedrene respectively. be to or to or with the where the plants Cupressus funebris wood oil is by the of and α-cedrene (16.9%), cedrol (7.6%), and β-cedrene (5.7%) were the major of C. funebris oil. The chemical composition of samples those of samples in China or (Adams and Li 2008, et al. 2006). the major of the three oils, and linalool are known to A. americanum as and J. communis components et al. in J. has been to be repellent to I. scapularis et al. and A. americanum nymphs et al. The oils of C. funebris and J. communis are to deet in their in A. but have deet and J. chinensis oil and lower and deet. The only oils, C. funebris and J. chinensis, tested for duration of repellency were americanum for 4 h after application. Deet repellency at 6 h at which time C. funebris and J. chinensis oils had to and The oils are not and can the of repellent they be into commercial repellent The of I. scapularis nymphs to be repelled by lower concentrations of repellent A. americanum nymphs has been for chemicals et al. 2004, and was in bioassays with the three oils. Because C. funebris and J. chinensis oils were not as repellents or toxicants against Ae. aegypti at the highest of these oils their major as repellents or insecticides for Ae. The oil of C. funebris was one of oils tested for repellency against Ae. aegypti et al. 2005), and to be at min post-application for 10% and there was only one where repellency was at min of the Juniperus species was tested by and that in a study with human the essential oil of J. communis source repelled of Ae. aegypti at min after application. the oil of J. communis ± was not as repellent as from mg/cm2 for in studies by et al. of the composition of the J. communis oil is α-pinene and linalool has been well as a repellent and of mosquitoes et al. et al. et al. et al. et al. the toxicity of cedarwood oil to However, they tested oil from the or cedar, Cedrus which is in the C. funebris, J. communis, and J. chinensis are in the Cupressaceae. the and Cupressaceae to the order is that an oil compounds a against a mosquito, Ae. and an EC95 against a tick, A. that the values obtained for a single synthetic repellent This the presence of at one repellent and repellent compounds that to produce the Panella et al. tested from six species of the Cupressaceae for toxicity to I. scapularis larvae and and that the extract from yellow cedar, was the extract against larvae, and the extract from cedar, Juniperus L., against nymphs. the compounds identified from yellow essential oil, the and were to I. scapularis nymphs et al. Dietrich et al. that and were to deet in their to I. populations of I. scapularis and A. americanum et al. 2009). The in and as anti-tick compounds et al. 2009) is an for continued on the compounds of J. communis, and for further in repellent and among the essential oils of Cupressaceae. This study was in by a from the Research and the of the We J. and A. S. USDA, ARS, and Laboratory, Beltsville Agricultural Research Center, Beltsville, for many of the tick bioassays. We are to USDA, ARS, Knipling-Bushland Livestock Insects Research Laboratory, Kerrville, for A. americanum for bioassays.