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
John F. Carroll,
USDA, ARS, Invasive Insect Biocontrol and Behavior Laboratory, Beltsville Agricultural Research Center, Beltsville, MD 20705, U.S.A. email@example.com
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.).
MATERIALS AND METHODS
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.
Essential oils, repellent, and insecticide
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.
GC/FID and GC/MS analysis
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.
Vertical filter paper bioassay
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.
Tick experimental design
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.
Mosquito repellent assay
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. Serial dilutions were then made analogously such that the concentrations on cloth for the remaining 1 ml solution were: 0.750, 0.375, 0.187, 0.094, 0.047, 0.023, 0.011, and 0.005 mg/cm2. Vials were sealed and stored at –4° C in a freezer until testing (normally <48 h). Each test involved removal of cloth from the vial and stapling it onto two sections of card stock (5 cm × 2.5 cm). Pieces of masking tape (2.5–5.0 cm long) secured the cloth onto the card stock. The card and cloth assembly was then placed on a drying rack for 3–5 min before testing.
A single test consisted of covering the hand of a volunteer with a soft-embossed long cuff poly glove (Atlantis Products, Mankato, MN), followed by a powder-free latex glove (Diamond Grip, Microflex Corporation, Reno, NV). A knee-high stocking (Leggs everyday knee highs, Winston-Salem, NC) was then placed over the gloved hand and arm. A plastic sleeve of polyvinyl was the final layer affixed over the stocking covered arm. The plastic sleeve was sealed around the arm by a Velcro™ strip. About half-way between the wrist and elbow a 4 × 8 cm opening in the sleeve allowed us to assess mosquito landing and biting behavior. Attractive odors emanated from the skin surface and attracted mosquitoes to the opening. During testing, this 32 cm2 open area was covered with extract-treated muslin cloth. The order in which treated cloths were tested was randomized among volunteers and randomized to minimize any variation due to day-to-day effects.
A test started when the arm, sleeve, and cloth were inserted into the mosquito cage. If fewer than four bites were received during the 1 min, the dosage of repellent on cloth was considered to have “passed.” A treatment in which five bites (out of 500 mosquitoes in the cage) occurred in 1 min was considered a failure. Normally, an intermediate dosage (e.g., 0.187 mg/cm2) was tested first. Depending on whether this concentration passed or failed, higher or lower treatment concentrations were evaluated with all subjects until each had pinpointed their individual concentration that produced the 1% (five bite) failure point. If the 1.500 mg/cm2 (or highest concentration) on cloth was not efficacious (>five bites in 1 min), then the minimum effective dosage (MED) was noted as ineffective at the highest concentration tested. Because the mosquitoes fatigue upon repeated exposure to repellent and attractant odors from the arm, a limit of ten successive tests were conducted, after which the caged mosquitoes were allowed a 15 min recovery period.
Three male volunteers (two tested twice) and a female volunteer participated in the studies of MED of the oils (USDA 1977). During a test, one or both volunteers wore a patch and tested each patch for 1 min intervals. Patches were rotated between the volunteers, thus, no patch was evaluated beyond 10 min after the 3-min drying period to avoid any bias that may result from evaporative loss of the treatment from the cloth during the duration of the test. The subjects provided written informed consent. The protocol was approved by the University of Florida Human Use Institutional Review Board-01 (Study #636–2005).
Mosquito larvicidal assay
Larval bioassays were performed as described in Pridgeon et al. (2009). Briefly, five Ae. aegypti 1st instar larvae were placed into individual wells of a 24-well plate containing 950 μl deionized water and 40 μl of a 2:1 suspension of alfalfa:pot belly pig chow. Chemicals to be tested were resuspended in DMSO (Sigma Cat. D8418), for a stock concentration of 50 μg/μl. Six concentrations in a two-fold serial dilution series in DMSO were tested for mortality by adding 10 μl of each concentration into the wells. After 24 h, the number of dead larvae was recorded. Serial dilutions were continued until 0% mortality was observed for each chemical. All concentrations were tested in triplicate. Controls included negative (untreated), carrier (DMSO), and positive (permethrin).
