Abstract: Background and Objectives: The continuous use of synthetic pesticides has increased the risk of ozone depletion, neurotoxicity, carcinogenicity, teratogenicity and mutagenic effects among non-target species and cross-resistance and multi-resistance in insects. These have created increased public awareness on human safety and possible environmental damage diverting attention towards other alternatives especially the use of plant products in stored-grain insect pest management. In the present study, essential oils of Allium sativum (A. sativum) and Aegle marmelos (A. marmelos) have been evaluated for their repellent, insecticidal, anti-ovipositional and acetylcholine esterase inhibitory activities against maize weevil, Sitophilus zeamais (S. zeamais). Materials and Methods: Garlic, Allium sativum and bel, Aegle marmelos essential oils have been isolated and evaluated for repellent, insecticidal, oviposition inhibitory and acetyl cholinesterase enzyme inhibitory activities in maize weevil, Sitophilus zeamais. One-way analysis of variance (ANOVA, p<0.01), correlation and linear regression analysis were used for data analysis. Results: In repellency assay, both essential oils showed repellent activity against S. zeamais adults. These essential oils caused toxicity in S. zeamais adults when applied by fumigation and contact methods. In fumigation toxicity assay, median lethal concentrations (LC50) recorded were 0.297 and 0.22 μL cm–3 air, 0.312 and 0.184 μL cm–3 air of A. sativum and A. marmelos oils after 24 and 48 h exposure of S. zeamais adults, respectively. In contact toxicity assay, median lethal concentrations (LC50) were found 0.208 and 0.116 μL cm–2 area and 0.227, 0.146 6,37 μL cm–2 area of A. sativum and A. marmelos oils after 24 and 48 h exposure of S. zeamais adults, respectively. Essential oils of A. sativum and A. marmelos oils were found to inhibit progeny production by inhibiting oviposition in S. zeamais adults when exposed to sub-lethal concentrations. Fumigation of S. zeamais with A. sativum and A. marmelos oils caused neurotoxicity by inhibiting acetylcholine esterase enzyme (AchE) activity. Conclusion: A. sativum and A. marmelos oils can be used as alternative in management of stored-grain insects.
INTRODUCTION
Insects damage grains under storage both qualitatively and quantitatively since the time immemorial. Several synthetic insecticides have been developed and formulated to protect stored grains from insect infestation. But the continuous use of these insecticides has been developing global problems like ozone depletion, carcinogenicity, teratogenicity and mutagenic effects among non-target species as well as cross-resistance and multi resistance among insect populations1-4. These public issues of human health and environmental damage have diverted attention towards other alternatives especially the use of phytochemicals in stored-grain insect pest management. Plant derived essential oils are complex mixtures of 20-60 compounds of different chemical nature in different concentrations5. Each essential oil is characterized by a specific essence due to two or three major compounds. The essential oil composition depends on plant parts used for extraction, extraction method, plant phenological stage, harvesting season, plant age, genotype of the plant, soil nature and environmental conditions6,7. These oils possess adulticidal, larvicidal and antifeedant activity, oviposition inhibitory activities, capacity to delay development and adult emergence8-11. These essential oil producing aromatic plants belong to families like Alliaceae, Apiaceae, Asteraceae, Cupressaceae, Lamiaceae, Lauraceae, Myrtaceae, Piperaceae, Poaceae, Rutaceae and Zingiberaceae. These essential oils have commercial application in food, cosmetic and pharmaceutical industries12.
Garlic, Allium sativum (Family: Alliaceae) is one of the most important ingredients of human food and Ayurvedic medicines since ancient time. Allicin, a key component of garlic reduces blood pressure by inhibiting angiotensin II and vasodilating effects13. Its various preparations have antidiabetic properties14. Its consumption protects human from cancer9. Garlic inhibits proliferation of atherosclerotic cells and other cell types as well as collagen synthesis and accumulation in the aorta15. Garlic preparations having allyl sulfides show antibacterial activity against both gram-negative and gram-positive bacteria like Bacillus, Clostridium, Escherichia, Klebsiella, Proteus, Salmonella, Staphylococcus and Streptococcus and antifungal activities against Candida albicans16,17. Diallyl sulfide and diallyl disulfide act as free radical scavangers by activating antioxidant enzymes like glutathione-s-transferase and catalase18. Alcoholic extract of garlic shows anthelmintic activity against Ascaris lumbricoldes.
