Abstract: Background: The uncontrolled use of synthetic chemicals is a great hazard for the environment and consumers. Methodology: In the present study, essential oil from leaves of Aegle marmelos was isolated by hydro-distillation and tested their insecticidal activity against Tribolium castaneum (Herbst). Results: The results showed that the essential oil of A. marmelos have fumigant toxicity, oviposition and developmental inhibitory activity against T. castaneum. The percentage mortality increased with increasing exposure time and concentration. The median lethal concentration (LC50) of A. marmelos essential oil at 48 h was 14.172 and 17.752 FL against larvae and adults of T. castaneum, respectively. The essential oil significantly reduced oviposition (F3,20 = 304.734) in adults and also reduced pupation (F3,20 = 137.442) and adult emergence (F3,20 = 225.619) in larvae when fumigated with sub-lethal concentration. The percent grains infection was reduced 83.66% at 60% of sub-lethal concentration of 24 h LC50. Fumigation of insect with sub-lethal concentration of A. marmelos essential oil inhibited AChE activity. Reduction in AChE activity was 81.48 and 54.32% of the control, after 24 h of fumigation with sub-lethal concentration. Conclusion: In conclusion, this essential oil probably induces toxicity in insect by inhibiting AChE activity.
INTRODUCTION
Stored-grain insect pests have been damaging economy by infesting agricultural products. The red flour beetle; Tribolium castaneum (Herbst) is one of the major pests of stored grains and grain products in the tropics. Synthetic chemical fumigants are commonly used to control stored product pests throughout the world, but these products caused adverse affect on the environment such as disturbances of the environment, increasing costs of application, pest resurgence, pest resistance to pesticides and lethal effects on non-target organisms in addition to direct toxicity to users and are hazardous to human health (Lee et al., 2004; Tapondjou et al., 2002). Therefore, considerable amount of investigations have been carried out in the last three decades to find alternative control methods of stored-grain insect pests (Morimoto et al., 2002; Park et al., 2002). Plant essential oils and their components have been shown to possess potential for development as new fumigants and they may have advantages over conventional fumigants in terms of low mammalian toxicity, rapid degradation and local availability (Isman, 2008). Essential oils derived from more than 75 plant species have been evaluated for fumigant toxicity against stored product insects so far (Rajendran and Sriranjini, 2008). Aegle marmelos (L.) Correa (Rutaceae), commonly known as Bael, is a sacred tree for Hindu religon, native to northern India, but is found widely throughout the Indian peninsula and in Ceylon, Burma, Thailand and Indo-China (Bailey, 1963). All parts of the tree namely, root, leaf, trunk, fruit and seed are used for treatment of many different diseases. The constituents of A. marmelos are used in heart diseases (Kakiuchi et al., 1991), inflammatory and wound healing (Udupa et al., 1994). Leaves of A. marmelos have been reported as hypoglycemic effect (Santhoshkumari and Devi, 1990; Sharma et al., 1996). The essential oil from the leaves of A. marmelos is known to exhibited antifungal properties (Rana et al., 1997). The repellent effect of A. marmelos essential oil has already determined against stored-grain pest T. castaneum and Sitophilus oryzae (Mishra and Tripathi, 2011).
Keeping these facts in mind, in the present investigation, essential oil of A. marmelos was tested as a fumigant to protect stored grain from insect pest Tribolium castaneum. The effect of A. marmelos oil applications on toxicity, chronic toxicity, oviposition and adult emergence of T. castaneum has also been determined.
MATERIALS AND METHODS
Plant collection and isolation of essential oil: Leaves of A. marmelos were collected from the local area of Gorakhpur districts of Uttar Pradesh, India. The specimens were identified and authenticated by the expert in the Department of Botany, D. D. U. Gorakhpur University, Gorakhpur. The leaves was dried in absence of sun light at room temperature (30±5°C) and grounded using a mixer. The essential oil was extracted by the method of Mishra et al. (2013).
Insect rearing: Larvae and adults of T. castaneum were obtained from Entomological Laboratory, D.D.U. Gorakhpur University, Gorakhpur laboratory stock cultures maintained in an incubator at 30±2°C, 75±5% RH and at photoperiod of 10:14 (L:D) without exposure to any insecticides. Tribolium castaneum adults were reared on mixed grain and flour of Triticum aestivum (L.) at 12-13% moisture content. Ten days old unsexed adults and 4th instars larvae of T. castaneum were used to determine the insecticidal property of A. marmelos essential oil.
Toxicity
Larval and adults toxicity: A serial dilution of four concentrations of essential oil (15, 20, 25 and 30 μL) was prepared in acetone (100 μL) and treated with larvae and adults of T. castaneum by the method of Mishra et al. (2012).
Oviposition inhibitory activity of essential oil: Oviposition inhibitory activity of A. marmelos essential oil was tested against T. castaneum by fumigation method of Mishra et al. (2014).
