Acute Toxicity of Plant Essential Oils to Scarab Larvae (Coleoptera: Scarabaeidae) and Their Analysis by Gas Chromatography-Mass Spectrometry

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ECOTOXICOLOGY Acute Toxicity of Plant Essential Oils to Scarab Larvae (Coleoptera: Scarabaeidae) and Their Analysis by Gas Chromatography-Mass Spectrometry

CHRISTOPHER M. RANGER,1,2,3 MICHAEL E. REDING,1,2 JASON B. OLIVER,4 1 4 1 JAMES J. MOYSEENKO, NADEER YOUSSEF, AND CHARLES R. KRAUSE

J. Econ. Entomol. 106(1): 159Ð167 (2013); DOI: http://dx.doi.org/10.1603/EC12319 ABSTRACT Larvae of scarab beetles (Coleoptera: Scarabaeidae) are important contaminant and root-herbivore pests of ornamental crops. To develop alternatives to conventional insecticides, 24 plant-based essential oils were tested for their acute toxicity against third instars of the Japanese beetle Popillia japonica Newman, European chafer Rhizotrogus majalis (Razoumowsky), oriental beetle Anomala orientalis (Waterhouse), and northern masked chafer Cyclocephala borealis Arrow. Diluted

solutions were topically applied to the thorax, which allowed for calculating LD50 and LD90 values associated with 1 d after treatment. A wide range in acute toxicity was observed across all four scarab species. Of the 24 oils tested, allyl isothiocyanate, cinnamon leaf, clove, garlic, and red thyme oils exhibited toxicity to all four species. Allyl isothiocyanate was the most toxic oil tested against the European chafer, and among the most toxic against the Japanese beetle, oriental beetle, and northern masked chafer. Red thyme was also comparatively toxic to the Japanese beetle, oriental beetle, European chafer, and northern masked chafer. InterspeciÞc variability in susceptibility to the essential oils was documented, with 12, 11, 8, and 6 of the 24 essential oils being toxic to the oriental beetle, Japanese beetle, European chafer, and northern masked chafer, respectively. Analysis of the active oils by gas chromatography-mass spectrometry revealed a diverse array of compounds, mostly con- sisting of mono- and sesquiterpenes. These results will aid in identifying active oils and their constituents for optimizing the development of plant essential oil mixtures for use against scarab larvae.

KEY WORDS essential oils, botanical insecticides, GC-MS, white grubs, scarab larvae

Root herbivory by the subterranean larvae of scarab found in Ohio include the European chafer Rhizotrogus beetles (Coleoptera: Scarabaeidae), commonly re- majalis (Razoumowsky) and oriental beetle Anomala ferred to as white grubs, can cause girdling of roots and orientalis (Waterhouse), along with the native species eventual stunting or death of nursery crops (Jackson northern masked chafer Cyclocephala borealis Arrow and Klein 2006). Scarab larvae are also important con- (Reding et al. 2004, Reding and Klein 2007). taminant pests of Þeld-grown and containerized nurs- To minimize the risk of introduction to uninfested ery crops. For instance, detection of Japanese beetle areas, the U.S. Department of Agriculture, National Popillia japonica Newman larvae in balled and bur- Plant Board, and the regulated industry participate in lapped nursery stock, container-grown plants, or grass a harmonization plan aimed at establishing quarantine sod can prevent such products from being shipped and certiÞcation requirements for the Japanese beetle within or outside of the United States (Potter and Held that are consistent with the National Plant Board Plant 2002, National Plant Board 2011). Other exotic species Quarantine, Nursery Inspection, and CertiÞcation Guidelines (Mannion et al. 2000, National Plant Board 2011). Immersing Þeld grown nursery stock in chlor- Mention of proprietary products or companies is included for the pyrifos (an organophosphate insecticide) or bifen- readerÕs convenience and does not imply any endorsement or pref- erential treatment by the USDA-ARS, The Ohio State University, or thrin (a pyrethroid insecticide) are currently the only Tennessee State University. certiÞed methods available to growers that need to 1 USDA-Agricultural Research Service, Application Technology treat within2dofshipping (Mannion et al. 2000, Research Unit, Horticultural Insects Research Laboratory, 1680 Mad- National Plant Board 2011). The large volume of in- ison Ave., Wooster, OH 44691. 2 Department of Entomology, Ohio Agricultural Research and De- secticidal solutions required to effectively immerse velopment Center, The Ohio State University, 1680 Madison Ave., root balls results in such procedures being difÞcult, Wooster, OH 44691. hazardous to applicators, and potentially phytotoxic to 3 Corresponding author, e-mail: [email protected]. certain tree species (Mannion et al. 2000). Neonic- 4 Tennessee State University, College of Agriculture, Human, and Natural Sciences, Otis L. Floyd Nursery Research Center, 472 Cadillac otinoids (e.g., imidacloprid and thiamethoxam) are Ln., McMinnville, TN 37110. also used as systemic preventative treatments for con-