Garlic bulbs contain a number of active compounds especially sulphur containing compounds which are responsible for the pharmacological activities. Steam distillation of garlic bulb produces essential oil containing diallyl, allyl methyl and dimethyl mono to hexa sulfide19. A. sativum essential oil extracted by steam distillation method have allyl methyl trisulfide (34.61%) and diallyl disulfide (31.65%) as major components20. Other components of low percentage are allyl methyl disulfide, diallyl sulfide, diallyl trisulfide and diallyl tetrasulfide. Douiri et al.21have reported that principal groups of components present in A. sativum essential oil are sulfur componunds represented mainly by trisulfides (57.4%) and disulfides (23.16). A. sativum essential oil contains 1,3-Dithiane, di-2-propenyl, 1-Propene,3,3’-thiobis, methyl 2-propenyl, 3-vinyl-1,3-dithiin, 2-vinyl-1,3-dithiin, di-2-propenyl, 3-vinyl-1,2 dithiin1-chloro-4-(1- ethoxy)-2-methylbut-2-ene, methyl 2-propenyl, diallyl disulfide, 3-vinyl-1,2 dithiin, methyl1-methyl-2-butenyl sulphide, octane 4-brom21. These components contribute to acaricidal22, antibacterial23, fungicidal24, insecticidal25, molluscicidal26, nematicidal27 and antiparasitic28 properties of garlic. Bel, Aegle marmelos (Family: Rutaceae) is a tree native to Northern India but also found throughout Ceylon, Burma, Bangladesh, Thailand and Indo-China29. It is traditionally used for treatment of various diseases such as dysentery, fever, diabetes, asthma, heart problems, ophthalmia, haemorrhoids, urinary problems, ulcer30,31. Moreover, the alcoholic leave extracts are used as antibacterial and antifungal activities32,33. The leaf extracts significantly inhibit the dermatophytic fungi like Trichophyton mentagrophytes, T. rubrum, Microsporum canis, M.gypseum and Epidermophyton floccosum34. The oil isolated from A. marmelos leaves of Cuba sources contains δ-cadinene (12.1%) and β-caryophyllene (10%) as major compounds35. On the other hand, the oil isolated from A. marmelos leaves is composed mainly of α-phellandrene (39.2%) and limonene (26.8%)36. The maize weevil, Sitophilus zeamais (Coleoptera: Curculionidae) is a major pest of maize in humid tropical areas around the world where maize is grown37. This species attacks both standing crops and stored cereal products including wheat, rice, sorghum, oats, barley, rye, buckwheat, peas and cotton seed38. The maize weevil also infests other types of stored, processed cereal products such as pasta, cassava and various coarse milled grains. In the present study, essential oils of A. sativum and A. marmelos have been evaluated for their repellent, insecticidal, anti-ovipositional and acetylcholine esterase inhibitory activities against maize weevil, S. zeamais.
MATERIALS AND METHODS
Essential oils: Dried A. sativum bulbs were purchased from the local market of Gorakhpur. Essential oil was isolated by crushing the bulb and hydrodistillation of crushed bulbs for 4 h in clevenger apparatus. Young and green leaves of A. marmelos were taken from the campus of M.G.P.G. College Gorakhpur (U.P.) and hydrodistilled for 4 h in clevenger apparatus. Essential oil was kept in Eppendorf tubes at 4°C till further use.
Insects: Maize weevil, S. zeamais was used to determine the insecticide nature of A. sativum and A. marmelos essential oils. The insects were reared on whole maize grain in the laboratory at 30±4°C, 75±5% RH and photoperiod of 10:14 (L:D) h.
Repellent activity: Repellency assay was performed in glass petri dishes (diameter 8.5 cm, height 1.2 cm). Test solutions of different dilutions (0.2, 0.4, 0.8 and 1.6% vol:vol) of A. sativum and A. marmelos were prepared in acetone. Whatman filter papers were cut into two halves and each test solution was applied to filter paper half as uniform as possible using micropipette. The other half of the filter paper was treated with acetone only. Essential oil treated and acetone treated halves were dried to evaporate the acetone completely. Both treated and untreated halves were then attached with cellophane tape in a manner so that seepage of the test samples from one half to other half can be avoided and placed at the bottom in each petri dish. Forty S. zeamais adults were released at the centre of the filter paper disc and the petri dish was covered and kept in dark. Six replicates were set for each concentration of essential oil. After 4 h of treatment, number of adults in treated and untreated halves was counted. Percent repellency (PR) was calculated using formula:
| C | = | Number of insects in the untreated halves |
| T | = | Number of insect in treated halves |
Preference index (PI) was calculated using the following formula:
PI values between -1.0 and -0.1 indicate repellant essential oil, - 0.1 to +0.1 neutral essential oil and + 0.1 to + 1.0 attractant essential oil.
