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J. stored Prod. Res. Vol. 34, No. 4, pp. 243±249, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0022-474X(98)00005-8 0022-474X/98 $19.00 + 0.00
Insecticidal Activity of Monoterpenes Against Rhyzopertha dominica (F.) and Tribolium castaneum (Herbst) H. T. PRATES1*, J. P. SANTOS1, J. M. WAQUIL1, J. D. FABRIS1, A. B. OLIVEIRA2, J. E. FOSTER3 EMBRAPA/Corn and Sorghum Research Center, Sete Lagoas, MG, Brazil, 2 Department of Chemistry, ICEx, UFMG, Belo Horizonte, MG, Brazil and 3Department of Entomology, UNL, Lincoln, NE, USA
1
(Accepted 29 January 1998)
AbstractÐSecondary metabolites play an important role in plant resistance to insects. The monocyclic monoterpenes 1,8-cineole (eucalyptol) and R-(+)-limonene have been considered economically important in this context. Cineole is a component of dierent species of Eucalyptus spp. and limonene is a constituent of Citrus spp. essential oils. These substances were bioassayed to determine possible fumigant, contact, and ingestion activity against Rhyzopertha dominica (F.), lesser grain borer, and Tribolium castaneum (Herbst), red ¯our beetle, which are important pests of stored grain. It was possible to show that both compounds are insecticidal against the two insects, mainly in the contact and/or ingestion action. # 1998 Elsevier Science Ltd. All rights reserved Key wordsÐnatural product, R-(+)-limonene, 1,8-cineole, stored-grain insects
INTRODUCTION
Rhyzopertha dominica (F.) and Tribolium castaneum (Herbst) cause large economic losses of stored wheat grain. The conventional way to control these insect pests has been with the use of insecticides, either directly applied to grains or by gas fumigation. However, concerns have arisen about the persistence of insecticide residues in grains which can be harmful to mammals. Some secondary metabolites of plants play an important role in plant±insect interaction, and are commonly responsible for plant resistance to insects (Mann, 1987). Some essential oils have acute toxicity, repellent action, feeding inhibition, or harmful eects on the reproductive system of insects. Additionally, secondary metabolites from higher plants have recently been used as pesticides or models for new synthetic pesticides as, for instance, toxaphene (insecticide and herbicide) and cinmethylin (herbicide) (Duke et al., 1988). These chemicals were developed from plant-derived products such as terpenoids that can be found in the essential oil secreted by the glandular trichomes of Artemisia (Compositae) or closely related genera. Pine oil, a by-product of the sulphate wood pulping industry, has the monoterpene a-terpineol among its major constituents. This substance protects living trees for up to 10 m by repelling three bark beetle *To whom all communications should be addressed: EMBRAPA/CNPMS, C.P. 151, 35701-970-Sete Lagoas, MG, Brazil. Fax: +55 31 7791088. E-mail:
[email protected] 243
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species (Nijholt et al., 1981). Alfaro et al. (1984) reported that pine oil is a feeding deterrent to Pissodes strobi (Peck) (Coleoptera: Curculionidae). Monoterpenes found in essential oil from dierent plants have insecticidal activity against termites (Baker and Walmsley, 1982). Coats et al. (1991) described fragrant volatile oils containing monoterpenes or their related compounds: alcohols, ketones, aldehydes, carboxylic acids, and oxides, such as d-limonene, b-myrcene, a-terpineol, linalool, and pulegone. These substances have toxicity to house ¯y (Musca domestica L.), German cockroach (Blatella germanica L.), rice weevil (Sitophilus oryzae L.), red ¯our beetle (Tribolium castaneum Herbst) and Southern corn rootworm (Diabrotica unidecimpunctata howardi Barber) (Rice and Coats, 1994a,b). A large reduction in the population of ticks (Boophilus microplus Canestrini) caused by the essential oil of the grass Melinis minuti¯ora (de Beauvois) was reported by Ayacardi et al. (1984). More recently, the GC/MS analysis of the M. minuti¯ora essential oil revealed a complex mixture of substances which included the monoterpene 1,8-cineole as a major active component against ticks (Prates, 1992; Prates et al., 1993). R-(+)-limonene (p-mentha-1,8-diene) and 1,8-cineole (1,3,3-trimethyl-2-oxabicycle[2.2.2.]octane) are natural products from plants with low toxicity to mammals. Limonene is a major component of Citrus spp. essential oils, and is also used as an ingredient of soaps, perfumes, and food additives (Karr and Coats, 1988). Cineole (or eucalyptol) is a constituent occurring in variable amounts in the essential oil of Eucalyptus spp. leaves (Gibson et al., 1991). The main purpose of this work was to test cineole and limonene as ecologically safe alternative insecticides, which could also be economically produced and used against R. dominica and T. castaneum. MATERIALS AND METHODS