Mosquito adult topical assay
Stock chemicals prepared from above were diluted into acetone for a final concentration of 6.25 μg/μl. Ten adult Ae. aegypti female mosquitoes, three to five days post-eclosion, were cold anaesthetized and placed on BioQuip chill table (Rancho Dominguez, CA) set at 4° C. Using a #1702 Gastight Hamilton syringe mounted onto a Hamilton PB600 repeating dispenser (Reno, NV), 0.5 μl of the test chemical was applied to the dorsal thorax of each insect, with a final dose of 3.125 μg per insect. After treatment, mosquitoes were placed in 3.5 oz (0.10 l) plastic cups, supplied with 10% sucrose solution, and maintained at 28° C and 80% relative humidity. All assays were performed in triplicate. Controls included negative (untreated), carrier (DMSO-acetone), and positive (permethrin).
The proportion of ticks in the vertical filter paper bioassay that were repelled was modeled in the generalized linear models framework (as binomial proportions) using the R statistical software glm function, with estimated EC50 and EC95 values computed using the dose.p function in the MASS package. A straight line relationship between the proportion repelled on the logit scale and concentration was obtained after a power transformation on concentration for some essential oils. When results from high concentrations were dropped, sometimes the proportion repelled was slightly lower in the highest concentration than at a previous lower concentration. With relatively few concentrations used, this artificially affects the modeling. The same methodology was used to model the mosquito larvicidal assay. Permethrin was compared to the essential oils in the adult mosquito topical assay data (also in a generalized linear models framework).
Essential oil composition
Essential oils from leaves of J. communis and wood oils from J. chinensis and C. funebris were characterized and identified by gas chromatography and gas chromatography/mass spectrometry. The relative percentages of the constituents are listed in Table 1. Forty-seven (95.5% total oil), 46 (72.6%), and 55 (66.8%) compounds were identified from J. communis, J. chinensis, and C. funebris essential oils, respectively. The major components of each species were: α-pinene (26.9%), α-terpineol (14.0%), linalool (10.9%), limonene (6.2%), and β-pinene (5.2%) for J. communis; cuparene (11.3%), δ-cadinene (7.8%), and α-cedrene (4.8%) for J. chinensis; α-cedrene (16.9%), cedrol (7.6%), β-cedrene (5.7%), and cuparene (5.4%) for C. funebris.
Table 1. Composition of essential oils of Juniperus communis, J. chinensis and Cupressus funebris.
All three essential oils were repellent to A. americanum and I. scapularis nymphs but were effective against I. scapularis nymphs at a much lower concentration than those needed to repel similar percentages of A. americanum. Figure 1 depicts dose-related responses of A. americanum nymphs to the three oils and to deet. The EC95 values of C. funebris, J. chinensis, and J. communis are 0.43, 0.92, and 0.51 mg oil/cm2 filter paper, respectively, whereas the EC95 for deet is 0.68 mg oil/cm2 filter paper (Table 2). Among the oils, the EC50 of J. communis (0.288 mg oil/cm2 filter paper) was the highest, but that of deet was 0.394 (Figure 1). Cupressus funebris oil, at 0.103 mg oil/cm2 filter paper, repelled all I. scapularis nymphs, and the same concentration of J. chinensis and J. communis oils repelled 90% and 73.3% respectively.
Table 2. Concentrations of essential oils of J. chinensis, J. communis, C. funebris, and deet estimated to repel 50 and 95% of A. americanum nymphs in vertical filter paper bioassays.
Concentrationa (Power Transformation)
aConcentrations as mg oil or deet/cm2 filter paper.
EC95 0.509 (none)
EC50 0.268 (sq rt)
EC95 0.917 (sq rt)
EC50 0.209 (none)
–3. 582 (±0.323)
EC95 0.465 (none)
EC50 0.394 (none)
EC95 0.683 (none)
The oils of C. funebris and J. chinensis, at 0.827 mg oil/cm2 filter paper, repelled ≥80%A. americanum 4 h after application (Table 3). Although repellent activity for both oils remained at 6 h post-application, it clearly showed diminished effectiveness.
Table 3. Percent of A. americanum nymphs repelled by C. funebris and J. chinensis essential oils, and deet at 0.827 mg/cm2 filter paper and ethanol control 2, 4, and 6 h after test solutions were applied to filter paper.
