CHEMICAL ECOLOGY
Bioactivity of Cedarwood Oil and Cedrol Against Arthropod Pests F. J. ELLER,1,2 R. K. VANDER MEER,3 R. W. BEHLE,4 L. B. FLOR-WEILER,4 5 AND DEBRA E. PALMQUIST
Environ. Entomol. 43(3): 762Ð766 (2014); DOI: http://dx.doi.org/10.1603/EN13270
ABSTRACT Heartwood samples from Juniperus virginiana L. were extracted with liquid carbon dioxide, and the bioactivity of carbon dioxide-derived cedarwood oil (CWO) toward several species of ants and cedrol toward ticks was determined. Repellency was tested for ants, and toxicity was tested for ticks. Ants in an outdoor bioassay were signiÞcantly repelled by the presence of CWO on a pole leading to a sugarÐwater solution. Similarly, CWO was a signiÞcant repellent barrier to red imported Þre ants and prevented them from Þnding a typical food source. Black-legged tick nymphs exhibited dosage-dependent mortality when exposed to cedrol and at the highest dosage (i.e., 6.3 mg/ml) tested, the cedrol killed 100% of the ticks. These repellency and toxicity results together demonstrate a clear potential for the use of CWO as a pest control agent. KEY WORDS cedarwood oil, repellency, toxicity, red imported Þre ant, black-legged tick
Eastern red cedar (Juniperus virginiana L.), western juniper (Juniperus occidentalis Hook.), and ashe juniper (Juniperus ashei J. Buchholz) (Cupressaceae) are very abundant conifers in the United States. The area covered by junipers has been expanding (Schmidt and Leatherberry 1995, Ganguli et al. 2008), and all three are often considered pest species because of their encroachment onto rangeland and pastures (Adams et al. 1988). In addition to its aromatic smell, junipers are known for their resistance to both microbial decay and termite attack. Because of this resistance, juniper has long been used for fence posts (Hemmerly 1970, Adams 2004). The antifungal (i.e., wood-rot fungi) activities of juniper heartwood extracts have recently been reported (Eller et al. 2010, Mun and Prewitt 2011). Juniper wood has been shown to be resistant to both Formosan (Morales-Ramos and Rojas 2001) and eastern subterranean (Carter and Smythe 1974, Arango et al. 2006) termites. Particleboard-chip panels made Mention of trade names or commercial products in this article is solely for the purpose of providing scientiÞc information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. 1 Functional Foods Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 North University St., Peoria, IL 61604. 2 Corresponding author, e-mail: [email protected]. 3 Imported Fire Ant and Household Insects Research Unit, Center for Medical, Agricultural, and Veterinary Entomology, Agricultural Research Service, United States Department of Agriculture, 1600 S.W. 23rd Dr., Gainesville, FL 32608. 4 Crop Bioprotection Research Unit, National Center for Agricultural, Agricultural Research Service, United States Department of Agriculture, 1815 North University St., Peoria, IL 61604. 5 Agricultural Research Service, United States Department of Agriculture, Midwest Area, 1815 North University St., Peoria, IL 61604.
from eastern red cedar are moderately resistant to termite damage (Kard et al. 2007), and Ko¨ se and Taylor (2012) recently reported heartwood and included sapwood of eastern red cedar were resistant to termites. The wood of eastern red cedar is also known for its toxicity and repellency to several species of insects including clothes moths (Huddle and Mills 1952), ßour beetles (Sighamony et al. 1984), and cockroaches (Appel and Mack 1989). Eastern red cedar mulch was also reported to be repellent to ants (Meissner and Silverman 2001, Thorvilson and Rudd 2001). However, the bioactivity of eastern red cedar extracts toward arthropods has not been extensively studied. Antitermite compounds have been extracted from eastern red cedar heartwood by organic solvent (i.e., acetone, pentane, hexane, or methanol) extraction (Carter and Smythe 1974, Carter 1976, Adams et al. 1988, McDaniel et al. 1989). An acetoneÐ hexaneÐ water extract of eastern red cedar signiÞcantly reduced termite attack when applied to southern pine by vacuum impregnation (McDaniel and Dunn 1994). Zhu et al. (2001) found that cedarwood oil (CWO) repelled termites. Interestingly, cedarwood and its extracts have also been demonstrated to induce oviposition by ladybird beetles (Boldyrev et al. 1969, Smith et al. 1973). Our laboratory has been investigating the extraction and composition of CWO, using supercritical and liquid carbon dioxide as well as pressurized solvents such as ethanol and hot water (Eller and King 2000, Eller and Taylor 2004). Carbon dioxide-derived CWO contains higher levels of cedrol and has an odor that more closely resembles that of eastern red cedar wood than does CWO obtained by steam distillation (Eller and King 2000). Carbon dioxide-derived CWO has been
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ELLER ET AL.: BIOACTIVITY OF CEDARWOOD OIL AND CEDROL
demonstrated to have antifungal, antitermite and antiinßammatory effects (Eller et al. 2010; Tumen et al. 2013a,b). CWO is considered safe and is approved as a food additive by the U.S. Food and Drug Administration (2013). The purpose of this research was to investigate potential uses of CWO against several economically important arthropods as a safe natural pest control agent. Evaluations included its use as a general ant repellent on hummingbird feeders, a repellent against red imported Þre ant, Solenopsis invicta Buren, and toxicity against black-legged ticks, Ixodes scapularis Say. Materials and Methods CWO Samples. Heartwood samples from eastern red cedar (Woodford Co., IL) were prepared from freshly cut trees. Sapwood was removed from the samples using a band saw, and heartwood sawdust was prepared using a compound miter saw. Sawdust samples were held in glass containers at room temperature before extraction. Extractions were made using liquid carbon dioxide (25⬚C and 10.3 Mpa), and the CO2 was depressurized into glass vials to collect the CWO as described previously (Eller et al. 2010). (⫹)-Cedrol was purchased from Aldrich (Milwaukee, WI). Ant Repellent–Hummingbird Feeder Bioassay. The experiment was set up as a paired test of untreated control versus a CWO barrier treatment. The hummingbird feeders (First Nature, Rogers, AR) used in this study consisted of a large inverted reservoir to provide a constant level of the sugarÐwater solution (1:4 by volume). The sugar solution was accessible through 10 holes in