0022-0493/13/0159Ð0167$04.00/0 ᭧ 2013 Entomological Society of America 160 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 1 trolling scarab larvae in ornamentals, along with in- (Simplers), clove (SigmaÐAldrich), eugenol (SigmaÐ corporating pyrethroids (e.g., bifenthrin or teßuthrin) Aldrich), fennel (SigmaÐAldrich), Þr balsam (Sim- into potting media or as a midsummer drench (Nielsen plers), garlic (SigmaÐAldrich), grapefruit (Simplers), and Cowles 1998, Potter and Held 2002). jojoba (Fluka Chemicals, Buchs, Switzerland), juni- The potentially deleterious environmental and per berry (Simplers), peppermint (SigmaÐAldrich), mammalian effects of conventional insecticides have red thyme (Simplers), rosemary (SigmaÐAldrich), encouraged efforts to identify environmentally sound sesame (SigmaÐAldrich), soybean (Fluka), spruce and sustainable alternatives. Biopesticides derived bark (Simplers), St. JohnÕs wort (Simplers), white from botanical sources are an alternative to synthetic pepper (Natural Green Aromatics, Richmond, Can- insecticides, and exhibit low environmental persis- ada), and wintergreen (SigmaÐAldrich). Mineral oil, tence and mammalian toxicity (Isman 2000). Plant- which is a nonplant oil containing C15 to C40 saturated derived essential oils are common components of bo- hydrocarbons, was also included as an oil control. tanical insecticides (Isman 2006). Such oils are most Stock formulations of each product were diluted in commonly collected from plant tissues using steam acetone to determine acute toxicity at various doses, distillation, and are mainly composed of terpenes, ben- thereby allowing for calculation of LD50 and LD90 zene derivatives, hydrocarbons, and other miscella- values. Densities (grams per milligram) were calcu- neous compounds (Ngoh et al. 1998, Tripathi et al. lated for each essential oil, and LD values are pre- 2009). Plant essential oils are a major source of bio- sented as mg of oil applied per larva (milligrams per logically active compounds with detrimental effects larva). Mean Ϯ SE larval weight was calculated using on insect biology, behavior, and physiology (Singh representative third instars (n ϭ 10 per species) of the and Upadhyay 1993, Isman 2006). Their rapid activity European chafer, Japanese beetle, northern masked and effects on neurotransmission indicate essential chafer, and oriental beetle. oils act primarily as neurotoxins (Rice and Coats 1994, Insects and Acute Toxicity Bioassays. The European Ngoh et al. 1998). chafer, Japanese beetle, northern masked chafer, and The ability of essential oils to act as contact and oriental beetle lay eggs in June and July in Ohio and fumigant toxins are best documented against stored grubs typically reach the third instar by mid-Septem- product pests while only a few studies have assessed ber. Grubs will continue feeding into October and the efÞcacy of botanical formulations against subter- then move downward within the soil as surface tem- ranean root herbivores. In particular, Scott et al. peratures decrease (Koppenho¨fer and Fuzy 2003). (2005) found a 2% botanical formulation of black pep- After overwintering, larvae will resume feeding until per, Piper nigrum L., exhibited low residual soil per- pupating in the late spring. In accordance with com- sistence and was as efÞcacious as the conventional mon procedures used for collecting white grubs as part insecticide diazinon for controlling the European cha- of efÞcacy trials (Koppenho¨fer and Fuzy 2003), third- fer. Ranger et al. (2009) assessed the toxicity of com- instars of each white grub species were collected dur- mercially available plant essential oil-based insecti- ing September to October from turf areas within Ohio. cides to third instars of the Japanese beetle, oriental Specimens were held in dishpans containing moist soil beetle, European chafer, and northern masked chafer. from each respective collection site at 5Ð6ЊC for 2Ð3 Of the eight botanical products tested, two exhibited mo to obtain postoverwintering third instars, as de- a comparatively high level of toxicity to each species. scribed by Ranger et al. (2009). Grubs held under However, blending essential oils or botanical extracts these conditions are more representative of spring from diverse botanical sources did not necessarily than fall specimens (Koppenho¨fer and Fuzy 2003), ensure enhanced toxicity compared with less complex but differences in susceptibilities to topically applied formulations (Ranger et al. 2009). Thus, understand- insecticides between preoverwintering and overwin- ing the activity of individual essential oil components tered grubs is unclear (Villani et al. 1988, Cowles and will ultimately aid in developing optimal mixtures. An Villani 1996). EfÞcacy of most insecticides decreases objective of our current study was to assess the acute as white grubs reach the third instar (Cowles and toxicity of 24 plant essential oils against larvae of the Villani 1996, Grewal et al. 2001). Japanese beetle, oriental beetle, European chafer, and Scarab larvae were removed from cold storage and northern masked chafer. A second objective was to allowed to acclimate for1hatroom temperature use gas chromatography-mass spectrometry (GC-MS) before use in bioassays. A Burkhard microapplicator to characterize the constituents of active essential oils (Burkard Manufacturing, Hertfordshire, England) to provide insight into structure-activity relationships. was then used to topically apply 1 ␮l of each dilution to the dorsal thorax of third instars of the European chafer, Japanese beetle, northern masked chafer, and Materials and Methods oriental beetle (see Tables 1Ð4 for the range of con- Essential Oils. Concentrated solutions of the fol- centrations tested). Preliminary bioassays were con- lowing plant oils were purchased for acute toxicity ducted to approximate the active range of concentra- tests: allyl isothiocyanate (SigmaÐAldrich, St. Louis, tions used during the Þnal bioassay for calculating

MO), black pepper (Simplers Botanical Co., Sebasto- LD50 and LD90 values (Hummelbrunner and Isman pol, CA), capsaicin (SigmaÐAldrich), cinnamalde- 2001). A minimum of four, but usually Þve to six hyde (Acros Organics, Morris Plains, NJ), cinnamon concentrations of each essential oil were tested bark (Simplers), cinnamon leaf (Simplers), citronella against each scarab species. The ranges of tested con- February 2013 RANGER ET AL.: TOXICITY OF ESSENTIAL OILS TO SCARAB LARVAE 161

