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Research ArticleReceived: 1 February 2011 Revised: 27 March 2011 Accepted: 28 March 2011 Published online in Wiley Online Library: 5 May 2011

(wileyonlinelibrary.com) DOI 10.1002/jsfa.4453

Efficacy of essential oils of Lippia alba (Mill.)N.E. Brown and Callistemon lanceolatus (Sm.)Sweet and their major constituents onmortality, oviposition and feeding behaviourof pulse beetle, Callosobruchus chinensis L.Ravindra Shukla, Priyanka Singh, Bhanu Prakash, Ashok Kumar,Prashant Kumar Mishra and Nawal Kishore Dubey∗

Abstract

BACKGROUND: Pulse beetle, Callosobruchus chinensis L., is the most destructive insect pest of pulses under storage in Asia andAfrica. Keeping in view the negative impacts of synthetic insecticides and the demands of botanical pesticides, the presentinvestigation explores the repellents, antifeedants, ovicidal, larvicidal and pupaecidal activity of two plant essential oils (EOs)and their major components, geranial and 1,8-cineole, when applied as fumigants for the management of the pulse beetle.

RESULTS: EO of Callistemon lanceolatus (Sm.) Sweet caused 100% repellency of pulse beetle in a Y-shaped olfactometer at adose of 150 µL, while Lippia alba (Mill.) N.E. Brown EO and 1,8-cineole showed 76 and 74.7% repellency at the same dose. At0.1 µL mL−1, both the oils and 1,8-cineole provided 100% insect mortality. The EO of C. lanceolatus was recorded as the mosteffective fumigant, showing 96.03% oviposition deterrency and 100% antifeedant activity at 0.1 µL mL−1. The LD50 of L. alba(11049.2 µL kg−1) and C. lanceolatus (14 626.3 µL kg−1) exhibited their favourable safety profiles when recorded on mice.

CONCLUSION: EOs of L. alba and C. lanceolatus exhibited significant biological activity on the mortality and reproductivebehaviour of pulse beetle. Based on their high LD50 values, the oils could be safely recommended as non-mammalian toxicfumigants in management strategies for pulse beetle.c© 2011 Society of Chemical Industry

Keywords: antifeedant; Callistemon lanceolatus; Callosobruchus chinensis; deterrency; essential oil; Lippia alba; repellency; oviposition

INTRODUCTIONLegumes are among the richest food sources of proteins andamino acids for human and animal nutrition.1 They are of specialsignificance to people in developing countries who can hardlyafford animal protein in adequate quantities.2 The full yieldpotential of pulses is seldom realised due to insect attack. Thougha number of insect pests of different groups cause varying degreeof losses to pulses, the pulse beetle, Callosobruchus chinensis L.(Coleoptera: Bruchidae), is one of the most destructive insectswhich alone is responsible for an average of 32–64% loss understorage in different parts of Asia and Africa.3,4 This bruchid beetlefrequently accounts for the greatest weight loss of legumes afterharvest due to the tunnelling activity of the larvae in the seed. Whensuch seeds are stored, the insect continues feeding, eventuallyemerges as an adult, and causes secondary infestation which,mostly, results in total destruction within 3–4 months.5

Indeed, the use of chemical insecticides like carbamates andorganophosphates is currently the most widely adopted methodfor pulse–legume protection against bruchid beetles.6 However,abusive utilisation of chemicals develops insect resistance andendangers the environment, which is polluted, and consumers

ingest treated grains that contain large quantities of pesticideresidues.7 This fact, combined with consumer demand for residue-free foods, prompted researchers to evaluate other alternativereduced-risk control methods for pulse–legume protection.

Recently, there has been growing interest in research concern-ing the possible use of plant extracts as alternatives to syntheticinsecticides. Plant essential oils (EOs) are among the best knownsubstances tested against insects. These compounds may act asfumigants,8 contact insecticides,9 repellents,10 and antifeedants,11

and may affect some biological parameters such as growth rate,life span and reproduction.12

Lippia alba (Mill.) N.E. Brown, an aromatic shrub (family:Verbenaceae), is a potential source of EO in India. Callistemonlanceolatus (Sm.) Sweet (family: Myrtaceae) commonly known

∗ Correspondence to: Nawal Kishore Dubey, Laboratory of Herbal Pesticides,Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi-221005, India. E-mail: nkdubey2@rediffmail.com

Laboratory of Herbal Pesticides, Centre of Advanced Study in Botany, BanarasHindu University, Varanasi-221005, India

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as ‘bottle-brush’ is commonly available in Indian gardens as anornamental tree.13

The present study was undertaken to investigate the effect ofEO vapours of the aforesaid plants and their prime components,geranial and 1,8-cineole, on C. chinensis adults, their egg hatchabil-ity and subsequent post-embryonic mortality. Antifeedant activityand non-mammalian toxicity of EOs were also determined.