the cover. A plastic hook on the top of the feeder is used to suspend the feeder. The hummingbird feeders were hung on a 1.63-m-tall black shepherdÕs hook (Enchanted Garden, Menards, Eau Claire, WI), and the traps were ⬇1.2 m from the ground. Twine (⬇1.5 mm in diameter and 30 cm in length) was wrapped Þve times around the base of the shepherdÕs hook ⬇15 cm from the ground and tied in a knot with the excess trimmed off. The concentration of the CWO in the string was ⬇500 mg/cm3. The control was left untreated while the treated twine had 250 l (⬇200 mg) of neat CWO extract applied to the twine. The two shepherdÕs hooks were placed 1 m apart at eight separate locations in Peoria and Woodford counties, IL. The feeders were checked daily for the presence of ants. When ants were Þrst detected, that replication was ended and each feeder was placed in a separate plastic bag and placed in a freezer (⫺10⬚C) to kill the ants present. The ants were then removed, counted, and preserved in 70% ethanol. The ants were identiÞed to genus. Imported Red Fire Ant Contact Repellency. The bioassay for contact repellency was similar to that described by Vander Meer et al. (1996). It was hypothesized that the CWO would act as a close-range repellent not as a volatile long-range repellent. The test tray was composed of a porcelain pan measuring 180 by 290 by 50 mm. The upper 3 mm of the pan was
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coated with Fluon (BioQuip Products, Rancho Dominguez, CA) to preclude ants from escaping. A petri dish nest cell (55 mm in diameter) was placed at one end of the pan. The petri dish had a 5-mm layer of Castone dental cement on the bottom that acted as a moisture reservoir. The lid of the petri dish had a hole placed in the center to allow ant access. To protect the bottom of the pan from contamination by the test materials, 2.5-cm2 pieces of aluminum foil were placed in the opposite end of the pan from the nest cell at each corner ⬇3.0 cm from the sides of the pan. No food or water was available to the ants during the bioassay. Fifty microliters of test material was introduced to the test chamber on a 2.0-cm2 piece of Whatman silicone-treated Þlter paper (cat. # 2200 125; Sigma-Aldrich, St. Louis, MO) and was randomly assigned and placed on one of the aluminum foil squares. The other aluminum square received a Þlter paper square with 50 l of pentane as a control. The solvent was allowed to evaporate to apparent dryness. Placed on top of each Þlter paper square was a small wad of cotton soaked in 10% sucrose to serve as a phagostimulant. Once test materials were in place, ⬇1 g of ants, Solenopsis invicta Buren (Hymenoptera: Formicidae) (starved for a minimum of 24 h) was placed in the nest cell and a stopwatch was started. The number of ants actively feeding on the treatment and control cotton balls was observed and recorded after a total of 5 min. The score for each bioassay replicate was the number of the 5-min values for control and treatment. Each experiment was replicated three times, each with a unique monogyne colony and in a different test tray. CWO at three concentrations (1 and 10% in pentane: wt:vol, and neat) was tested against pentane controls. Cedrol at 50% (wt:vol in pentane) was tested against a pentane control. Black-Legged Tick Contact Toxicity. Tick Colony. Unfed nymphs (⬇2Ð3 wk since molting) of the black-legged tick, I. scapularis (Acari: Ixodidae), were procured from the Tick Rearing Facility, National Tick Research and Education Resource, Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK. Before toxicological bioassays, nymphs were held in four-dram vials contained in a desiccator with potassium sulfate solution to maintain high relative humidity (RH; ⬎90%), at room temperature (22Ð24⬚C), with a photoperiod of 16:8 (L:D) h. Nymphs were allowed to acclimate to our laboratory conditions at least 24 h before starting the toxicological assay. Coating of Vials and Bioassays. Different concentrations of cedrol were tested on unfed nymphs of I. scapularis, in laboratory bioassays. Three serial dilutions (10⫻) of cedrol were made with hexane ranging from 6.3 to 0.063 mg/ml. Coating of vials with cedrol concentrations in hexane was done following the method of Panella et al. (2005) with modiÞcations. Each four-dram size vial (27.25 by 67 mm, Fisherbrand, Fisher ScientiÞc, Pittsburgh, PA) was coated evenly on the inside by adding 1 ml of the appropriate solution of cedrol in hexane. Vials were placed on their side on a roller (Bellco Biotechnology, Bellco Glass,
ENVIRONMENTAL ENTOMOLOGY
Inc., Vineland, NJ) and allowed to dry in a fume hood for 15Ð20 min or until hexane had completely evaporated. There were three replications (i.e., vials) of each treatment concentration, and three additional vials were treated with hexane only as a control treatment. The coated vials had only a very thin coating of oil, which did not appear to impair the movement of the tick nymphs. For the bioassay, 10 unfed nymphs were introduced into each vial and the vials were then capped with a piece of cotton fabric secured with a rubber band and Þnally covered with aluminum foil. Vials were placed in the desiccator with potassium sulfate solution to maintain high RH (⬎90%), at room temperature (22Ð 24⬚C), with a photoperiod of 16:8 (L:D)h. Thus, 30 tick nymphs were exposed to each cedrol concentration. Tick mortality was recorded 24 and 48 h after exposure to the cedrol-coated vials. Ticks were considered alive when they exhibited normal behavior, and considered moribund or dead when they were incapable of movement, failed to maintain normal posture, exhibited uncoordinated movement, were unable to right themselves, or showed no sign of life (motionless). Statistical Analyses. A sign test was used to test the probability of obtaining the observed results in the ant repellentÐ hummingbird feeder bioassay, and a oneway analysis of variance (ANOVA) was used to compare the number of ants at the CWO treatment with the number of ants at the control after square-root transformation of the count data plus 1 (Statistix 7.0, Analytical Software, Tallahassee, FL). A 2 analysis was used to compare number of red imported Þre ants at the CWO treatments with the number of red imported Þre ants at the controls. Probit analysis with logistic distribution was used to analyze probability of tick mortality based on cedrol concentration (SAS version 9.3 @ 2002Ð2110 [SAS Institute Inc., Cary, NC]).