Bioassays to determine the insecticidal activity of cineole and limonene consisted of tests for fumigation, contact, and contact and/or ingestion actions. In all assays, 20 individual adults of R. dominica and T. castaneum, obtained from cultures maintained in the laboratory were used in each of three replicate assays. A control test was prepared the same way but no impregnating substance was used. Insect mortality was observed by using a stereomicroscope when needed. All tests were carried out at room temperature (26 2 18C) in the Entomology Laboratory for Stored Grain Pest Control of EMBRAPA/Milho e Sorgo at Sete Lagoas, MG, Brazil. All substances under test in this work were analytical grade, supplied by Aldrich. The fumigation test was based on the procedure described by Karr and Coats (1988), with some modi®cations. A 2 liter tightly closed glass container was used as a fumigation chamber. Sixty-three milligrams of limonene and 59 mg of cineole were separately placed in a watch glass to evaporate, in such a way that the vapour was con®ned in the chamber. The atmosphere inside the fumigation chamber was homogenized by stirring the inner atmosphere with a magnetic stirrer. Insects were exposed to the gas by putting them inside a wire screen cage hanging from a metal support. The fumigant activity was measured 24 h after exposing the insects to the gas. The contact eect was evaluated by treating a 7 cm diameter (38.5 cm2) Whatman No. 1 ®lter paper with the substances dissolved in acetone. The treated ®lter paper was placed on top of a three-pin-point support for 2 min to allow the acetone to evaporate before putting it inside a Petri dish. Glass rings (5.0 cm internal diameter and 2.5 cm high) treated on the inside with a ®ne powder (caulin) to prevent the insects from climbing the glass ring wall were used to force them to stay on the ®lter paper. The glass rings were also covered with a ®ne cloth to prevent the insects from ¯ying away. The ®lter papers were treated by evenly spreading doses of the substances diluted in acetone at the ratios (substance : acetone) of 10:0, 8:2, 6:4, 4:6, 2:8, 1:9, 0:10. One dose of limonene or one of cineole means 21.00 mg and 19.67 mg, respectively. Also, it means the application of limonene at concentrations of 5.50, 4.40, 3.30, 2.20, 1.10, 0.55 and 0 mg/cm2 and cineole at concentrations of 5.10, 4.08, 3.06, 2.04, 1.02, 0.51 and 0 mg/cm2. The contact and/or ingestion activity was evaluated by infesting samples of treated wheat grains of 20 g, stored in a 30 ml glass vial, using the same doses as above. The applied concentrations of limonene were 10.50, 8.40, 6.30, 4.20, 2.10, 1.05 and 0 mg/g and of cineole were 9.80, 7.84, 5.88, 3.80, 1.96, 0.98 and 0 mg/g. The substances were allowed to penetrate the insects,
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Table 1. Sigmoidal (Boltzman) curve ®tted parameters, and LC (lethal concentration) for 50, 90 and 99% insect population mortality Cineole R. dominica (Fig. 1) Parameters A2 W x0 y1 LC50 (x1) y2 LC90 (x2) y3 LC99 (x3)
Limonene
T. castaneum (Fig. 2)
R. dominica (Fig. 3)
T. castaneum (Fig. 4)
Filter paper Wheat grain Filter paper Wheat grain Filter paper* Wheat grain Filter paper Wheat grain 92.6 0.6 3.1 50 3.2 90 5.1 Ð Ð
100 0.4 0.7 50 0.7 Ð Ð 99 2.4
100 0.3 2.7 50 2.7 90.0 3.4 Ð Ð
100.0 0.0 1 50 1 Ð Ð 99 1.4
Ð Ð Ð Ð Ð Ð Ð Ð Ð
99.5 0.2 1.7 50 1.7 Ð Ð 99 2.9
98.1 0.7 2.3 50 2.3 90 4 Ð Ð
100.2 0.3 0.4 50 0.4 Ð Ð 99 1.8
*Not enough data to run the curve ®tting. A1=lowest eciency (mortality, %) value, in the present case equal to 0%; A2=highest eciency (mortality, %) value, up to 100%; x0=center of Boltzman distribution; W = width of Boltzman distribution; x = independent variable, observed doses (1 dose of cineole = 19.67 mg, 1 dose of limonene = 21.00 mg); y = dependent variable, expected mortality (%); LC50=lethal concentration of tested substance to kill 50% population; LC90=lethal concentration of tested substance to kill 90% population; LC99=lethal concentration of tested substance to kill 99% population