MATERIALS AND METHODSRearing of test insectsAdults of C. chinensis were obtained from naturally infestedchickpea seeds from a local market in Varanasi, India. The beetleswere reared on clean and uninfested chickpea (Cicer arietinum L.var. Radha). Two hundred adult insects were released in 500 gchickpea seeds in a Kilner jar capped with muslin cloth to ensureventilation. The jar was kept at a controlled temperature (30±2 ◦C)and relative humidity (70 ± 5%). After 48 h, the adults wereremoved and the jar was left for 25 days to obtain adult beetles ofthe same age for the experiment.

Plant materials and extraction of essential oilsL. alba, growing wild in the premises, and C. lanceolatus, plantedin the botanical garden of Banaras Hindu University, Varanasi,were identified by morphological features with the help of theFlora of BHU Campus13 and the voucher specimens (LHP/Ver-21/2008 and LHP/Myr/13/2008) were deposited at the Laboratoryof Herbal Pesticides, Banaras Hindu University, Varanasi. Geranial(monoterpene aldehyde) and 1,8-cineole (monoterpene ether)were procured from Genuine Chemical Co., Mumbai, India.

Fresh plant leaves (200 g) of L. alba and C. lanceolatus werecollected in June and subjected to hydrodistillation (4 h) usinga Clevenger-type apparatus.14 The yield (mL kg−1) of EOs wasaveraged over four experiments and calculated on the basisof plant material fresh weight. The yield of EOs was 1.6 and7.6 mL kg−1 for L. alba and C. lanceolatus, respectively.

Repellent activity of essential oils and their constituentsagainst C. chinensisThe in vitro insect repellent activity of L. alba and C. lanceolatus EOsand their major constituents, geranial and 1,8-cineole, respectively,were studied against C. chinensis. The behaviour of C. chinensiswas observed in a glass Y- shaped olfactometer with a commonarm of 24 cm length, and two arms A and B, each of 25 cm length.The repellency experiments were carried out in a climatic roomat 30 ± 2 ◦C and a light intensity of 37.3 lux. Unsexed bruchidinsects (3 to 7 days old) were used in the experiments. A cottonswab (1 cm diameter) was soaked separately in 10, 50, 100 and150 µL of the EOs and kept in the experimental arm. A cottonswab soaked in an equal amount of distilled water was kept in thecontrol arm. The apparatus was then attached to a suction pumpto create a vacuum. Thirty test insects were introduced separatelyinto the base of the stem of the Y-arm of the olfactometer. After30 min the number of insects in the experimental and control armwas counted. The repellency (%) was calculated according to theequation:

repellency = C − E

T× 100

where C is the number of insects in the control arm, E is the numberof insects in the experimental arm, and T is the total number ofinsects released.

Fumigant toxicity of essential oils/constituents on adultC. chinensisThe fumigant activities of EOs and their constituents againstadults of C. chinensis were determined via impregnated paperassays following the method of Park and colleagues,15 with somemodifications. Plastic jars of 500 mL capacity with screw lids wereused as exposure chambers. Samples of 6.25, 12.5, 25 and 50 µL ofeach essential oil/compound were dissolved separately in 0.25 mLacetone and applied to a circular filter paper (Whatman No. 1, 3 cmdiameter).The treated filter paper discs were then introduced intothe plastic jars (500 mL capacity) to achieve final concentrations of0.012, 0.025, 0.050 and 0.100 µL mL−1, respectively, with respectto volume of the jars. Controls received only 25 mL acetone. Afterallowing the acetone to evaporate by keeping the jars in a fumechamber for 10 min at room temperature, the filter paper wasattached to the inner surface of the screw lid of the jar usingadhesive tape. Twenty bruchid beetles (2 to 4 days old) werereleased into each jar containing 50 g chickpea seeds. Doses ofeach compound were used for exposure periods of 12 and 24 h.After exposure to the oil compound vapours, the insects wereseparated from the treatment jars and numbers of dead and aliveinsects were counted. Knocked-down beetles were regarded asdead if they did not show any response after exposure to gentleheat from a 60 W lamp. Only active insects were transferred tofresh 15 g chickpea seeds and their further mortality was recorded.Final mortality was calculated after 72 h following Abbot’s formulaof percent corrected mortality:16

P = T − C

100 − C× 100

where P is the % corrected mortality, T is the % kill in the treatmentgroup, and C is the % kill in the control.