Results and Discussion Ant Repellent–Hummingbird Feeder Bioassay. Ants were Þrst detected at the feeders at 2Ð10 d after placement in the Þeld. Ants were found at the hummingbird feeders at seven of the eight control replicates, whereas no ants were found at the hummingbird feeders treated with CWO (n ⫽ 8). The P value for this result was P ⫽ 0.0078 (Sign test; n ⫽ 7). The total number of ants captured at the controls ranged from 8 to 112, and the mean number of ants found on the feeders was 30.7 (n ⫽ 8). The ANOVA indicated that signiÞcantly more ants were captured at the control feeders than the feeders with the CWO (F1,14 ⫽ 14.1; P ⫽ 0.0021). Five genera of ants were identiÞed from the seven sites and included Camponotus (at two sites), Formica (at two sites), and Crematogaster (one site), Lasius (one site), and Tapinoma sessile Say (at one site). These results indicate that the ants were much more likely to be found on the untreated sugar source than the sugar source with the CWO barrier and this experiment demonstrated 100% exclusion of
Mean Number of Fire Ants
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50 45 40 35 30 25 20 15 10 5 0
Vol. 43, no. 3 b
Cedarwood Oil Control
b
b a a
a 1%
10%
Neat
Treatment
Fig. 1. Effect of CWO on number of Þre ants on food source. SigniÞcantly more Þre ants were on control than CWO at all three levels (P ⬍ 0.0001, P ⬍ 0.0007, and P ⬍ 0.0001, respectively for 1%, 10% and Neat using 2).
ants with CWO without the use of synthetic pesticides. A CWO-impregnated barrier could be an alternative to ant deterrent products for hummingbird feeders currently in the market. Commercial products include “barrier” type products such as AntGuard, which contains the synthetic insecticide permethrin. Other types of products are water-Þlled moats (e.g., Trap-It), which rely on water, which is subject to evaporation, to prevent contact with the attractive source. Because the most abundant component of CO2-derived CWO is cedrol (Eller and King 2000) and cedrol has a relatively high melting point (i.e., 86 Ð 87⬚C; Merck Index, 1989), CWO might be expected to last over the course of a season outdoors. A CWObased ant deterrent could be a natural long-lasting, inexpensive, safe, and effective means to exclude ants from otherwise attractive sources, such as hummingbird feeders as well as other uses in structural pest control. Red Imported Fire Ant Contact Repellency. The results of the red imported Þre ant contact CWO repellency tests are shown in Fig. 1. At every dosage, the CWO treatment had signiÞcantly fewer ants on the food source than did the control. Although there was not a clear dosageÐresponse of CWO concentration and repellency, during the 5-min test period, the 100% CWO had a mean (SE) of only 1.3 (0.9) ants on the food source compared with ⬇40 (6.0) for the corresponding control (Fig. 1). The major component of CWO is cedrol (⬇50% of total). When cedrol alone was tested against a control, a mean (SE) of only 31.2 (2.7) red imported Þre ants were at the cedrol treatment compared with a mean (SE) of 68.8 (2.7) red imported Þre ants at the control. The cedrol treatment had signiÞcantly fewer red imported Þre ants than did the control (P ⬍ 0.0001 using 2). The 50% cedrol treatment matches the amount of cedrol in the CWO. Although 50% cedrol is a signiÞcant red imported Þre ant repellent, it does not account for all the activity from the CWO. Other compounds in the CWO that contribute to CWO repellency against red imported Þre ants will be the subjects of future investigations. Essential oils have previously been demonstrated to be repellent to red imported Þre ants. Mint oil
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ELLER ET AL.: BIOACTIVITY OF CEDARWOOD OIL AND CEDROL
Table 1. Mean percentage black-legged tick mortality after exposure to cedrol after 24 and 48 h Cedrol concentration (mg/vial) 0 (control) 0.063 mg/ml 0.63 mg/ml 6.3 mg/ml
Exposure duration 24h (%)
48h (%)
3.3c 63.3b 83.3ab 100a
3.3b 76.7a 93.3a 100a
n ⫽ 3 reps of 10 ticks (I. scapularis nymphs) each. Means in a column without letters in common differ signiÞcantly based on overlap of 95% Þducial limits on concentration from Probit analysis of mortality.
(Appel et al. 2004), an essential oil product from China (Chen 2009), the sesquiterpenes callicarpenal and intermedeol (Chen et al. 2008), and compounds from cloves (Kaße and Shih 2013), have all been shown to be repellent to red imported Þre ants. In addition, Anderson et al. (2002) reported a water suspension from Juniperus wood was repellent to red imported Þre ants. In this study, the CO2-derived CWO from J. virginiana was also repellent to red imported Þre ants. The potential uses for repelling red imported Þre ants could include exclusion from homes as well as structures such as electrical systems. Black-Legged Tick Mortality. The results of the tick mortality tests are shown in Table 1. Only 3.3% of the tick nymphs in the control treatment were dead after either 24 or 48 h. However, for those exposed to the cedrol, a high percentage was dead after as little as 24 h. Nymphs exhibited very high mortality (i.e., ⬎60%) when exposed to cedrol in glass vials after both 24 and 48 h compared with only 3% mortality for those exposed to controls. Mortality increased with dosage, and toxic effects were observed sooner in ticks exposed to higher concentrations based on Probit analysis of the data. Previously, essential oils have been shown to be effective against I. scapularis. Flor-Weiler et al. (2011) reported nootkatone was toxic to I. scapularis, and Elias et al. (2013) reported a rosemary-based acaricide was effective against I. scapularis. Chen et al. (2005) reported that vetiver oil and nootkatone were repellent to both ants and ticks, and Dolan et al. (2009) suggested the natural products, nootkatone and carvacrol, could be alternative control products to conventional synthetic acaricides. Carroll et al. (2011) reported that the essential oil from Juniperus chinensis L. was repellent to I. scapularis. Our results demonstrate that CWO or cedrol from J. virginiana could also be used as an alternative to conventional synthetic acaricides for I. scapularis. The possible uses for CWO in insect management beyond the species investigated here could be much broader. In addition to its potential beneÞts in insect control, the use of CWO would also use an underutilized abundant domestic natural resource.