Table 1. Plant essential oils exhibiting acute toxicity to larvae of the Japanese beetle

a Ϯ a a Product Range of doses tested Slope SE P LD50 95% CI LD90 95% CI Allyl Isothiocyanate 0.05Ð0.31 1.3 Ϯ 0.3 0.001 0.16a 0.12Ð0.19 0.42b 0.29Ð0.86 Cinnamaldehyde 0.17Ð0.94 1.0 Ϯ 0.2 0.001 0.46bcd 0.35Ð0.62 1.69d 1.07Ð4.99 Cinnamon bark 0.30Ð0.88 1.8 Ϯ 0.4 0.001 0.61de 0.51Ð0.74 1.22cd 0.93Ð2.29 Cinnamon leaf 0.20Ð0.69 2.2 Ϯ 0.4 0.001 0.35bc 0.3Ð0.4 0.62b 0.52Ð0.84 Citronella 0.43Ð0.81 3.0 Ϯ 0.6 0.001 0.55d 0.48Ð0.6 0.83bcd 0.73Ð1.1 Clove 0.07Ð0.74 1.7 Ϯ 0.3 0.001 0.34bc 0.28Ð0.42 0.73bcd 0.57Ð1.13 Eugenol 0.33Ð0.94 1.5 Ϯ 0.4 0.001 0.32abc 0.16Ð0.41 0.77bcd 0.61Ð1.29 Garlic 0.05Ð0.54 1.7 Ϯ 0.3 0.001 0.29b 0.23Ð0.36 0.62bc 0.48Ð1.01 Red thyme 0.09Ð0.23 3.8 Ϯ 0.6 0.001 0.14a 0.13Ð0.16 0.2a 0.18Ð0.23 Rosemary 0.53Ð0.82 4.5 Ϯ 1.0 0.001 0.77e 0.72Ð0.84 1.02cd 0.90Ð1.36 Wintergreen 0.18Ð1.11 1.6 Ϯ 0.3 0.001 0.48cd 0.39Ð0.57 1.04bcd 0.82Ð1.61

Ͻ LD50 and LD90 values within a column followed by the same letter are not signiÞcantly different (P 0.05) as based on nonoverlap of the 95% CI. a Range of doses tested, LD50 and LD90 values are expressed as milligrams of essential oil per larva. Only active essential oils are presented; see Materials and Methods for a list of all plant essential oils tested. centrations are presented in Tables 1Ð4. At least three jector was maintained at a 1:20 split ratio for the concentrations associated with 15Ð85% mortality and duration of the analysis. A capillary nonpolar Varian at least one concentration below 15% and/or above VF-5MS column (0.25 ␮m ϫ 30 m ϫ 0.25 ␮m; 5% 85% mortality were used to minimize variance and phenyl-methyl) was used for analysis according to the increase the precision of parameter estimates used following program: 40Ð246ЊCat3ЊC/min. Helium was during probit analyses (Finney 1964, Vandenberg and used as a carrier gas at 1 ml/min. A Varian 2200 mass Soper 1979, Farnham 1998). Inactive products were spectral detector was operated in electron impact characterized by oils that did not exhibit any toxicity mode with a scan range of 14Ð415 m/z. Data acqui- after topical application of 1 ␮l of concentrated, un- sition by the mass spectrometer was delayed for the diluted solution. After treatment, individual larvae Þrst 2 min after injection. System control was accom- were placed in transparent plastic cups (30 ml vol- plished with Star Chromatography Workstation soft- ume) containing 20 g of moist sand that were capped ware (Star Toolbar, version 6.8; Varian). A fresh meth- to retain moisture. Cups were held at 22ЊC, 50Ð55% ylene chloride sample was analyzed in between each relative humidity (RH), 0:24 h (L:D) and mortality essential oil analysis. Compound identiÞcations are was measured 1 d after treatment (DAT). Live, based on Adams (2007), the National Institute of Stan- healthy grubs were able to actively crawl after being dards and Technology (NIST) MS database, and/or disturbed; moribund grubs were discolored, hump- conÞrmed by comparing mass spectral fragmentation backed, lethargic, and unable to crawl. Twenty larvae patterns and retention times with authentic standards. were used for each concentration of each essential oil. Statistics. Percentage mortality of the Japanese bee- GC-MS Analysis. Dilutions (0.1%) of the aforemen- tle, oriental beetle, European chafer, and northern tioned oils were made in methylene chloride for anal- masked chafer were corrected for untreated control ysis by GC-MS. A Varian (Palo Alto, CA) CP-8400 mortality using AbbottÕs formula (Abbott 1925), but autosampler was used to inject 1 ␮l into the injection untreated control mortality was Ͻ20% for all bioas- port of a Varian CP-3800 gas chromatograph, which says. Probit analysis was used to determine slopes and consisted of a deactivated glass liner with a 0.75 mm LD50 and LD90 values at one DAT (Finney 1971, internal diameter and a single tapered end. The in- Minitab 2003). SigniÞcant differences in LD50 and