Effect of essential oils/constituents on the reproduction ofC. chinensisOviposition deterrent activityAliquots of 6.25, 12.5, 25 and 50 µL of each test compound(oils/components) dissolved in 0.25 mL acetone were applied sep-arately to circular Whatman No.1 filter papers (3 cm diameter) togenerate concentrations of 0.012, 0.025, 0.050 and 0.100 µL mL−1,respectively, in plastic jars (500 mL). A similar amount of distilledwater was used instead of the oils to dissolve in 0.25 mL acetonefor the controls. After allowing the acetone to evaporate, the filterpaper was attached to the inner surface of the lid of the jar usingadhesive tape.

Females of C. chinensis are characterised by a larger abdomen.Ten male and 10 female bruchid beetles were released into eachjar containing 50 g fresh chickpea seeds. An exposure periodof 24 h was used for each treatment. After exposure to the oilvapours, the insects were separated from the treatment jars andchickpea seeds were placed in clean and untreated jars for 3 daysfor the prominent appearance of eggs laid on seeds. The totalnumber of eggs was counted on the treated and control seeds.The % deterrency of oviposition was calculated according to theequation:17

deterrency = NC − NT

NC× 100

where NC is the number of eggs laid on control seeds, and NT isthe number of eggs laid on treated seeds.

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Effects on eggs and reproductive stages of C. chinensisTo study the influence of oil vapours on larvae and pupae insidethe seeds, the procedure according to Ketoh and colleagues wasadopted.18 Adult male and female insects (100) were collectedfrom the stock culture and were put together in Petri dishes for4 days in order to ensure mating. Thereafter, the insects wereplaced in a 1000 mL plastic jar containing 250 g chickpea seedsand left for 24 h for oviposition. The seeds with laid eggs werethen collected and placed in Petri dishes until the eggs reachedan appropriate age for the experiment. The tests were carried outon different lots of chickpea seeds containing unhatched eggs(1 day old) and their post-embryonic developed stages. The post-embryonic stages were obtained 6–10 days after oviposition forLI –LII larvae, 10–14 days for LIII –LIV larvae and 16–20 days forpupae.19

One hundred chickpea seeds each with one egg or oneindividual developing inside the seed, were sampled from a cultureof 1, 6, 10 and 16 days old. They were exposed separately to 0.050and 0.100 µL mL−1 EOs and their constituents in closed plasticcontainers, for 48 h following the impregnated paper assay.15 Thetreated seeds of each lot were then removed and transferred toclean plastic containers. These were incubated at 30 ± 2 ◦C untiladult emergence. Ovicidal, larvicidal or pupaecidal activities ofdifferent treatments were calculated in the form of deterrency tof1 adult emergence as follows:

deterrency = NC − NT

NC× 100

where NC is the number of adults that emerged from the controlseeds, and NT is the number of adults that emerged from thetreated seeds.

Antifeedant activity of essential oils/constituents againstC. chinensisThe experiment was performed following the method given byKumar et al.20 Two doses (200 and 300 µL) of the treatmentswere soaked separately in a cotton swab and stick on the innerside of the lid of plastic jar (2 L) to create fumigation of 0.10and 0.15 µL mL−1 concentration. Two kilograms of uninfestedchickpea seeds (variety, Radha; moisture content, 11–13%) wereplaced inside the fumigated jars. Fifty adult bruchids werereleased into each treatment and the jars were made airtight.Chickpea seeds with 50 bruchids and without any treatmentwere prepared as a control set. After 24 months of storage in atemperature–humidity control cabinet (32 ± 2 ◦C and 80 ± 5%relative humidity), the efficacy of EOs was determined on the basisof % grain damage and % weight loss of treated and control seeds.The legume damage was assessed by observing emergence holeson the surface of the legume. The weight loss (%) of the sampleswas calculated on a fresh weight basis:

weight loss = WI − W

WI× 100

where WI is weight of the pulses before the experiment and W isthe weight of the pulses at the end of the experiment.