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Acknowledgments The authors thank Allard Cosse, Steven Vaughn, Ray Holloway, Amber Durham, and Karen Ray for their assistance with conducting Þeld tests; and Michele Hosack and David Milne for red imported Þre ant technical assistance. The Þeld-collected ants were identiÞed by Michael W. Gates (Systematic Entomology Laboratory, Agriculture Research Service, USDA, Beltsville, MD). Gregory Akerman provided the eastern red cedar samples.
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Elias, S. P., C. B. Lubelczyk, P. W. Rand, J. K. Staples, T. W. St Amand, C. S. Stubbs, E. H. Lacombe, L. B. Smith, and R. P. Smith, Jr. 2013. Effect of a botanical acaricide on Ixodes scapularis (Acari: Ixodidae) and nontarget arthropods. J. Med. Entomol. 50: 126 Ð136. Eller, F. J., and J. W. King. 2000. Supercritical carbon dioxide extraction of cedarwood oil: a study of extraction parameters and oil characteristics. Phytochem. Anal. 11: 226 Ð231. Eller, F. J., and S. L. Taylor. 2004. Pressurized ßuids for extraction of cedarwood oil from Juniperus virginiana. J. Agric. Food Chem. 52: 2335Ð2338. Eller, F. J., C. A. Clausen, F. Green, and S. L. Taylor. 2010. Critical ßuid extraction of Juniperus virginiana L. and bioactivity of extracts against subterranean termites and wood-rot fungi. Ind. Crops Prod. 32: 481Ð 485. Flor-Weiler, L. B., R. W. Behle, and K. C. Stafford, III. 2011. Susceptibility of four tick species, Amblyomma americanum, Dermacentor variabilis, Ixodes scapularis, and Rhipicephalus sanguineus (Acari: Ixodidae), to nootkatone from essential oil of grapefruit. J. Med. Entomol. 48: 322Ð 326. Ganguli, A. C., D. M. Engle, P. M. Mayer, and E. C. Hellgren. 2008. Plant community diversity and composition provide little resistance to Juniperus encroachment. Botany 86: 1416 Ð1426. Hemmerly, T. E. 1970. Economic uses of Eastern red cedar. Econ. Bot. 24: 39 Ð 41. Huddle, H. B., and A. P. Mills. 1952. The toxicity of cedar oil vapors to clothes moths. J. Econ. Entomol. 45: 40 Ð 43. Kafle, L., and C. J. Shih. 2013. Toxicity and repellency of compounds from clove (Syzygium aromaticum) to red imported Þre ants (Solenopsis invicta) (Hymenoptera: Formicidae). Ecotoxicology 106: 131Ð135. Kard, B., S. Hiziroglu, and M. E. Payton. 2007. Resistance of redcedar panels to damage by subterranean termites (Isoptera: Rhinotermitidae). For. Prod. J. 57: 74 Ð79. Ko¨ se, C., and A. M. Taylor. 2012. Evaluation of mold and termite resistance of included sapwood in eastern redcedar. Wood Fiber Sci. 44: 319 Ð324. McDaniel, C. A., and B. S. Dunn. 1994. Can wood extractives be used as wood protectants? Proc. Symp. Curr. Res. Chem. Sci. Gen. Tech. Rep. Soc. 101: 61Ð 63. McDaniel, C. A., J. A. Klocke, and M. F. Balandrin. 1989. Major antitermitic wood extractive components from eastern red cedar, Juniperus virginiana. Mater. Org. 24: 301Ð331. Meissner, H. E., and J. Silverman. 2001. Effects of aromatic cedar mulch on the Argentine ant and the odorous house ant (Hymenoptera: Formicidae). J. Econ. Entomol. 94: 1526 Ð1531.