Table 2. Plant essential oils exhibiting acute toxicity to larvae of the oriental beetle

a Ϯ a a Product Range of doses tested Slope SE P LD50 95% CI LD90 95% CI Ally Isothiocyanate 0.05Ð0.31 1.8 Ϯ 0.4 0.001 0.19a 0.16Ð0.23 0.38a 0.29Ð0.65 Cinnamon bark 0.25Ð0.88 1.8 Ϯ 0.4 0.001 0.67de 0.57Ð0.81 1.39cd 1.06Ð2.64 Cinnamon leaf 0.29Ð0.88 0.8 Ϯ 0.4 0.03 0.92de 0.64Ð76.78 4.0d 2.0Ð3.6 ϫ 108 Citronella 0.26Ð0.81 2.1 Ϯ 0.7 0.003 0.89e 0.75Ð1.73 1.68cd 1.13Ð10.03 Clove 0.14Ð0.85 1.2 Ϯ 0.3 0.001 0.62cde 0.49Ð0.85 1.75cd 1.15Ð4.56 Eugenol 0.16Ð0.88 0.9 Ϯ 0.2 0.001 0.55bcde 0.41Ð0.83 2.26cd 1.26Ð12.62 Garlic 0.10Ð0.75 1.7 Ϯ 0.3 0.001 0.43bc 0.35Ð0.52 0.89bc 0.70Ð1.41 Grapefruit 0.26Ð0.76 1.6 Ϯ 0.7 0.018 1.09e 0.80Ð20.06 2.43cd 1.28Ð2279.11 Peppermint 0.42Ð0.80 2.1 Ϯ 0.6 0.001 0.57cd 0.48Ð0.66 1.06bcd 0.85Ð2.25 Red thyme 0.17Ð0.77 2.3 Ϯ 0.4 0.001 0.38b 0.32Ð0.43 0.66ab 0.55Ð0.86 Rosemary 0.53Ð0.82 3.5 Ϯ 0.9 0.001 0.79e 0.74Ð0.94 1.16cd 0.97Ð2.01 Wintergreen 0.06Ð0.79 1.5 Ϯ 0.5 0.001 0.81de 0.62Ð1.62 1.94cd 1.16Ð14.11

Table 3. Plant essential oils exhibiting acute toxicity to larvae of the European chafer

a Ϯ a a Product Range of doses tested Slope SE P LD50 95% CI LD90 95% CI Allyl Isothiocyanate 0.10Ð0.51 3.8 Ϯ 0.7 0.001 0.33a 0.29Ð0.36 0.46a 0.41Ð0.56 Cinnamon leaf 0.20Ð0.88 0.9 Ϯ 0.4 0.017 2.00c 1.00Ð350.00 7.00c 2.0Ð6.24 ϫ 105 Clove 0.21Ð0.85 1.1 Ϯ 0.4 0.003 1.06c 0.78Ð3.19 3.33c 1.66Ð83.23 Eugenol 0.33Ð0.89 5.7 Ϯ 2.8 0.04 0.97c 0.88Ð33.16 1.22bc 1.00Ð4383.18 Fennel 0.28Ð0.85 1.8 Ϯ 0.6 0.004 1.06c 0.85Ð2.86 2.20bc 1.35Ð28.86 Garlic 0.21Ð0.96 3.1 Ϯ 1.3 0.019 1.04c 0.91Ð3.01 1.57bc 1.17Ð38.79 Red thyme 0.13Ð0.70 2.1 Ϯ 0.4 0.001 0.48b 0.41Ð0.57 0.89b 0.72Ð1.36 Wintergreen 0.18Ð1.11 2.3 Ϯ 0.4 0.001 0.60b 0.52Ð0.69 1.05b 0.88Ð1.46

␮ LD90 values were based on nonoverlap of the 95% CI. of 1 l, undiluted acetone or mineral oil were not toxic Larval weights associated with the four white grub to any of the scarab larvae (data not shown). species were compared using one-way analysis of vari- Acute toxicity against third instars of the Japanese ance (ANOVA) and TukeyÕs honestly signiÞcant dif- beetle was associated with 11 of the 24 plant essential ference (HSD) at ␣ ϭ 0.05 was used to test for dif- oils, namely, allyl isothiocyanate, cinnamaldehyde, ferences between means. cinnamon bark, cinnamon leaf, citronella, clove, eugenol, garlic, red thyme, rosemary, and wintergreen (Table 1). The two lowest LD values were associ- Results ϭ ϭ ated with red thyme (LD50 0.14 mg; LD90 0.2 ϭ Acute Toxicity Bioassays. Allyl isothiocyanate, cin- mg) and allyl isothiocyanate (LD50 0.16 mg; ϭ namon leaf, clove, garlic, red thyme, and wintergreen LD90 0.42 mg). were toxic to all four species of scarab larvae (Tables Toxicity against larvae of the oriental beetle was 1Ð4). The following 13 oils were toxic to at least one associated with 12 of the 24 plant essential oils, namely, species of scarab larvae: allyl isothiocyanate, cinnam- allyl isothiocyanate, cinnamon bark, cinnamon leaf, aldehyde, cinnamon bark, cinnamon leaf, citronella, citronella, clove, eugenol, garlic, grapefruit, pepper- clove, eugenol, fennel, garlic, peppermint, red thyme, mint, red thyme, rosemary, and wintergreen (Table rosemary, and wintergreen (Tables 1Ð4). Allyl iso- 2). The two lowest LD values were associated with ϭ ϭ thiocyanate was the most toxic oil tested against the allyl isothiocyanate (LD50 0.19 mg; LD90 0.38 mg) ϭ ϭ ϭ ϭ European chafer (LD50 0.33 mg per larva; LD90 and red thyme (LD50 0.38 mg; LD90 0.66 mg). 0.46 mg; Table 3), and among the most toxic against the Activity against third instar European chafers was ϭ ϭ Japanese beetle (LD50 0.16 mg; LD90 0.42 mg; associated with eight of the 24 plant essential oils, ϭ ϭ Table 1), oriental beetle (LD50 0.19 mg; LD90 0.38 namely, allyl isothiocyanate cinnamon leaf, clove, eu- ϭ mg; Table 2), and northern masked chafer (LD50 genol, fennel, garlic, red thyme, and wintergreen (Ta- ϭ 0.23 mg; LD90 0.51 mg; Table 4). Red thyme was also ble 3). The two lowest LD values in ascending order ϭ among the most toxic essential oils tested against the were associated with allyl isothiocyanate (LD50 0.3 ϭ ϭ ϭ ϭ Japanese beetle (LD50 0.14 mg; LD90 0.2 mg; mg; LD90 0.5 mg) and red thyme (LD50 0.5 mg; ϭ ϭ ϭ Table 1), oriental beetle (LD50 0.38 mg; LD90 0.66 LD90 0.9 mg). ϭ mg; Table 2), European chafer (LD50 0.48 mg; Acute toxicity against third instars of the northern ϭ LD90 0.89 mg; Table 3), and northern masked chafer masked chafer was associated with 6 of the 24 plant ϭ ϭ (LD50 0.38 mg; LD90 0.83 mg; Table 4). Inactive essential oils, namely, allyl isothiocyanate, cinnamon products were characterized by oils that did not ex- leaf, clove, garlic, red thyme, and wintergreen (Table