The antifeedant action of the treatments as fumigants wasobserved by calculating the feeding deterrent index (FDI, %):21

FDI = C − T

T× 100

where, C is the weight loss of control seeds, and T is the weightloss of treated seeds.

Determination of LD50 of essential oils against miceThe safety limits for C. lanceolatus and L. alba EOs were determinedthrough LD50 experiments on mice of the same sex and same age(Mus musculus L., average weight 50.0 g, age 3 months). Micewere procured from the Institute of Medical Sciences, BanarasHindu University, Varanasi, and were kept in a laboratory undercontrolled environmental conditions (30 ± 2 ◦C) for a week toacclimatise them before performing the LD50 experiments. LD50

represents the lethal dose of oil per unit weight which kills 50%of the population of test animals.22 A stock solution of distilledwater and Tween-80 (2 : 1) was prepared. For the toxicity trials, theanimals were divided randomly into groups of 12 animals in eachgroup. Different amounts (0.2–3.2 mL) of each EO were addedseparately in requisite amounts of stock solution to obtain a finaldose of 4 mL. Each animal was given 4 mL of the solution, orally,with the help of a fine syringe and catherator. Equal amountsof distilled water in place of oil was mixed in stock solutionand administered orally to mice of the control sets. The mice ofcontrol and treatment sets were kept in separate mouse cages.The mortality of animals was observed up to 14 days and the LD50

values for the EOs were calculated by probit analysis.23

Statistical analysisTests for repellency, fumigant toxicity, oviposition deterrency,ovicidal activity and antifeedant activity were performed intriplicate and data presented are mean ± SE. The means werecompared by one-way ANOVA and Tukey’s multiple comparisontests using software SPSS, version 11.5.

RESULTSThe repellent behaviour of C. chinensis increased with the increasein the doses of EOs or their constituents (Table 1). One hundred %repellency of C. chinensis was observed at 150 µL of C. lanceolatusoil. However, at a similar dose, Lippia oil showed 76% repellencywhile 1,8-cineole and geranial exhibited 74.7% and 63% repellantactivity. C. lanceolatus oil caused >50% bruchid repellency at only50 µL.

Table 2 shows the fumigant toxicity of EOs and their majorcompounds on adults of C. chinensis. Bruchids were found to besusceptible to all four treatments. At a dose of 0.1 µL mL−1, all thetreatments provided 100% mortality, except geranial which caused82.5% mortality, after 24 h. C. lanceolatus oil caused absolutemortality at 0.025, 0.05 and 0.1 µL mL−1. 1,8-Cineole was found to

Table 1. Repellent activity (%) of essential oils and their constituents(at various doses) against C. chinensis

Dose of essential oil/constituent (µL)

Treatment 10 50 100 150

L. alba EO 20.0 ± 2.5c 34.3 ± 3.1bc 51.3 ± 2.8cd 76.0 ± 3.3b

C. lanceolatusEO

35.3 ± 1.7a 50.7 ± 4.3a 86.0 ± 4.3a 100 ± 0.0a

Geranial 19.0 ± 2.9c 27.3 ± 2.7c 48.3 ± 3.7cd 63.0 ± 2.5c

1,8-cineole 23.3 ± 4.3abc 35.3 ± 2.8bc 55.7 ± 2.7cd 74.7 ± 3.7b

% values are mean (n = 3) ± SE. The means followed by letter (a) arenot significantly different from each other according to ANOVA andTukey’s comparison tests.

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Table 2. Fumigant toxicity of essential oils/compounds againstadults of C. chinensis