through contact with the cuticle (palps) and probably also with at least the legs, or other body parts, as the insects moved through the grain, and by ingesting treated particles of grain. The insects could, independently or simultaneously, be aected by these two mechanisms. Observations on the survival rate of insects were made 24 h after contact with the treated ®lter papers or grains. Data were transformed using the Abbott (1925) formula and least-squares ®tted to a Boltzman sigmoidal model of equation (1): y
A1 ÿ A2 A2 1 e
xÿx0 =w
1
where A1=lowest eciency (mortality, %) value (in the present case A1=0); A2=highest eciency (mortality, %) value (maximum, 100%); x0=center of the Boltzman distribution; w = width of the Boltzman distribution; x = independent variable (dose of cineole or limonene); y = dependent variable (insect mortality, %). For a given expected value of y, the dose can be drawn from equation (1). The eective concentration (LC) to kill 50 and 90% of the insect population in the ®lter paper test, and to kill 50 (LC50) and 99% (LC99) of the insects in the contact and/or ingestion activity test were calculated by assuming y = 50 and y = 99, respectively, in equation (2), provided that A2 and x0 are known ®tted parameters (Table 1). y :
2 x x0 w ln A2 ÿ y
RESULTS AND DISCUSSION
Fumigant activity The fumigant activity of cineole and limonene, evaluated by the eciency (mortality, %) to kill R. dominica and T. castaneum are presented in Table 2. The analysis of variance revealed signi®cant dierences between the treatments. Cineole was more eective against R. dominica than against T. castaneum. Also, cineole was slightly more eective than limonene against R. dominica. Limonene had about the same eect on mortality (e95%, Table 2) of both insects, but it was more eective against T. castaneum than cineole (58.3%). Although we did not look either at penetration of substances into grain bulk or adsorption of chemicals by wheat, these results are good evidence that the toxicity of the evaporated substance is sucient to knock down and kill all insects, in a period of time as short as 24 h. The present results agree with
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H. T. Prates et al. Table 2. Fumigant activity of 1,8-cineole and R-(+)-limonene, against two insect pests of stored grains Insect mortality* (%)
Insect species
Cineole
R. dominica T. castaneum
100.0 Aa$ 58.3 Ba
Limonene 96.7Aa 94.9 Ab
*Mortality calculated according to Abbott (1925). $Capital letters for columns and small ones for the rows compare the statistical signi®cance of the means at 5% probability
those reported by Karr and Coats (1988), in the sense that limonene has high toxic fumigant activity against cockroachs and rice weevils. Contact activity with treated ®lter paper The contact eciency of cineole against R. dominica and T. castaneum is illustrated in Figs 1 and 2, respectively. Results with limonene are shown in Figs 3 and 4. The LC for 50, 90 and 99% insect population mortality are shown in Table 1. Cineole applied in dierent proportions to acetone was slightly more toxic to T. castaneum than to R. dominica. The LC50 and LC90 for cineole against R. dominica estimated from equation (2) was 1.64 mg/cm2 and 2.61 mg/cm2, respectively. For T. castaneum the LC50 and LC90 was 2.7 and 3.4 doses (1 dose = 19.67 mg), or 1.38 mg/cm2 and 1.74 mg/cm2, respectively. LC50 and LC90 for limonene against T. castaneum were found to be 2.3 and 4.0 doses (1 dose = 21.00 mg), or 1.25 mg/cm2 and 2.18 mg/cm2, respectively. Corresponding values for limonene against R. dominica were not assessed, as the mortality curve did not reach the upper plateau (Fig. 3). This means that, quantitatively, limonene has a much higher LC50 and LC90 to R. dominica than cineole, and, as a general trend, cineole showed higher potential to control both insects than did limonene. Contact and/or ingestion activity with treated wheat grains The eciency of the contact and/or ingestion activity of cineole is illustrated in Figs 1 and 2, and that of limonene is shown in Figs 3 and 4. The observed eectiveness of cineole was equiv-
Fig. 1. Mortality of R. dominica caused by dierent doses (1 dose = 19.67 mg) of 1,8-cineole.