hibit any toxicity within 24 h after topical application 4). The two lowest LD50 andLD90 values were asso- ␮ ϭ ϭ of 1 l of concentrated, undiluted solution. At a dose ciated with red thyme (LD50 0.4 mg; LD90 0.8 mg)

Table 4. Plant essential oils exhibiting acute toxicity to larvae of the northern masked chafer

a Ϯ a a Product Range of doses tested Slope SE P LD50 95% CI LD90 95% CI Allyl Isothiocyanate 0.10Ð0.51 1.6 Ϯ 0.3 0.001 0.23a 0.19Ð0.28 0.51a 0.40Ð0.79 Cinnamon leaf 0.29Ð0.93 1.3 Ϯ 0.4 0.004 1.23c 0.87Ð6.33 3.44d 1.65Ð165.12 Clove 0.29Ð0.85 2.2 Ϯ 0.6 0.001 0.78c 0.68Ð0.96 1.39bcd 1.08Ð2.71 Garlic 0.21Ð0.96 1.1 Ϯ 0.3 0.001 1.06c 0.79Ð2.52 3.39cd 1.74Ð43.90 Red thyme 0.13Ð0.77 1.7 Ϯ 0.3 0.001 0.38b 0.32Ð0.46 0.83ab 0.66Ð1.27 Wintergreen 0.36Ð1.16 1.0 Ϯ 0.3 0.004 1.15c 0.86Ð2.93 4.21d 2.06Ð151.67

Table 5. Constituents of essential oils exhibiting acute toxicity against scarab larvae

Relative composition (%)a Compound Retention Allyl time isothiocyanante Cinnamaldehyde Cinnamon Cinnamon Citronella Clove Eugenol Garlic Peppermint Red Rosemary Wintergreen allyl bark leaf thyme