% Mortality

Treatment Dose (µL mL−1) 12 h∗ 24 h∗

L. alba EO 0.012 17.8 ± 2.8d 19.7 ± 3.2d

0.025 31.3 ± 4.2c 42.7 ± 4.0c

0.050 40.0 ± 2.0b 58.6 ± 2.6b

0.100 69.2 ± 3.3a 100 ± 0.0a

C. lanceolatus EO 0.012 34.5 ± 2.5d 91.7 ± 2.6b

0.025 54.3 ± 4.2c 100 ± 0.0a

0.050 86.6 ± 1.7b 100 ± 0.0a

0.100 100 ± 0.0a 100 ± 0.0a

Geranial 0.012 16.5 ± 2.1d 18.9 ± 2.3d

0.025 21.7 ± 3.1c 37.9 ± 3.4c

0.050 37.1 ± 2.7b 58.0 ± 1.2b

0.100 61.6 ± 3.7a 82.5 ± 0.0a

1,8-cineole 0.012 16.2 ± 1.7d 18.2 ± 1.7c

0.025 22.7 ± 3.3c 40.0 ± 2.0b

0.050 61.9 ± 4.1b 100 ± 0.0a

0.100 100 ± 0.0a 100 ± 0.0a

∗ Exposure time of fumigation.% values are mean (n = 3) ± SE. The means followed by letter (a) arenot significantly different from each other according to ANOVA andTukey’s comparison tests.

be more effective compared to Lippia oil and geranial as it alsocaused 100% mortality at 0.05 and 0.1 µL mL−1.

The % deterrency of oviposition increased with increasing con-centrations of all the treatments (Table 3). Significant differences(P < 0.05) in the number of laid eggs were found betweentreated and untreated seeds and among different treatments. At0.1 µL mL−1, the essential oil of C. lanceolatus allowed the bruchidsto lay only 12 eggs as compared to the control (302 eggs) andproved to be the most effective treatments, with 96% deterrency.Lippia oil (66.8%) and 1,8-cineole (65.8%) also showed remarkableactivity.

The toxic effect of the two oils and their major constituentson eggs of different ages is illustrated in Table 4. In all cases, eggmortality increased when the concentration was doubled. Theearly embryonic stages (eggs and neonate larvae) were foundmore susceptible than older stages (mature larvae and pupae).The ovicidal activity of oils/constituents was found between 34

and 76%, whereas larvicidal activity ranged between 10 and 77%.A reduced range of toxicity (6–39%) of the oils/components wasrecorded for pupae. C. lanceolatus EO was the most effectivetreatment, causing 75.93% ovicidal, 77.18% larvicidal (LI/LII),49.06% (LIII/LIV) and 39.68% pupaecidal activity.

Table 5 shows the deterrent effects of EOs and their constituentson the feeding behaviour of C. chinensis. All the treatmentsshowed significantly better results than the controls. A 100%infestation of bruchid beetles was observed in control seedswhere an emergence hole was found in almost every seed. Onthe other hand, only 0.03% damage occurred in seeds treatedwith 1,8-cineole. The EO of L. alba and its major component,geranial, showed comparatively poor results, with 7.3% and 8.3%,respectively, seed damage found in their treatments. A reductionof 8.2% was recorded in fresh weight of control chickpea, whereasless than 0.7% weight loss was calculated in all the treatments.In C. lanceolatus oil treatment, 0% seed damage and weightloss and thus 100% antifeedant activity was recorded at bothconcentrations used. At 0.15 µL mL−1, both the EOs showed betteractivity in terms of % FDI than their major components.

The LD50 of L. alba and C. lanceolatus EOs was calculated as 11049.2 and 14 626.3 µL kg−1 body weight, respectively.

DISCUSSIONThe present results show that the EOs of L. alba and C. lanceolatusand their two pure major components have varying degrees ofinsecticidal activity against C. chinensis adults and larvae. Theiractivity increased with increasing doses and exposure times.These significantly protected the chickpea seeds from insectdamage.

A number of reports are available which indicate that EO activityis the result of their inherent biologically active components andtheir different modes of action, either synergistic or antagonistic.22

Hence, the major component of each essential oil, along with therespective EO, was tested in the present investigation to correlatethe efficacy of the EO either by the synergism between the allexisting components or only due to the major component.24,25

Based on previous investigations, the major components of L.alba and C. lanceolatus EOs, geranial (22.2%)26 and 1,8-cineole(60.75%),27 were selected for comparative study with EO todetermine their impact on toxicity. Both the EOs caused morerepellency and mortality than their major components, whichis contrary to most of the earlier reports where the primecomponents showed better activity than the EO. 1,8-Cineole andterpinen-4-ol were found to be more toxic against Sitophilus

Table 3. Oviposition deterrent effects∗ of essential oils/compounds (at various doses) against of C. chinensis

Dose (µL mL−1)