Fumigant toxicity: Formulations of A. sativum and A. marmelos essential oils (10, 15, 20 and 25 μL mL–1) were made by using acetone as solvent. Ten adults taken from the laboratory culture were placed with 2 g of wheat grains in glass petri dish (diameter 8.5 cm, height 1.2 cm). Filter paper strip (2 cm diameter) was treated with essential oil formulations and left for 2 min for evaporation of acetone. Treated filter paper was pasted on the undercover of petri dish, air tightened with parafilm and kept in dark in conditions applied for rearing of insect. Six replicates were set for each concentration of essential oil and control. After 24 and 48 h of fumigation, mortality in adults was recorded.
Contact toxicity: Formulations of A. sativum and A. marmelos essential oils (10, 15, 20 and 25 μL mL–1 solvent) were made in acetone, applied on bottom surface of glass petri dish (diameter 8.5 cm, height 1.2 cm) and left for 2 min for evaporation of acetone. Ten adults taken from the laboratory culture were released at the centre of petri dish, covered and kept in dark in conditions applied for rearing of insect. After 24 and 48 h of fumigation, mortality was recorded.
Oviposition inhibitory effect: Ten S. zeamais adults of mixed sex were fumigated with sublethal concentrations viz. 40 and 80% of 24 h LC50 and 48 h LC50 of A. sativum and A. marmelos essential oils for 24 and 48 h, respectively and reared on wheat grain in a 250 mL plastic box for 10 days. After 45 days, adults were discarded and number of F1 progeny was counted. Six replicates were set for each concentration of essential oils and control.
Acetylcholine esterase enzyme (AChE) activity determination: S. zeamais adults were fumigated with two sublethal concentrations viz. 40 and 80% of 24 h LC50 of A. sativum and A. marmelos oils as in fumigant toxicity assay. After 24 h of fumigation, adults were used for determination of acetylcholine esterase enzyme activity39. Fumigated insects were homogenized in phosphate buffer saline (50 mM, pH 8) and centrifuged. Supernatant was used as the acetylcholine esterase source. To 0.1 mL of enzyme source, added 0.1 mL substrate acetyl thiocholine iodide (ATChI) (0.5 mM), 0.05 mL chromogenic reagent 5,5-Dithiobis 2-Nitrobenzoic acid (DTNB) (0.33 mM) and 1.45 mL phosphate buffer (50 mM, pH 8). Acetylcholine esterase enzyme activity was determined by measuring changes in the optical density at 412 nm by incubating the reaction mixture for 3 min at 25°C. Enzyme activity was expressed as mmol of ‘SH’ hydrolysed min–1 mg–1 protein.
Data analysis: Median lethal concentration (LC50) was calculated using POLO programme40. One-way analysis of variance (ANOVA, p<0.01), correlation and linear regression analysis were conducted to define concentration-response relationship41.
RESULTS
Repellent activity: Maximum repellency was observed at 0.8% concentrations of A. sativum and A. marmelos essential oils (Table 1). Maximum preference index (PI) was found at 0.8% concentrations of A. sativum and A. marmelos essential oils (Table 1). A. sativum and A. marmelos essential oils showed significant (F = 198.58 for A. sativum and F = 201.64 for A. marmelos p<0.01) repellency against S. zeamais adults.
Fumigant toxicity: Fumigation of A. sativum and A. marmelos essential oils caused toxicity by vapour action. Median lethal concentrations (LC50) were 0.297, 0.22 μL cm–3 and 0.312, 0.184 μL cm–3 air for A. sativum and A. marmelos essential oils after 24 and 48 h of exposure, respectively (Table 2). Regression analysis showed concentration-dependent mortality in S. zeamais adults against A. sativum and A. marmelos essential oils (F = 257.33 for 24 h and 314.67 for 48 h for A. sativum essential oil and F = 213.64 for 24 h and 257.84 for 48 h for A. marmelos essential oil, p<0.01) (Table 3). The index of significancy of potency estimation, p-value indicates that the mean value is within the limits of all probabilities (p<0.1, 0.5 and 0.01) as it is less than 0.5. Values of t-ratio greater than 1.6 indicate that the regression is significant. Values of heterogeneity factor less than 1.0 denotes that model fits the data adequate.
Contact toxicity: A. sativum and A. marmelos essential oils caused contact toxicity in S. zeamais adults. Median lethal concentrations (LC50) were 0.208 and 0.116 μL cm–2, and 0.227 and 0.146 μL cm–2 area for A. sativum and A. marmelos essential oils after 24 and 48 h of exposure, respectively (Table 2). Regression analysis showed concentration-dependent mortality in S. zeamais adults (F = 249.34 for 24 h and 279.87 for 48 h for A. sativum essential oil and F = 216.39 for 24 h and 226.51 for 48 h for A. marmelos essential oil, p<0.01) (Table 3).
Oviposition inhibition: Fumigation of S. zeamais adults with A. sativum and A. marmelos essential oils significantly reduced oviposition potential. Maximum reduction in oviposition was 43.9 and 49.63% and 22.6 and 33.27% of the control when S. zeamais adults were fumigated with 80% of 24 h LC50 and 48 h LC50 of A. sativum and A. marmelos essential oils, respectively (p<0.01, Table 4).