Diallyl sulÞde 4.41 Ñ Ñ Ñ Ñ Ñ Ñ Ñ 6.5 Ñ Ñ Ñ Ñ Allyl isothiocyanate 5.14 100 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ ␣-pinene 6.38 Ñ Ñ Ñ 0.4 Ñ Ñ Ñ Ñ 0.3 0.6 18.6 Ñ (ϩ)-camphene 6.93 Ñ Ñ Ñ 0.2 Ñ Ñ Ñ Ñ Ñ 0.6 8.3 Ñ ␤-pinene 7.92 Ñ Ñ Ñ 0.2 Ñ Ñ Ñ Ñ 0.5 0.1 4.8 Ñ Myrcene 8.47 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 0.9 0.7 Ñ 3-carene 9.04 Ñ Ñ 0.1 0.5 Ñ Ñ Ñ Ñ Ñ 0.4 0.2 Ñ ␣-terpinene 9.47 Ñ Ñ 0.1 0.1 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Cymene 9.82 Ñ Ñ 0.6 0.5 Ñ Ñ Ñ Ñ 0.6 21.5 1.8 Ñ Limonene 9.99 Ñ Ñ 0.7 0.4 2.2 Ñ Ñ Ñ 1.0 0.6 4.0 Ñ Eucalyptol 10.08 Ñ Ñ Ñ 0.1 Ñ Ñ Ñ Ñ 4.4 0.4 27.4 Ñ ␥-terpinene 11.27 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 1.7 0.4 Ñ Methyl allyl disulÞde 12.24 Ñ Ñ Ñ Ñ Ñ Ñ Ñ 12.9 Ñ Ñ Ñ Ñ Linalool 13.01 Ñ Ñ 2.1 1.3 Ñ Ñ Ñ Ñ Ñ 3.0 0.5 Ñ (E)-sabinene hydrate 13.12 Ñ Ñ Ñ Ñ 0.7 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Camphor 15.09 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 0.5 20.3 Ñ Isopulegol 15.12 Ñ Ñ Ñ Ñ 2.0 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Citronellal 15.46 Ñ Ñ Ñ Ñ 28.1 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Menthone 15.58 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 21.2 Ñ Ñ Ñ Isoborneol 15.87 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 1.5 Ñ Isomenthone 15.95 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 3.7 Ñ Ñ Ñ Neomenthol 16.19 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 4.3 Ñ Ñ Ñ Borneol 16.27 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 2.9 Ñ Menthol 16.68 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 49.3 Ñ Ñ Ñ Terpinen-4-ol 16.58 Ñ Ñ 0.7 0.1 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ ␣-terpineol 17.29 Ñ Ñ 0.8 0.2 Ñ Ñ Ñ Ñ Ñ 0.2 1.8 Ñ Methyl salicylate 17.43 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 100 Citronellol 18.81 Ñ Ñ Ñ Ñ 10.6 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Carvotanacetone 19.26 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 0.6 Ñ Ñ Ñ Geraniol 19.87 Ñ Ñ Ñ Ñ 16.9 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Piperitone 19.99 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 0.4 Ñ Ñ Ñ (E)-myrtanol 20.68 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 0.3 Ñ Ñ Ñ (E)-cinnamaldehyde 20.90 Ñ 100 65.1 0.3 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Isobornyl acetate 21.16 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 3.6 Ñ Safrole 21.43 Ñ Ñ Ñ 1.1 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Neomenthyl acetate 21.45 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 8.0 Ñ Ñ Ñ Thymol 21.83 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ 58.7 Ñ Ñ Diallyl disulÞde 21.95 Ñ Ñ Ñ Ñ Ñ Ñ Ñ 35.2 Ñ Ñ Ñ Ñ Citronellyl acetate 23.98 Ñ Ñ Ñ Ñ 3.6 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Eugenol 24.34 Ñ Ñ 8.6 84.0 Ñ 78.4 100 Ñ Ñ Ñ Ñ Ñ ␣-copaene 25.04 Ñ Ñ Ñ 0.5 0.3 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Geranyl acetate 25.21 Ñ Ñ Ñ Ñ 3.6 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Methyl allyl trisulÞde 25.23 Ñ Ñ Ñ Ñ Ñ Ñ Ñ 5.6 Ñ Ñ Ñ Ñ ␤-panasinsene 25.37 Ñ Ñ Ñ Ñ 4.5 Ñ Ñ Ñ Ñ Ñ Ñ Ñ (E)-caryophyllene 26.51 Ñ Ñ 3.1 2.8 0.2 6.0 Ñ Ñ 0.8 0.7 2.3 Ñ (Z)-prenyl limonene 27.97 Ñ Ñ 0.6 0.4 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Cinnamyl acetate 28.04 Ñ Ñ 14.1 0.9 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ ␣-humulene 28.07 Ñ Ñ Ñ Ñ Ñ 1.0 Ñ Ñ Ñ Ñ Ñ Ñ ␥-(E)-bisabolene 30.55 Ñ Ñ Ñ 0.1 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Nopsan-4-ol 30.80 Ñ Ñ Ñ 2.7 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Eugenol acetate 30.96 Ñ Ñ Ñ Ñ Ñ 13.1 Ñ Ñ Ñ Ñ Ñ Ñ Germacrene B 31.59 Ñ Ñ Ñ Ñ 7.7 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Diallyl trisulÞde 31.70 Ñ Ñ Ñ Ñ Ñ Ñ Ñ 20.0 Ñ Ñ Ñ Ñ Caryophyllene oxide 32.92 Ñ Ñ 0.6 0.5 0.2 1.4 Ñ Ñ 2.2 1.5 Ñ Ñ Benzyl benzoate 39.84 Ñ Ñ 1.2 2.5 Ñ Ñ Ñ Ñ Ñ Ñ Ñ Ñ Other compounds Ñ Ñ 1.7 0.3 19.4 0.2 Ñ 19.8 2.4 7.9 0.9 Ñ

a Values within a column represent the percentage composition of individual compounds for each essential oil product tested. Ñ ϭ Compound not detected.