Insecticidal activity of monoterpenes
247
Fig. 2. Mortality of T. castaneum caused by dierent doses (1 dose = 19.67 mg) of 1,8-cineole.
alent for both insects. However, the limonene was more eective for T. castaneum than for R. dominica. The combined eect caused maximum mortality of nearly 100%. LC50 and LC99 of cineole against R. dominica were 0.7 and 2.4 doses, corresponding to the concentrations of 0.69 mg/g and 236 mg/g, respectively. For T. castaneum the LC50 and LC99 were 1.0 and 1.4 doses (0.98 mg/g and 1.38 mg/g, respectively). The corresponding results of
Fig. 3. Mortality of R. dominica caused by dierent doses (1 dose = 21.00 mg) of R(+)-limonene.
248
H. T. Prates et al.
Fig. 4. Mortality of T. castaneum caused by dierent doses (1 dose = 21.00 mg) of R(+)-limonene.
LC50 and LC99 for limonene against R. dominica were 1.7 and 1.4 doses (1.78 mg/g and 1.47 mg/g). Limonene was more eective against T. castaneum and the LC50 and LC99 were, respectively, 0.4 dose and 1.8 doses (0.42 mg/g and 1.89 mg/g). The mortality on ®lter paper was slightly lower than that on treated grains. A possible reason for this result would be that insects may take up the toxic substance either through the cuticle or by eating (oral intake) when exposed to the treated grains. Limonene and cineole showed signi®cant contact and/or ingestion and fumigant action. The glass vials used to test for contact and/or ingestion activity with wheat grain in open air were slender, which could cause a slow release of the compound, which would be retained for a sucient time to form a fumigant atmosphere, possibly causing mortality by this means, even before the contact and/or ingestion action had taken place. If so, the observed mortality cannot be credited to the contact and/or ingestion eect only. Based on the present results it can be concluded that: (1) the monoterpenes cineole and limonene, which are components of essential oils found in leaves of Eucalyptus globulus LabillardieÁre, E. camaldulensis Dehnhardt and E. cameroni Blakely & McKie, and in peel of Citrus aurantium L. and Citrus limonum Risso have signi®cant insecticidal eect, being lethal to both R. dominica and T. castaneum; (2) these substances are toxic by penetrating the insect body via the (a) the respiratory system (fumigant eect), (b) the cuticle (contact eect) or (c) the digestive system (ingestion eect); (3) the limonene was more eective for T. castaneum than for R. dominica and (4) the test with treated wheat grain was somewhat more sensitive than that with the ®lter paper. REFERENCES Abbott W. S. (1925) A method of computing the eectiveness of an insecticide. Journal of Economic Entomology 18, 265±266. Alfaro R. I., Borden J. H., Harris L. J., Nijholt W. W. and McMullen L. H. (1984) Pine oil, a feeding deterrent for the white pine weevil, Pissodes strobi (Coleoptera: Curculionidae). Canadian Entomologist 116, 41±44. Ayacardi E., Benevides E., Garcia O., Mateus G., Henao F. and Zuluaga F. N. (1984) Boophilus microplus tick burdens on grazing cattle in Colombia. Tropical Animal Health and Production 16, 78±84.
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