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Morales-Ramos, J., and M. G. Rojas. 2001. Nutritional ecology of the Formosan subterranean termite (Isoptera: Rhinotermitidae): feeding response to commercial wood species. J. Econ. Entomol. 94: 516 Ð523. Mun, S. P., and L. Prewitt. 2011. Antifungal activity of organic extracts from Juniperus virginiana heartwood against wood decay fungi. For. Prod. J. 61: 443Ð 449. Panella, N. A., M. C. Dolan, J. J. Karchesy, Y. Xiong, J. Peralta-Cruz, M. Khasawneh, J. A. Monteneiri, and G. O. Maupin. 2005. Use of novel compounds for pest control: Insecticidal and acaricidal activity of essential oil components from heartwood of Alaska yellow cedar. J. Med. Entomol. 42: 352Ð358. Schmidt, T. L., and E. C. Leatherberry. 1995. The expansion of Eastern red cedar in the lower Midwest. North. J. Appl. Forestry 12: 180 Ð183. Sighamony, S., I. Anees, T. Chanrakaka, and Z. Osmani. 1984. Natural products as repellents for Tribolium castaneum Herbst. Int. Pest Control 26: 156 Ð157. Smith, B. C., A. N. Starratt, and R. P. Bodnaryk. 1973. Oviposition responses of Coleomegilla maculata lengi (Coleoptera: Coccinellidae) to the wood and extracts of Juniperus virginiana and to various chemicals. Ann. Entomol. Soc. Am. 66: 452Ð 456. Thorvilson, H., and B. Rudd. 2001. Are landscaping mulches repellent to red imported Þre ants? Southwestern Entomol. 26: 195Ð202. Tumen, I., F. J. Eller, C. A. Clausen, and J. A. Teel. 2013a. Antifungal activity of heartwood extracts from three Juniperus species. Bioresources 8: 12Ð20. Tumen, I., I. Suntar, F. J. Eller, H. Keles, and E. K. Akkol. 2013b. Topical wound-healing effects and phytochemical composition of heartwood essential oils of Juniperus virginiana L., Juniperus occidentalis Hook., and Juniperus ashei Buchholz. J. Med. Food. 16: 48 Ð55. U.S. Food and Drug Administration. 2013. Code of Federal Regulations Title 21, Chapter I, Subchapter B, Part 172, Subpart F, Flavoring Agents and Related Substances. Sec. 172.515 Synthetic ßavoring substances and adjuvants. Food and Drug Administration Department of Health and Human Services, Silver Spring, MD. Vander Meer, R. K., W. A. Banks, and C. S. Lofgren. 1996 Dec 24. Repellents for ants. U.S. patent 5,587,401. Zhu, B.C.R., G. Henderson, F. Chen, H. Fei, and R. A. Laine. 2001. Evaluation of vetiver oil and seven insect-active essential oils against the Formosan subterranean termite. J. Chem. Ecol. 27: 1617Ð1625. Received 23 September 2013; accepted 21 February 2014.
Bioactivity of Cedarwood Oil and Cedrol Against Arthropod Pests F. J. ELLER,1,2 R. K. VANDER MEER,3 R. W. BEHLE,4 L. B. FLOR-WEILER,4 5 AND DEBRA E. PALMQUIST
Environ. Entomol. 43(3): 762Ð766 (2014); DOI: http://dx.doi.org/10.1603/EN13270
ABSTRACT Heartwood samples from Juniperus virginiana L. were extracted with liquid carbon dioxide, and the bioactivity of carbon dioxide-derived cedarwood oil (CWO) toward several species of ants and cedrol toward ticks was determined. Repellency was tested for ants, and toxicity was tested for ticks. Ants in an outdoor bioassay were signiÞcantly repelled by the presence of CWO on a pole leading to a sugarÐwater solution. Similarly, CWO was a signiÞcant repellent barrier to red imported Þre ants and prevented them from Þnding a typical food source. Black-legged tick nymphs exhibited dosage-dependent mortality when exposed to cedrol and at the highest dosage (i.e., 6.3 mg/ml) tested, the cedrol killed 100% of the ticks. These repellency and toxicity results together demonstrate a clear potential for the use of CWO as a pest control agent. KEY WORDS cedarwood oil, repellency, toxicity, red imported Þre ant, black-legged tick
Eastern red cedar (Juniperus virginiana L.), western juniper (Juniperus occidentalis Hook.), and ashe juniper (Juniperus ashei J. Buchholz) (Cupressaceae) are very abundant conifers in the United States. The area covered by junipers has been expanding (Schmidt and Leatherberry 1995, Ganguli et al. 2008), and all three are often considered pest species because of their encroachment onto rangeland and pastures (Adams et al. 1988). In addition to its aromatic smell, junipers are known for their resistance to both microbial decay and termite attack. Because of this resistance, juniper has long been used for fence posts (Hemmerly 1970, Adams 2004). The antifungal (i.e., wood-rot fungi) activities of juniper heartwood extracts have recently been reported (Eller et al. 2010, Mun and Prewitt 2011). Juniper wood has been shown to be resistant to both Formosan (Morales-Ramos and Rojas 2001) and eastern subterranean (Carter and Smythe 1974, Arango et al. 2006) termites. Particleboard-chip panels made Mention of trade names or commercial products in this article is solely for the purpose of providing scientiÞc information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. 1 Functional Foods Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 North University St., Peoria, IL 61604. 2 Corresponding author, e-mail: [email protected]. 3 Imported Fire Ant and Household Insects Research Unit, Center for Medical, Agricultural, and Veterinary Entomology, Agricultural Research Service, United States Department of Agriculture, 1600 S.W. 23rd Dr., Gainesville, FL 32608. 4 Crop Bioprotection Research Unit, National Center for Agricultural, Agricultural Research Service, United States Department of Agriculture, 1815 North University St., Peoria, IL 61604. 5 Agricultural Research Service, United States Department of Agriculture, Midwest Area, 1815 North University St., Peoria, IL 61604.