% (mean ±SD)c
% (mean ± SD)
% (mean ± SD)
aFour replicates tested. bThree replicates tested. cMean per replicate of ten ticks.
C. funebris oil
J. chinensis oil
Neither the oil of C. funebris nor that of J. chinensis consistently repelled female Ae. aegypti mosquitoes from biting through cloth treated at the highest dosage tested (1.500 mg/cm2). However, the oil of J. communis had a MED of 0.057 ± 0.013 mg/cm2 (Table 4).
Table 4. Minimum effective doses (MED)a of J. chinensis, J. communis, and C. funebris essential oils tested on human volunteers against Ae. aegypti mosquitoes.
aMED in mg/cm2.. bIf ≥5 bites in 1 min exposure to 500 mosquitoes then that compound/dose was considered a failure. cVolunteer 4 was a female, the other three volunteers males (volunteers 1 and 2 tested twice).
Mortalities of 100, 80, and 67% at 250 ppm and 80, 27, and 40% at 125 ppm in Ae. aegypti larvae were observed for J. chinensis, C. funebris, and J. communis oils, respectively. The concentrations required to produce these levels of mortality by these oils are significantly higher than the 0.25 ppb of the commonly used insecticide, permethrin (synthetic pyrethroid), which produced 67% (SEM 5.2%) mortality. The LD50s and LD95s of the oils and deet for Ae. aegypti larvae are presented in Table 5. Mortality of adult Ae. aegypti was also negligible, 3.3% at a dose of 3.125 ug oil/insect, for all three oils.
Table 5. Doses of essential oils of J. communis, C. funebris, and permethrin estimated to kill 50 and 95% of Ae. aegypti larvae. There were not enough intermediate concentrations for J. chinensis to calculate the ED50 and ED95. Intercept and slope values (±SE) are given for a generalized linear model (with proportion, the dependent variable, on the logit scale).
Concentrationa (Power Transformation)
aTen larvae tested for each of six concentrations for each oil, and seven concentrations for permethrin.
LD50 163.6 ppm (sqrt)
LD95 440.3 ppm (sqrt)
LD50 263.2 ppm (none)
LD95 298.9 ppm (none)
LD50 0.1894 ppb (sqrt)
LD95 0.4088 ppb (sqrt)
α-Pinene predominates in most of the leaf oils of J. communis from countries of the eastern hemisphere (Butkiene et al. 2006). In contrast, major components of the oil of J. chinensis that we analyzed were cuparene (11.3%), δ-cadinene (7.8%), α-cedrene (4.8%), and cedrol (3.2%). The main components of wood essential oils of J. chinensis cultivated at Waco, TX, and collected from Japan were found by Adams and Li (2008) to be cis-thujopsene (28.4, 8.4%), cedrol (13.7, 39.4%), widrol (9.2, 2.0%), α-cedrene (3.6, 3.1%), and β-cedrene (3.5, 0.6%), respectively. These differences could be due to distillation variation or to ecological or climatic variables associated with the localities where the plants grew.
Cupressus funebris wood oil is characterized by the occurrence of four components: cedrol, α-cedrene, β-cedrene, and thujopsene. In our study, α-cedrene (16.9%), cedrol (7.6%), and β-cedrene (5.7%) were the major constituents of C. funebris oil. The chemical composition of our samples resembled those of samples originating in China or Vietnam (Adams and Li 2008, Duquesnoy et al. 2006).
Among the major constituents of the three oils, α-terpineol (14.0%J. communis) and linalool (10.9%J. communis) are known to repel A. americanum nymphs, as do carveol and carvone, less plentiful J. communis components (Weldon et al. 2011). Terpinen-4-ol (1.6% in J. communis) has been found to be somewhat repellent to I. scapularis (Dietrich et al. 2006) and A. americanum nymphs (Weldon et al. 2011).