Determination of developmental inhibitory activity of essential oil: Developmental inhibitory activity of A. marmelos essential oil was tested against 4th instars larvae of T. castaneum. Twenty larvae of T. castaneum were places in 2 g of wheat flour and grains in glass petri dish. A paper strip (2 cm2) treated with 40, 60 and 80% sub-lethal concentration of 24 h LC50 of essential oil in acetone was pasted on the inner surface of the cover of each petri dish. Another paper strip (2 cm2) was treated with absolute acetone only was used as control. All the closed petri dishes were kept in dark and six replicates were set for each concentration. After fumigation of glass petri dishes for 72 h as was done in larvicidal assay and then the treated larvae were transferred to fresh wheat flour in other petri dish. Number of survived larvae, transformed pupae from treated larvae and emerged adults from transformed pupae were recorded. Six replicates were set for each concentration.
Feeding deterrence activity: Chronic activity of A. marmelos essential oil was tested against adults of T. castaneum by contraction method with two sub-lethal concentrations of essential oil prepared in acetone (30 and 60% of 24 h LC50) (Mishra and Tripathi, 2011; Mishra et al., 2012) by using micropipette. The treated filter papers were dried to evaporate the solvent completely. The treated filter paper placed at the bottom in glass petri dish (height 15 mm×radius 45 mm). Ten adults taken from the laboratory culture (1-2 week old) were placed with 5 g of wheat flour and grain in petri dish. Flour and grains were spread uniformly along the whole surface of the petri dish. All the closed petri dishes were kept in dark and six replicates were set for each concentration. After 30 days, percent grains damage by stored-grain insect pests were recorded. The grain damage was determined by counting feeding injuries and emergence holes on the surface of the grains. Feeding deterrent was calculated using the feeding deterrent index following farmula:
where, C and T is the weight loss in the controls and in the fumigated sets, respectively.
Acetylcholinesterase enzyme (AChE) activity determination: Acetylcholinesterase activity of T. castaneum was measured by the method of Ellman et al. (1961). Adults of T. castaneum were fumigated with two sub-lethal concentrations (40 and 80% of 24 h LC50) of A. marmelos essential oil as done in toxicity assay. Six replicates were set up for each concentration. Control animals were taken as such without any treatment. After 24 h of fumigation, adults were utilized for the measurement of AChE activity in the treated as well as the control group. Enzyme activity has been expressed as μmol "SH" hydrolyzed min–1 mg–1 protein.
Data analysis: Lethal concentration (LC50), lower and upper confidence limits (LCL-UCL), slope value, t-ratio, g-value, heterogeneity factor and chi-square value were calculated using computer software of Robertson et al. (2007). Correlation and linear regression analysis were conducted to define all dose-response relationships (Sokal and Rohlf, 1973). Analysis of variance was performed to test the equality of regression coefficient (Sokal and Rohlf, 1973). Student t-test was performed to test the significant changes in enzyme activity with respect to control (Armitage et al., 2001).
RESULTS
Fumigant toxicity of the essential oil from leaves of A. marmelos against larvae and adults of T. castaneum gradually increased with increasing exposure time and concentration (p<0.01). The essential oil from A. marmelos killed the larvae and adults of T. castaneum by fumigation action. The medium Lethal Concentration (LC50) of larvae of T. castaneum was 16.870 μL at 24 h and 14.172 μL at 48 h whereas, 19.656 and 17.752 μL against adults of T. castaneum at 24 and 48 h, respectively (Table 1).
The t-ratio values were greater than 1.96, indicating a significant regression of each dose response line. The heterogeneity factor was less than 1.0, demonstrating that the log-dose-probit lines are within the 95% confidence limits and thus the model fittest the data. Value of g less than 0.5 indicated that mean was within the limit at all probability levels of 90, 95 and 95%.
The regression analysis showed a concentration dependent significant correlation of the oil with larval mortality of T. castaneum (F3,20 = 15.432, p<0.01), (F3,20 = 22.667, p<0.01) and adults of T. castaneum (F3,20 = 24.583, p<0.01), (F3,20 = 26.958, p<0.01) at 24 and 48 h exposure, respectively. The oviposition was reduced to 60.96, 49.43 and 40.15% when adults were fumigated with 40, 60 and 80% sub lethal concentration of essential oil, respectively (Table 2). The reduction in oviposition potential of T. castaneum was significant (F3,20 = 304.734) when fumigated with sub lethal concentration of A. marmelos. Pupation was reduced to 78.88, 62.49 and 45.18% when 4th instars larvae were fumigated with 40, 60 and 80% sub lethal concentrations of essential oil, respectively. Similarly, adult emergence was reduced to 66.67, 46.48 and 35.33% when 4th in stars larvae were fumigated with different sub lethal concentration of essential oil (Table 3). The decrease in pupation (F3,20 = 137.442) and adult emergence (F3,20 = 225.619) was increased significantly with increased the concentration of essential oil.