from eastern red cedar are moderately resistant to termite damage (Kard et al. 2007), and Ko¨ se and Taylor (2012) recently reported heartwood and included sapwood of eastern red cedar were resistant to termites. The wood of eastern red cedar is also known for its toxicity and repellency to several species of insects including clothes moths (Huddle and Mills 1952), ßour beetles (Sighamony et al. 1984), and cockroaches (Appel and Mack 1989). Eastern red cedar mulch was also reported to be repellent to ants (Meissner and Silverman 2001, Thorvilson and Rudd 2001). However, the bioactivity of eastern red cedar extracts toward arthropods has not been extensively studied. Antitermite compounds have been extracted from eastern red cedar heartwood by organic solvent (i.e., acetone, pentane, hexane, or methanol) extraction (Carter and Smythe 1974, Carter 1976, Adams et al. 1988, McDaniel et al. 1989). An acetoneÐ hexaneÐ water extract of eastern red cedar signiÞcantly reduced termite attack when applied to southern pine by vacuum impregnation (McDaniel and Dunn 1994). Zhu et al. (2001) found that cedarwood oil (CWO) repelled termites. Interestingly, cedarwood and its extracts have also been demonstrated to induce oviposition by ladybird beetles (Boldyrev et al. 1969, Smith et al. 1973). Our laboratory has been investigating the extraction and composition of CWO, using supercritical and liquid carbon dioxide as well as pressurized solvents such as ethanol and hot water (Eller and King 2000, Eller and Taylor 2004). Carbon dioxide-derived CWO contains higher levels of cedrol and has an odor that more closely resembles that of eastern red cedar wood than does CWO obtained by steam distillation (Eller and King 2000). Carbon dioxide-derived CWO has been
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ELLER ET AL.: BIOACTIVITY OF CEDARWOOD OIL AND CEDROL
demonstrated to have antifungal, antitermite and antiinßammatory effects (Eller et al. 2010; Tumen et al. 2013a,b). CWO is considered safe and is approved as a food additive by the U.S. Food and Drug Administration (2013). The purpose of this research was to investigate potential uses of CWO against several economically important arthropods as a safe natural pest control agent. Evaluations included its use as a general ant repellent on hummingbird feeders, a repellent against red imported Þre ant, Solenopsis invicta Buren, and toxicity against black-legged ticks, Ixodes scapularis Say. Materials and Methods CWO Samples. Heartwood samples from eastern red cedar (Woodford Co., IL) were prepared from freshly cut trees. Sapwood was removed from the samples using a band saw, and heartwood sawdust was prepared using a compound miter saw. Sawdust samples were held in glass containers at room temperature before extraction. Extractions were made using liquid carbon dioxide (25⬚C and 10.3 Mpa), and the CO2 was depressurized into glass vials to collect the CWO as described previously (Eller et al. 2010). (⫹)-Cedrol was purchased from Aldrich (Milwaukee, WI). Ant Repellent–Hummingbird Feeder Bioassay. The experiment was set up as a paired test of untreated control versus a CWO barrier treatment. The hummingbird feeders (First Nature, Rogers, AR) used in this study consisted of a large inverted reservoir to provide a constant level of the sugarÐwater solution (1:4 by volume). The sugar solution was accessible through 10 holes in the cover. A plastic hook on the top of the feeder is used to suspend the feeder. The hummingbird feeders were hung on a 1.63-m-tall black shepherdÕs hook (Enchanted Garden, Menards, Eau Claire, WI), and the traps were ⬇1.2 m from the ground. Twine (⬇1.5 mm in diameter and 30 cm in length) was wrapped Þve times around the base of the shepherdÕs hook ⬇15 cm from the ground and tied in a knot with the excess trimmed off. The concentration of the CWO in the string was ⬇500 mg/cm3. The control was left untreated while the treated twine had 250 l (⬇200 mg) of neat CWO extract applied to the twine. The two shepherdÕs hooks were placed 1 m apart at eight separate locations in Peoria and Woodford counties, IL. The feeders were checked daily for the presence of ants. When ants were Þrst detected, that replication was ended and each feeder was placed in a separate plastic bag and placed in a freezer (⫺10⬚C) to kill the ants present. The ants were then removed, counted, and preserved in 70% ethanol. The ants were identiÞed to genus. Imported Red Fire Ant Contact Repellency. The bioassay for contact repellency was similar to that described by Vander Meer et al. (1996). It was hypothesized that the CWO would act as a close-range repellent not as a volatile long-range repellent. The test tray was composed of a porcelain pan measuring 180 by 290 by 50 mm. The upper 3 mm of the pan was
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coated with Fluon (BioQuip Products, Rancho Dominguez, CA) to preclude ants from escaping. A petri dish nest cell (55 mm in diameter) was placed at one end of the pan. The petri dish had a 5-mm layer of Castone dental cement on the bottom that acted as a moisture reservoir. The lid of the petri dish had a hole placed in the center to allow ant access. To protect the bottom of the pan from contamination by the test materials, 2.5-cm2 pieces of aluminum foil were placed in the opposite end of the pan from the nest cell at each corner ⬇3.0 cm from the sides of the pan. No food or water was available to the ants during the bioassay. Fifty microliters of test material was introduced to the test chamber on a 2.0-cm2 piece of Whatman silicone-treated Þlter paper (cat. # 2200 125; Sigma-Aldrich, St. Louis, MO) and was randomly assigned and placed on one of the aluminum foil squares. The other aluminum square received a Þlter paper square with 50 l of pentane as a control. The solvent was allowed to evaporate to apparent dryness. Placed on top of each Þlter paper square was a small wad of cotton soaked in 10% sucrose to serve as a phagostimulant. Once test materials were in place, ⬇1 g of ants, Solenopsis invicta Buren (Hymenoptera: Formicidae) (starved for a minimum of 24 h) was placed in the nest cell and a stopwatch was started. The number of ants actively feeding on the treatment and control cotton balls was observed and recorded after a total of 5 min. The score for each bioassay replicate was the number of the 5-min values for control and treatment. Each experiment was replicated three times, each with a unique monogyne colony and in a different test tray. CWO at three concentrations (1 and 10% in pentane: wt:vol, and neat) was tested against pentane controls. Cedrol at 50% (wt:vol in pentane) was tested against a pentane control. Black-Legged Tick Contact Toxicity. Tick Colony. Unfed nymphs (⬇2Ð3 wk since molting) of the black-legged tick, I. scapularis (Acari: Ixodidae), were procured from the Tick Rearing Facility, National Tick Research and Education Resource, Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK. Before toxicological bioassays, nymphs were held in four-dram vials contained in a desiccator with potassium sulfate solution to maintain high relative humidity (RH; ⬎90%), at room temperature (22Ð24⬚C), with a photoperiod of 16:8 (L:D) h. Nymphs were allowed to acclimate to our laboratory conditions at least 24 h before starting the toxicological assay. Coating of Vials and Bioassays. Different concentrations of cedrol were tested on unfed nymphs of I. scapularis, in laboratory bioassays. Three serial dilutions (10⫻) of cedrol were made with hexane ranging from 6.3 to 0.063 mg/ml. Coating of vials with cedrol concentrations in hexane was done following the method of Panella et al. (2005) with modiÞcations. Each four-dram size vial (27.25 by 67 mm, Fisherbrand, Fisher ScientiÞc, Pittsburgh, PA) was coated evenly on the inside by adding 1 ml of the appropriate solution of cedrol in hexane. Vials were placed on their side on a roller (Bellco Biotechnology, Bellco Glass,