ϭ ϭ and allyl isothiocyanate (LD50 0.6 mg; LD90 0.5 scarab larvae (Table 5). As previously noted, allyl mg) (Table 4). isothiocyanate and red thyme essential oils were con- The mean Ϯ SE weight of representative third in- sistently the two most toxic essential oils tested against stars was determined for the oriental beetle (0.15 Ϯ the four white grub species. Allyl isothiocyanate, an 0.01 g), Japanese beetle (0.21 Ϯ 0.02 g), European organosulfur compound, was conÞrmed by GC-MS to chafer (0.37 Ϯ 0.02 g), and northern masked chafer be the sole constituent of this oil. Allyl isothiocyanate (0.42 Ϯ 0.02 g). Japanese beetle and Oriental beetle was not detected in any of the remaining active oils. third instars weighed signiÞcantly less than European Red thyme essential oil was mainly comprised of thy- chafer and northern masked chafer third instars, but mol and cymene, while linalool, ␥-terpinene, and differences were not detected between the Japanese caryophyllene oxide were minor components. A num- beetle and Oriental beetle, or the European chafer and ber of compounds unidentiÞed as parts of our study northern masked chafer (F ϭ 45.71; df ϭ 3, 37; P Ͻ were also associated with red thyme essential oil. 0.0001). Cinnamaldehyde was also the only compound de- GC-MS Analyses. Collectively, a total of 55 com- tected by GC-MS within this oil. Cinnamon bark es- pounds were associated with the 12 plant essential oils sential oil was mainly comprised of (E)-cinnamalde- that exhibited acute toxicity to at least one species of hyde, cinnamyl acetate, and eugenol. Eugenol was also 164 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 1 a major component of cinnamon leaf oil, along with tically with the major constituents through a variety of (E)-caryophyllene, nopsan-4-ol, and benzyl benzoate mechanisms (Hummelbrunner and Isman 2001). as minor components. Clove essential oil was also Characterizing the key synergists within complex mix- mainly comprised of eugenol. Eugenol acetate and tures could thereby lead to the development of more (E)-caryophyllene were also major components of effective control and allow for lower rates to be used clove essential oil, along with caryophyllene oxide and (Hummelbrunner and Isman 2001). ␣-humulene as minor components. Previous studies involving commercially available Citronella was mainly comprised of citronellal, ge- botanical insecticides against scarab larvae found that raniol, citronellol, germacrene B, ␤-panasinsene, ge- blending extracts from diverse botanical sources can ranyl acetate, and citronellyl acetate. A number of be advantageous, but it did not necessarily ensure compounds unidentiÞed as parts of our study were adequate or enhanced biological activity (Ranger et also associated with citronella. al. 2009). Thus, the efÞcacy of essential oils from var- Garlic essential oil contained a variety of organo- ious botanical sources should be considered when sulfur compounds, with the major compounds being attempting to prepare optimal mixtures. For instance, tentatively identiÞed as diallyl disulÞde, diallyl trisul- the commercially available botanical insecticides Ar- Þde, methyl allyl disulÞde, diallyl sulÞde, and methyl morex (sesame, garlic, clove, rosemary, and white allyl trisulÞde. A number of compounds unidentiÞed pepper oils; Soil Technologies Inc., FairÞeld, IA) and as parts of our study were also associated with garlic Veggie Pharm (coconut, soybean, garlic, and pepper- essential oil. mint oils; Pharm Solutions, Port Townsend, WA) were Peppermint essential oil was mainly comprised of both prepared with plant essential oils from diverse menthol, menthone, neomenthyl acetate, eucalyp- botanical sources, but were the most and least active tol, and neomenthol. Minor components included formulations tested against these scarab larvae, re- isoborneol, caryophyllene oxide, and limonene. spectively (Ranger et al. 2009). Armorex has also been Methyl salicylate was the only compound associated characterized as including mustard essential oil (Bry- with wintergreen essential oil. Rosemary essential ant and Bite 2003, Copping 2004, CABI 2012), which oil was mainly comprised of eucalyptol, camphor, would provide a source of allyl isothiocyanate (Ji- ␣-pinene, and (ϩ)-camphene, while ␤-pinene, li- rovetz et al. 2002). Subsequent analysis determined monene, isobornyl acetate, and (E)-caryophyllene Armorex was mainly comprised of allyl isothiocyanate were minor components. (Ranger et al. 2011). Allyl isothiocyanate is an organosulfur compound produced by plants within the Brassicaceae, with over Discussion 20 different isothiocyanates having been identiÞed The aforementioned techniques were effectively from various Brassica species (Brown and Morra used to characterize the acute toxicity of a diverse 1996). Allyl isothiocyanate and related isothiocya- array of plant essential oils against third instars of the nates are well known to exhibit potent insecticidal Japanese beetle, oriental beetle, European chafer, activity (Williams et al. 1993, Borek et al. 1998). In and/or northern masked chafer. Six of the 24 plant particular, soil amended with Brassica juncea L. tissue essential oils were toxic to all four species, namely, was toxic to larvae of masked chafer beetles (Cyclo- allyl isothiocyanate, cinnamon leaf, clove, garlic, red cephala spp.) and allyl isothiocyanate levels were pos- thyme, and wintergreen. The following 13 oils were itively correlated with larval mortality (Noble et al. toxic to at least one species of scarab larvae: allyl 2002). Borek et al. (1995) tested the contact toxicity isothiocyanate, cinnamaldehyde, cinnamon bark, cin- of methyl, propyl, allyl, phenyl, benzyl, and 2- phe- namon leaf, citronella, clove, eugenol, fennel, garlic, nylethyl isothiocyanates to eggs of the black vine wee- peppermint, red thyme, rosemary, and wintergreen. vil, Otiorhynchus sulcatus (F.). All of the isothiocya- Allyl isothiocyanate and red thyme were consistently nates tested were toxic, but those containing aromatic among the two most toxic plant essential oils to all four moieties (i.e., phenyl, benzyl, and 2-phenylethyl) species of white grubs. were considerably more toxic than the aliphatic iso- The ability for quasi-synergism, potentiation, and thiocyanates (i.e., methyl, propyl, and allyl). synergism to occur among constituents of botanical Similar to allyl isothiocyanate, red thyme essential extracts has been well documented to result in en- oil was also among the most toxic essential oils to all hanced biological activity (Stark and Walter 1995, four species of scarab larvae. Thyme essential oil has Bekele and Hassanali 2001, Hummelbrunner and Is- exhibited insecticidal activity against the tobacco cut- man 2001, Scott et al. 2002, Miresmailli et al. 2006, worm Spodoptera litura (Isman et al. 2001) and a Isman et al. 2008). Quasi-synergism is characterized by mushroom-infesting ßy Lycoriella mali (Fitch) (Choi increased toxicity because of increased cuticular pen- et al. 2006). Essential oils from Thymus species mainly etration (Sun and Johnson 1972). Potentiation in- contain thymol, ␳-cymene, carvacrol, and aromatic volves an inactive compound increasing the effective- monoterpenes (Choi et al. 2006, Kordali et al. 2008). ness of an active compound, while synergism consists Our GC-MS analysis of red thyme essential oil also of an active compound increasing the effectiveness of detected thymol and cymene as major constituents, another active compound (Axelrad et al. 2002). Plant along with and a variety of other mono- and sesquit- chemical defenses usually occur as a blend of com- erpenes as minor components. Thymol has previously pounds and the minor constituents may act synergis- exhibited insecticidal activity against insects such as S. February 2013 RANGER ET AL.: TOXICITY OF ESSENTIAL OILS TO SCARAB LARVAE 165 litura (Isman et al. 2001), Drosophila melanogaster mitter in invertebrates (Orchard 1982, Roeder 1999, (Meigen) (Enan 2001), and S. oryzae (Rozman et al. Enan 2001, Price and Berry 2006). 