Acetylcholine esterase enzyme (AChE) activity: Fumigation of A. sativum and A. marmelos essential oils against S. zeamais adults significantly reduced AChE activity.
| Table 1: | Repellency of A. sativum and A. marmelos essential oils against S. zeamais adults |
*Percent repellency (PR) was calculated as: PR = (C-T)/(C+T) ×100, Where C = Number of insects in the untreated halves and T = Number of insect in treated halves,
**Preference index (PI) was calculated as: PI = (percentage of insects in treated halves - percentage of insects in untreated halves)/(percentage of insects in treated halves+percentage of insects in untreated halves). PI value between -1.0 to -0.1 indicates repellant essential oil, -0.1 to +0.1 neutral essential oil and +0.1 to +1.0 attractant essential oil | |
| Table 2: | Fumigant and contact toxicity of A. sativum and A. marmelos essential oils against S. zeamais adults |
| aμL cm–3 for fumigant and μL cm–2 for contact toxicity | |
| Table 3: | Regression analysis of fumigant and contact toxicity of A. sativum and A. marmelos essential oils against S. zeamais adults |
| Table 4: | Oviposition inhibitory activities of A. sativum and A. marmelos essential oils in S. zeamais |
**F-values significant (p<0.01), Values in parentheses indicate per cent change with respect to control taken as 100% | |
| Table 5: | Effect of A. sativum and A. marmelos essential oils on acetylcholine esterase enzyme (AChE) activity in S. zeamais adults |
*Enzyme activity was expressed as mol of ‘SH’ hydrolysed min–1mg–1 protein, **F-values significant (p<0.01), Values in parentheses indicate percent change with respect to control taken as 100% |
|
Maximum reduction in AChE activity was 28.25 and 39.02% of control when S. zeamais adults were fumigated with 80% of 24 h LC50 of A. sativum and A. marmelos essential oils, respectively (F = 253.66 for A. sativum and F = 207.7 for A. marmelos essential oils, p<0.01) (Table 5).
DISCUSSION
Essential oils of plant origin have found its wide applicability in stored grain insect pest management programmes9-11,42-46. Besides essential oils, its individual components have also been known for its effectiveness against insect pests. Linalool and linalyl acetate show toxicity in rice weevils47. Menthol, methonene, limonene, α-pipene, β-pipene, β-caryophyllene and linalool show lethality and AChE inhibitory activities44,48. In present study, toxic, oviposition and AChE inhibitory activities of A. sativum and A. marmelos essential oils were studied in S. zeamais. Both essential oils repelled S. zeamais adults. A. sativum and A. marmelos essential oils caused lethality in S. zeamais adults. Reduction in progeny production in S. zeamais was observed when treated with A. sativum and A. marmelos oils which may reduce damage by the insect. Similar results have been observed in Callosobruchus chinensis and Tribolium castaneum49-51. Fumigation with A. sativum and A. marmelos essential oils significantly reduced AChE activity in S. zeamais adults. Essential oil monoterpenes have been reported to interfere with AChE activity in S. oryzae and T. castaneum50,52. The rapid action of essential oils in insects indicates is neurotoxic mode of action. These interference with the neuromodulator octopamine53 or GABA-gated chloride channels54. Several essential oil and its components act on the octopaminergic system of insects. Octopamine is a neurotransmitter, neurohormone and circulating neurohormone-neuromodulator and its disruption results in total breakdown of the nervous system55. Thus, the octopaminergic system of insects represents a target for insect control. Low molecular weight terpenoids are too lipophilic to be soluble in the haemolymph after crossing the cuticle and the proposed route of entry is tracheae56. Most insecticides bind to receptor proteins in the insect and interrupt normal neurotransmission leading to paralysis and death. Recent evidence suggests that low molecular weight terpenoids with different structures may also bind to target sites on receptors that modulate nervous activity55.
CONCLUSION
Use of essential oils as an alternative in insect pest management programmes is a sustainable alternative as they can be obtained from nature. Essential oils can be used as contact toxicity, fumigant toxicity, repellent, oviposition inhibitory and developmental inhibitory agents. These act on various levels in the insects so possibility of generating resistance is low. Thus, A. sativum and A. marmelos essential oils can be used as an alternative of synthetic insecticides in the stored-grain insect pest management.
SIGNIFICANCE STATEMENTS
This study determined the insecticidal and acetylcholine esterase inhibitory properties of A. sativum and A. marmelos essential oils in maize weevil, Sitophilus zeamais. The findings of this study helps in the preparation of essential oil based formulations for the management of stored grain insect pests.