Treatment 0.012 0.025 0.050 0.100

L. alba EO 14.76 (258.0 ± 5.0)bc 19.72 (243.0 ± 5.7)b 41.42 (177.3 ± 5.8)b 66.86 (100.3 ± 6.1)c

C. lanceolatus EO 41.19 (178.0 ± 7.0)d 67.85 (97.3 ± 3.70)e 86.12 (42.0 ± 5.0)e 96.03 (12.0 ± 2.3)e

Geranial 7.49 (280.0 ± 5.0)ab 14.10 (260.0 ± 4.9)b 34.58 (198.7 ± 4.0)b 56.29 (132.7 ± 10.3)b

1,8-cineole 18.85 (246.7 ± 4.7)c 35.81 (194.3 ± 5.2)c 57.05 (130.0 ± 4.0)c 65.87 (103.3 ± 4.3)b

Control (302.7 ± 7.8)a (302.7 ± 7.8)a (302.7 ± 7.8)a (302.7 ± 7.8)a

∗ % Oviposition deterrency was measured as the mean number of eggs laid ± SE.% values are mean (n = 3). The means followed by the same letter in the same column are not significantly different from each other according toANOVA and Tukey’s comparison tests.

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Table 4. Effects of essential oils/constituents on eggs and reproductive stages of C. chinensis

Ovicidal/larvicidal/pupaecidal activity (%)

Treatment Dose (µL mL−1) Eggs LI/LII Larvae LIII/LIV Larvae Pupae

L. alba EO 0.05 34.30 (21.0 ± 1.0)b 31.25 (22.0 ± 1.0)b 15.62 (27.0 ± 1.0)c 15.62 (27.0 ± 0.7)c

0.10 49.06 (16.3 ± 0.3)de 50.93 (15.7 ± 1.8)ef 22.81 (24.7 ± 0.3)e 21.87 (25.0 ± 0.5)d

C. lanceolatus EO 0.05 72.81 (8.7 ± 0.7)g 69.68 (9.7 ± 1.2)h 42.81 (18.3 ± 1.3)h 39.68 (19.3 ± 2.3)f

0.10 75.93 (7.7 ± 0.7)h 77.18 (7.3 ± 0.3)i 49.06 (16.3 ± 1.7)i 39.68 (19.3 ± 0.3)f

Geranial 0.05 38.43 (19.7 ± 1.3)c 35.31 (20.7 ± 0.3)c 10.31 (28.7 ± 1.3)b 6.25 (30.0 ± 1.1)b

0.10 38.43 (19.7 ± 0.3)c 41.56 (18.7 ± 1.3)d 19.68 (25.7 ± 0.3)de 14.68 (27.3 ± 1.3)c

1,8-cineole 0.05 50.93 (15.7 ± 1.8)e 53.12 (15.0 ± 1.5)f 27.18 (23.3 ± 0.7)f 16.56 (26.7 ± 0.7)c

0.10 60.31 (12.7 ± 0.7)f 59.37 (13.0 ± 0.5)g 32.18 (21.7 ± 0.3)g 28.12 (23.0 ± 2.0)e

Control 0.0 – (32.0 ± 2.3)a – (32.0 ± 2.3)a – (32.0 ± 2.3)a – (32.0 ± 2.3)a

Values in parentheses are mean number (n = 3) of f1 emerged ± SE.The means followed by letter (a) are not significantly different from each other according to ANOVA and Tukey’s comparison tests.

Table 5. Feeding deterrence of essential oils/constituents against C. chinensis on chickpea seeds

Concentration of 0.10 µL mL−1 Concentration of 0.15 µL mL−1

Treatment % seed damage % weight loss % FDI % seed damage % weight loss % FDI

L. alba EO 7.3 ± 0.60b 0.41 ± 0.01bc 95.00 2.8 ± 0.40c 0.26 ± 0.02b 96.82

C. lanceolatus EO 0.0 ± 0.0c 0.0 ± 0.0c 100 0.0 ± 0.0d 0.0 ± 0.0b 100

Geranial 8.3 ± 0.20b 0.74 ± 0.06b 90.97 4.7 ± 0.62b 0.33 ± 0.10b 95.97

1,8-cineole 0.18 ± 0.01c 0.07 ± 0.01c 99.14 0.03 ± 0.01d 0.006 ± 0.003b 99.92

Control 100 ± 0.0a 8.2 ± 0.21a – 100 ± 0.0a 8.2 ± 0.21a –

% values for seed damage and weight loss are mean (n = 3) ± SE.The means followed by same letter in the same column are not significantly different from each other according to ANOVA and Tukey’s comparisontests.

granarius adults, in comparison to Artemesia absinthium oil.28

Hence, the toxicity of L. alba and C. lanceolatus can not beattributed to their major components only. Other minor and/ortrace compound(s) may cause possible synergistic effect ofcompound(s) in the oils.