2007). Isman et al. (2001) felt the oxygenated mono- Black pepper essential oil was not found to be terpenes thymol and carvacrol were most likely the acutely toxic to any of the four white grub species main compounds responsible for the toxicity of es- tested as part of our current study, but Scott et al. sential oils from Thymus and Satureia species to S. (2005) found a botanical formulation prepared by litura. extracting black pepper with ethyl acetate:water (1:1) Given the demonstrated toxicity of allyl isothiocya- was relatively toxic to larvae of the European chafer. nate and red thyme essential oils to scarab larvae, Our acute toxicity methods included topical applica- additional structure-activity studies are warranted. tion of black pepper essential oil in 1 ␮l doses to the Characterizing synergistic relationships among key white grubs, while Scott et al. (2005) treated soil plant essential oils and individual compounds against containing European chafer white grubs with 100 ml scarab larvae would also be useful. Such information of black pepper extracts at varying concentrations. will be critical for preparing mixtures from diverse Differences in the activity of black pepper between botanical sources to achieve optimal levels of activity our two studies may be attributed to differences in against these pests. However, interspeciÞc variability delivery methods, concentrations of active com- in the susceptibility of different scarab species to plant pounds in the oils, or tissue preparations (i.e., solvent essential oils may provide challenges in developing extraction vs. distillation). Thus, additional experi- optimal mixtures for multiple species and implement- ments are required to determine if certain essential ing their use for managing these pests in ornamental oils inactive as part of our acute toxicity experiments crops. exhibit promising activity when used a drench or im- Variability was documented in the susceptibility of mersion treatment. the four scarab species to the plant essential oils, with Because immersing root balls of nursery stock in 12, 11, 8, and 6 of the 24 essential oils tested being toxic chlorpyrifos or bifenthrin is performed by growers to to the oriental beetle, Japanese beetle, European cha- treat for contaminating white grubs before shipment fer, and northern masked chafer, respectively. Inter- (Mannion et al. 2000, National Plant Board 2011), a speciÞc differences in susceptibilities were also ob- similar protocol is currently being tested under Þeld served to the ecdysone agonist halofenozide, which conditions with plant-based essential oils (J.B.O., un- was highly toxic under Þeld and laboratory conditions published data). As noted by Mannion et al. (2000), to Japanese beetle larvae, less toxic to oriental beetle immersing root balls of nursery stock in solutions of larvae, and least toxic to the European chafer (Cowles conventional insecticides can be difÞcult, messy, and and Villani 1996, Cowles et al. 1999). Villani et al. potentially hazardous. Alternative products should (1988) also found interspeciÞc differences in suscep- therefore be examined and essential oil-based insec- tibilities among scarab larvae to Þve soil insecticides, with larvae of the European chafer generally being the ticides may help to alleviate these risks. Field exper- least susceptible species compared with the Japanese iments are underway to assess efÞcacy associated with beetle and oriental beetle. immersing root balls of Þeld grown nursery stock in InterspeciÞc differences in susceptibilities may be plant essential oil-based insecticides (J.B.O., unpub- attributed to differences in larval weights among the lished data). Because phytotoxicity issues can result white grub species. Notably, our current study found from immersing root balls in conventional and botan- third instars of the oriental beetle and Japanese beetle ically based insecticides (Mannion et al. 2000), addi- weighed signiÞcantly less than European chafer and tional studies are also needed to identify trees most northern masked chafer third instars. Differences in vulnerable to phytotoxicity. activities of detoxiÞcation enzymes among the white It should be noted that plant-based essential oils grub species may also explain the interspeciÞc vari- typically exhibit low environmental persistence and ability observed as part of our current study and pre- are mostly nontoxic to mammals, birds, and Þsh (Is- vious ones (Villani et al. 1988, Cowles and Villani 1996, man 2000, Isman 2006). Certain essential oils are even Cowles et al. 1999). Unlike the oriental beetle and exempt from toxicity data requirements as part of the European chafer, third instars of the Japanese beetle registration process with the U.S. Environmental Pro- and northern masked chafer enter a true diapause tection Agency (Isman 2000). However, pollinators (Cowles and Villani 1996, Grewal et al. 2001), which and natural enemies are vulnerable to poisoning by may have also provided a physiological basis for dif- essential oil based products, and exceptions do exist ferences in observed susceptibility. Additional studies regarding the mammalian toxicity of certain plant- accounting for differences in larval weights and com- based essential oils (Isman 2006, Maia and Moore paring activities of detoxiÞcation enzymes among the 2011). In particular, allyl isothiocyanate is a common four white grub species could provide useful insight additive in many foods and a naturally occurring ßavor into the basis for this phenomenon. Various essential ingredient in mustard, horseradish, and wasabi, but is oil compounds have been demonstrated to have a also a lachrymator, exhibits a potent ability to break speciÞc effect on octopamine receptors and thereby chromosomes, and is effective at unusually low levels block octopamine, which is a biogenic monoamine in transforming and mutating animal cells (Dunnick et that is structurally similar to noradrenaline and acts as al. 1982, Ishidate et al. 1988, Kasamaki et al. 1987, Ames a neurohormone, neuromodulator, and neurotrans- et al. 1990). Therefore, such impacts could pose lim- 166 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 1 itations to using allyl isothiocyanates and related com- Farnham, A. W. 1998. The mode of action of piperonyl pounds for developing botanically based insecticides. butoxide with reference to studying pesticide resistance, pp. 199Ð213. In D. G. Jones (ed.), Piperonyl Butoxide: The Insecticide Synergist. 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