ENVIRONMENTAL ENTOMOLOGY
Inc., Vineland, NJ) and allowed to dry in a fume hood for 15Ð20 min or until hexane had completely evaporated. There were three replications (i.e., vials) of each treatment concentration, and three additional vials were treated with hexane only as a control treatment. The coated vials had only a very thin coating of oil, which did not appear to impair the movement of the tick nymphs. For the bioassay, 10 unfed nymphs were introduced into each vial and the vials were then capped with a piece of cotton fabric secured with a rubber band and Þnally covered with aluminum foil. Vials were placed in the desiccator with potassium sulfate solution to maintain high RH (⬎90%), at room temperature (22Ð 24⬚C), with a photoperiod of 16:8 (L:D)h. Thus, 30 tick nymphs were exposed to each cedrol concentration. Tick mortality was recorded 24 and 48 h after exposure to the cedrol-coated vials. Ticks were considered alive when they exhibited normal behavior, and considered moribund or dead when they were incapable of movement, failed to maintain normal posture, exhibited uncoordinated movement, were unable to right themselves, or showed no sign of life (motionless). Statistical Analyses. A sign test was used to test the probability of obtaining the observed results in the ant repellentÐ hummingbird feeder bioassay, and a oneway analysis of variance (ANOVA) was used to compare the number of ants at the CWO treatment with the number of ants at the control after square-root transformation of the count data plus 1 (Statistix 7.0, Analytical Software, Tallahassee, FL). A 2 analysis was used to compare number of red imported Þre ants at the CWO treatments with the number of red imported Þre ants at the controls. Probit analysis with logistic distribution was used to analyze probability of tick mortality based on cedrol concentration (SAS version 9.3 @ 2002Ð2110 [SAS Institute Inc., Cary, NC]).
Results and Discussion Ant Repellent–Hummingbird Feeder Bioassay. Ants were Þrst detected at the feeders at 2Ð10 d after placement in the Þeld. Ants were found at the hummingbird feeders at seven of the eight control replicates, whereas no ants were found at the hummingbird feeders treated with CWO (n ⫽ 8). The P value for this result was P ⫽ 0.0078 (Sign test; n ⫽ 7). The total number of ants captured at the controls ranged from 8 to 112, and the mean number of ants found on the feeders was 30.7 (n ⫽ 8). The ANOVA indicated that signiÞcantly more ants were captured at the control feeders than the feeders with the CWO (F1,14 ⫽ 14.1; P ⫽ 0.0021). Five genera of ants were identiÞed from the seven sites and included Camponotus (at two sites), Formica (at two sites), and Crematogaster (one site), Lasius (one site), and Tapinoma sessile Say (at one site). These results indicate that the ants were much more likely to be found on the untreated sugar source than the sugar source with the CWO barrier and this experiment demonstrated 100% exclusion of
Mean Number of Fire Ants
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50 45 40 35 30 25 20 15 10 5 0
Vol. 43, no. 3 b
Cedarwood Oil Control
b
b a a
a 1%
10%
Neat
Treatment
Fig. 1. Effect of CWO on number of Þre ants on food source. SigniÞcantly more Þre ants were on control than CWO at all three levels (P ⬍ 0.0001, P ⬍ 0.0007, and P ⬍ 0.0001, respectively for 1%, 10% and Neat using 2).
ants with CWO without the use of synthetic pesticides. A CWO-impregnated barrier could be an alternative to ant deterrent products for hummingbird feeders currently in the market. Commercial products include “barrier” type products such as AntGuard, which contains the synthetic insecticide permethrin. Other types of products are water-Þlled moats (e.g., Trap-It), which rely on water, which is subject to evaporation, to prevent contact with the attractive source. Because the most abundant component of CO2-derived CWO is cedrol (Eller and King 2000) and cedrol has a relatively high melting point (i.e., 86 Ð 87⬚C; Merck Index, 1989), CWO might be expected to last over the course of a season outdoors. A CWObased ant deterrent could be a natural long-lasting, inexpensive, safe, and effective means to exclude ants from otherwise attractive sources, such as hummingbird feeders as well as other uses in structural pest control. Red Imported Fire Ant Contact Repellency. The results of the red imported Þre ant contact CWO repellency tests are shown in Fig. 1. At every dosage, the CWO treatment had signiÞcantly fewer ants on the food source than did the control. Although there was not a clear dosageÐresponse of CWO concentration and repellency, during the 5-min test period, the 100% CWO had a mean (SE) of only 1.3 (0.9) ants on the food source compared with ⬇40 (6.0) for the corresponding control (Fig. 1). The major component of CWO is cedrol (⬇50% of total). When cedrol alone was tested against a control, a mean (SE) of only 31.2 (2.7) red imported Þre ants were at the cedrol treatment compared with a mean (SE) of 68.8 (2.7) red imported Þre ants at the control. The cedrol treatment had signiÞcantly fewer red imported Þre ants than did the control (P ⬍ 0.0001 using 2). The 50% cedrol treatment matches the amount of cedrol in the CWO. Although 50% cedrol is a signiÞcant red imported Þre ant repellent, it does not account for all the activity from the CWO. Other compounds in the CWO that contribute to CWO repellency against red imported Þre ants will be the subjects of future investigations. Essential oils have previously been demonstrated to be repellent to red imported Þre ants. Mint oil
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Table 1. Mean percentage black-legged tick mortality after exposure to cedrol after 24 and 48 h Cedrol concentration (mg/vial) 0 (control) 0.063 mg/ml 0.63 mg/ml 6.3 mg/ml
Exposure duration 24h (%)
48h (%)
3.3c 63.3b 83.3ab 100a
3.3b 76.7a 93.3a 100a
n ⫽ 3 reps of 10 ticks (I. scapularis nymphs) each. Means in a column without letters in common differ signiÞcantly based on overlap of 95% Þducial limits on concentration from Probit analysis of mortality.