The oils of C. funebris and J. communis are comparable to deet in their effectiveness in repelling A. americanum, but have steeper dose response curves than deet and J. chinensis oil (Figure 1) and lower EC50s and EC95s than deet. The only oils, C. funebris and J. chinensis, tested for duration of repellency were effective (≥80%A. americanum repelled) for 4 h after application. Deet retained 90% repellency at 6 h post-application, at which time C. funebris and J. chinensis oils had declined to 67.7 and 46.7% repellency. The oils are clearly not ephemeral and formulation chemistry can extend the longevity of repellent activity should they be developed into commercial repellent products.
The tendency of I. scapularis nymphs to be repelled by considerably lower concentrations of repellent than A. americanum nymphs has been reported for several chemicals (Carroll et al. 2004, 2005, 2007), and was again observed in our bioassays with the three oils.
Because C. funebris and J. chinensis oils were not efficacious as repellents or toxicants against Ae. aegypti at the highest concentrations, neither of these oils nor their major constituents merit additional examination as repellents or insecticides for Ae. aegypti. The oil of C. funebris was one of 38 oils tested for repellency against Ae. aegypti (Trongtokit et al. 2005), and found to be ineffective at 30 min post-application for 10% and 50% dilutions; there was only one case where repellency was noted at 30 min post-application. Neither of the Juniperus species reported here was tested by Trongtokit (2005). Amer and Mehlkorn (2006) reported that in a study with human volunteers, the essential oil of J. communis (plant source Austria) repelled 43.2% of Ae. aegypti at 210 min after application.
In our study, the oil of J. communis (0.057 ± 0.013 mg/cm2) was not quite as repellent as technical (85%) deet, whose MED typically ranged from 0.005–0.023 mg/cm2 for volunteers participating in recent studies by Tabanca et al. (2010). Just over 50% of the composition of the J. communis oil is α-pinene (27%), α-terpineol (14%), and linalool (11%). Linalool has been well characterized as a repellent and attraction-inhibitor of mosquitoes (Hwang et al. 1985, Traboulsi et al. 2005, Park et al. 2005, Kline et al. 2003). Singh et al. (1984) reported the toxicity of cedarwood oil to mosquitoes. However, they tested oil from the deodar or Himalayan cedar, Cedrus deodara Roxb., which is classified in the Pinaceae, whereas C. funebris, J. communis, and J. chinensis are in the Cupressaceae. Both the Pinaceae and Cupressaceae belong to the order Pinales.
Perhaps most encouraging is that an oil (J. communis) comprising multiple compounds exhibited a MED against a mosquito, Ae. aegypti, and an EC95 against a tick, A. americanum, that resembled the values obtained for a single pure synthetic repellent (deet). This indicates the presence of at least one good repellent and probably multiple repellent compounds that act synergistically to produce the observed repellency. Panella et al. (1997) tested extracts from six species of the Cupressaceae for toxicity to I. scapularis larvae and nymphs, and found that the extract from Alaska yellow cedar, Chamaecyparis nootkatensis (D. Don) Spach, was the most effective extract against larvae, and the extract from eastern red cedar, Juniperus virginiana L., most effective against nymphs. Among the compounds identified from Alaska yellow cedar essential oil, the sesquiterpene nootkatone and monoterpene carvacrol were found notably toxic to I. scapularis nymphs (Panella et al. 2005). Dietrich et al. (2006) found that nootkatone, and carvacrol were similar to deet in their capacity to repel I. scapularis. In field tests, nootkatone effectively suppressed populations of I. scapularis and A. americanum (Dolan et al. 2009). The progress made in developing nootkatone and carvacrol as anti-tick compounds (Dolan et al. 2009) is an incentive for continued research on the constituent compounds of J. communis, and for further efforts in repellent and toxicant discovery among the essential oils (and constituents) of Cupressaceae.
This study was supported in part by a grant from the Deployed War-Fighter Protection (DWFP) Research Program and the U.S. Department of Defense through the Armed Forces Pest Management Board (AFPMB). We thank J. L. McCrary and A. S. Khan, USDA, ARS, Invasive Insect Biocontrol and Behavior Laboratory, Beltsville Agricultural Research Center, Beltsville, MD, for carefully performing many of the tick bioassays. We are grateful to USDA, ARS, Knipling-Bushland U.S. Livestock Insects Research Laboratory, Kerrville, TX, for providing A. americanum for bioassays.