Plant extracts contain metabolic compounds that show repel-lent, antifeedant, sterilisation and toxic effects in insects.9 Thereare many reviews dealing with the use of EOs against insect pestsof stored products,29 while other reviews deal only with monoter-penoids, the chief components of EOs.30 Despite these reports,there is a paucity of information on plant products that show fu-migant action against insect eggs in stored products, and againstintracotyledonal immature stages such as larvae and pupae. Dur-ing fumigant toxicity, adult bruchids were susceptible to all the fourtreatments. At 0.1 µL mL−1, both the EOs and the monoterpeneether (1,8-cineole) provided 100% mortality within 24 h.

Little is known about the physiological actions of EOs oninsects, but treatments with various EOs or their monoterpeneconstituents cause symptoms that suggest a neurotoxic modeof action.31 It interrupts the functioning of a neuromodulator,octopamine, which results in total breakdown of the nervoussystem in insects. Therefore, the octopaminergic system of insectsrepresents a bio-rational target for insect control. The lack ofoctopamine receptors in vertebrates likely accounts for theprofound mammalian selectivity of EOs as insecticides.31 Other

studies on EOs indicate inhibition of acetylcholinesterase enzymeactivity as the major site of action.32

At 0.1 µL mL−1, the EO of C. lanceolatus allowed the bruchidsto lay only 12 eggs as compared to untreated control seeds with302 eggs and thus showed 96% oviposition deterrency. Two typesof oviposition inhibition occurred: (1) the females died beforelaying their eggs, and (2) the failure of live females to lay manyeggs. Regnault-Roger and Hamraoui (1995) observed that reducedfecundity was attributed to a complex of mortality and deterrency,but it was not possible to separate the contribution of each factorto the final result. Terpenoids in EOs are considered as semio-chemicals which alter the behaviour of insect pests34 and thusmight be responsible for strong oviposition deterrency in presentinvestigation.

In the present study, the early embryonic stages, such aseggs (34–77%) and neonate larvae (31–77%), were found tobe more susceptible than older stages, such as mature larvae(10–49%) and pupae (6–39%). The results obtained are similarto those observed for eggs, larvae and pupae of Acanthoscelidesobtectus.35 – 37

Ovicidal activity of EOs is only apparent when the targetnervous system begins to develop.38 Alternatively, changes inthe permeability of the chorion and/or vitelline membrane mayoccur during embryogenesis and may facilitate the diffusion of EOvapours into older eggs so that vital physiological and biochemicalprocesses are affected.39 These factors have been considered to

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play a dominant role in the susceptibility of eggs of many insectspecies to different ovicides.38

Some of the purified terpenoid constituents of EOs aremoderately toxic to mammals, but, with few exceptions, theoils themselves or products based on oils are mostly non-toxicto mammals, birds and fish.40,41 The high LD50 of L. alba andC. lanceolatus EOs strengthens their non-mammalian toxicity.

Furthermore, unlike conventional insecticides that are based ona single active ingredient, plant-derived EOs comprise an array ofchemical compounds which act concertedly on both behaviouraland physiological processes. Thus the chances of pests developingresistance to such substances are less likely.42

On the basis of the unique properties of the two EOs,with repellent activity, fumigant toxicity, oviposition deterrentactivity, ovicidal activity, larvicidal activity, antifeedant activityand favourable safety limits, we can therefore recommend thepotential exploitation of EOs of L. alba and C. lanceolatus asfumigants in insect management strategies for grains/pulses.Theraw materials for extraction of the essential oils are easily availablesince the plants of L. alba and C. lanceolatus grow luxuriantly inIndia and the the yield of EOs was also high (1.6 and 7.6 mL kg−1,respectively). The essential oils may be extracted in bulk byusing hydrodistillation. Hence, the application of essential oilsas fumigants in management of pulse beetle and other storageinsect pests would be economical. However, further studies areneeded to increase our understanding of the effective use of thesetechnologies on a wide range of pests in commercial stores.

ACKNOWLEDGEMENTSThis work was financially supported by Council of Scientific andIndustrial Research (CSIR), New Delhi, India

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