(Appel et al. 2004), an essential oil product from China (Chen 2009), the sesquiterpenes callicarpenal and intermedeol (Chen et al. 2008), and compounds from cloves (Kaße and Shih 2013), have all been shown to be repellent to red imported Þre ants. In addition, Anderson et al. (2002) reported a water suspension from Juniperus wood was repellent to red imported Þre ants. In this study, the CO2-derived CWO from J. virginiana was also repellent to red imported Þre ants. The potential uses for repelling red imported Þre ants could include exclusion from homes as well as structures such as electrical systems. Black-Legged Tick Mortality. The results of the tick mortality tests are shown in Table 1. Only 3.3% of the tick nymphs in the control treatment were dead after either 24 or 48 h. However, for those exposed to the cedrol, a high percentage was dead after as little as 24 h. Nymphs exhibited very high mortality (i.e., ⬎60%) when exposed to cedrol in glass vials after both 24 and 48 h compared with only 3% mortality for those exposed to controls. Mortality increased with dosage, and toxic effects were observed sooner in ticks exposed to higher concentrations based on Probit analysis of the data. Previously, essential oils have been shown to be effective against I. scapularis. Flor-Weiler et al. (2011) reported nootkatone was toxic to I. scapularis, and Elias et al. (2013) reported a rosemary-based acaricide was effective against I. scapularis. Chen et al. (2005) reported that vetiver oil and nootkatone were repellent to both ants and ticks, and Dolan et al. (2009) suggested the natural products, nootkatone and carvacrol, could be alternative control products to conventional synthetic acaricides. Carroll et al. (2011) reported that the essential oil from Juniperus chinensis L. was repellent to I. scapularis. Our results demonstrate that CWO or cedrol from J. virginiana could also be used as an alternative to conventional synthetic acaricides for I. scapularis. The possible uses for CWO in insect management beyond the species investigated here could be much broader. In addition to its potential beneÞts in insect control, the use of CWO would also use an underutilized abundant domestic natural resource.
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Acknowledgments The authors thank Allard Cosse, Steven Vaughn, Ray Holloway, Amber Durham, and Karen Ray for their assistance with conducting Þeld tests; and Michele Hosack and David Milne for red imported Þre ant technical assistance. The Þeld-collected ants were identiÞed by Michael W. Gates (Systematic Entomology Laboratory, Agriculture Research Service, USDA, Beltsville, MD). Gregory Akerman provided the eastern red cedar samples.
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Morales-Ramos, J., and M. G. Rojas. 2001. Nutritional ecology of the Formosan subterranean termite (Isoptera: Rhinotermitidae): feeding response to commercial wood species. J. Econ. Entomol. 94: 516 Ð523. Mun, S. P., and L. Prewitt. 2011. Antifungal activity of organic extracts from Juniperus virginiana heartwood against wood decay fungi. For. Prod. J. 61: 443Ð 449. Panella, N. A., M. C. Dolan, J. J. Karchesy, Y. Xiong, J. Peralta-Cruz, M. Khasawneh, J. A. Monteneiri, and G. O. Maupin. 2005. Use of novel compounds for pest control: Insecticidal and acaricidal activity of essential oil components from heartwood of Alaska yellow cedar. J. Med. Entomol. 42: 352Ð358. Schmidt, T. L., and E. C. Leatherberry. 1995. The expansion of Eastern red cedar in the lower Midwest. North. J. Appl. Forestry 12: 180 Ð183. Sighamony, S., I. Anees, T. Chanrakaka, and Z. Osmani. 1984. Natural products as repellents for Tribolium castaneum Herbst. Int. Pest Control 26: 156 Ð157. Smith, B. C., A. N. Starratt, and R. P. Bodnaryk. 1973. Oviposition responses of Coleomegilla maculata lengi (Coleoptera: Coccinellidae) to the wood and extracts of Juniperus virginiana and to various chemicals. Ann. Entomol. Soc. Am. 66: 452Ð 456. Thorvilson, H., and B. Rudd. 2001. Are landscaping mulches repellent to red imported Þre ants? Southwestern Entomol. 26: 195Ð202. Tumen, I., F. J. Eller, C. A. Clausen, and J. A. Teel. 2013a. Antifungal activity of heartwood extracts from three Juniperus species. Bioresources 8: 12Ð20. Tumen, I., I. Suntar, F. J. Eller, H. Keles, and E. K. Akkol. 2013b. Topical wound-healing effects and phytochemical composition of heartwood essential oils of Juniperus virginiana L., Juniperus occidentalis Hook., and Juniperus ashei Buchholz. J. Med. Food. 16: 48 Ð55. U.S. Food and Drug Administration. 2013. Code of Federal Regulations Title 21, Chapter I, Subchapter B, Part 172, Subpart F, Flavoring Agents and Related Substances. Sec. 172.515 Synthetic ßavoring substances and adjuvants. Food and Drug Administration Department of Health and Human Services, Silver Spring, MD. Vander Meer, R. K., W. A. Banks, and C. S. Lofgren. 1996 Dec 24. Repellents for ants. U.S. patent 5,587,401. Zhu, B.C.R., G. Henderson, F. Chen, H. Fei, and R. A. Laine. 2001. Evaluation of vetiver oil and seven insect-active essential oils against the Formosan subterranean termite. J. Chem. Ecol. 27: 1617Ð1625. Received 23 September 2013; accepted 21 February 2014.