The percent grains infection was reduced by A. marmelos essential oils against T. castaneum was 51.82 and 83.66% at 30 and 60% of sub-lethal concentration of 24 h LC50, respectively (Table 4). The regression analysis indicated that percent damage grain reduction by adults of T. castaneum by essential oil showed a significant negative correlation with concentration when fumigated with A. marmelos essential oil (F = 659.63) (Table 5).
Fumigation of T. castaneum adults with two sub lethal concentration 40 and 80% of 24 h LC50 of A. marmelos essential oil significantly reduced AChE activity.
| Table 1: | Summary of Aegle marmelos essential oil toxicity assays against larvae and adult of Tribolium casteneum |
aLC50 represent the median lethal concentration, bUCL and LCL represent upper confidence limit and lower confidence limit. cg-value, t-ratio and heterogeneity were significant at all probability levels (90, 95 and 99%) | |
| Table 2: | Effect of fumigation of Aegle marmelos essential oil on oviposition of stored-grain insect pest Tribolium castaneum |
| ODIa (%) was calculated as 100 (C-T)/(C+T), where C and T represent the number of eggs/larvae produced in the control and in the test, respectively | |
| Table 3: | Effect of Aegle marmelos essential oil on development (pupation and adult emergence) of stored-grain insect pest Tribolium castaneum |
| Values in parentheses represent percent with respect to control taken as 100% | |
| Table 4: | Effect of fumigation of Aegle marmelos essential oil on damage caused by stored-grain insect pest Tribolium castaneum |
| Table 5: | Regression parameters of lethal, sub lethal and chronic activity on stored-grain insect pest Tribolium castaneum with Aegle marmelos essential oils by fumigation method |
Regression analysis was performed between different concentrations of essential oil and responses of the insect pest. *Significant at 99% probability level. *F-values were significant at all probability levels (90, 95 and 99%), *df: 3,20; **df: 2,20 | |
| Table 6: | Effect of 40 and 80% of 24h LC50 of Aegle marmelos essential oil on acetylcholinesterase enzyme (AChE) activity in Tribolium castaneum |
Enzyme activity was expressed as μ mol of ‘SH’ hydrolyzed min‾1 mg–1 protein. Values indicate Mean±SD of four replicates. Values in parentheses indicate per cent change with respect to control taken as 100%. #Paired t-test was applied. *Significant (p<0.01) |
|
Reduction in AChE activity was 81.48 and 54.32% of the control after 24 h fumigation with 40 and 80% of 24 h LC50, respectively (Table 6).
DISCUSSION
Essential oils are the best known plant products tested against insects pests (Papachristos and Stamopoulos, 2002; Formisano et al., 2008; Chaubey, 2013). Plant volatile essential oils are a group of botanical insecticide that has recently been commercialized in the United States (Isman, 2008). Many essential oils and their constituents have been studied to possess potential as alternative compounds to currently used insect-control agents (Shaaya et al., 1997; Huang et al., 2000; Lee et al., 2004; Batish et al., 2008; Sahaf et al., 2008; Cosimi et al., 2009; Nerio et al., 2009). In the present investigation the essential oil of A. marmelos was found to be effective against larvae and adults of T. castaneum. The present investigation was supported with the result of Mishra and Tripathi (2011) who investigated the repellent activity of A. marmelos essential oil against stored-grain insect pests. In Gas Chromatography Mass Spectrophotometry (GC/MS) analysis, the essential oil of A. marmelos show different chemical components. The leaf essential oil of A. marmelos contain 15 compounds, including seven monotorpene hydrocarbons, three oxygenated monoterpenes, four sesquiterpene hydrocarbons and one phenolic compound in all of this limonene was the main constituent (Kaur et al., 2006). The insecticidal constituents of many essential oils are mainly monoterpenoids (Coats et al., 1991; Regnault-Roger and Hamraoui, 1995; Kim et al., 2003). Monoterpenoids have been reported to inhibit reproduction of stored insects at several steps of the cycle. The mode of toxicity for monoterpenoids is believed to be via competitive inhibition of acetylcholinesterase (Ryan and Byrne, 1988). On other hand the volatile oil of Pyrenacantha staudtii also contains limonene which is reported to be an active insecticidal constituent that exerts a toxic effect on coleopterans (Tapondjou et al., 2002). In the present study limonene was the major component of the oil of A. marmelos and it may be responsible for fumigant toxicity, oviposition deterrent, inhibition of adult development of test insect. Aegle marmelos oil was mixture of a number of components that would reduce the chances for the development of insect resistance, which could be due to the diffusion of the gene selection process (Davies, 1992; Begon et al., 1999). However, concerns for residue of essential oil pesticides on food grains should be mitigated by the growing body of evidence that many essential constituents acquired through the diet are actually beneficial to human health (Huang et al., 1994). The insecticidal activity of the essential oil would be dependent on the active chemical constituents and the gross sensitivity of the target pest to the active chemical principles (Obeng-Ofori et al., 1997). Most of the essential oil components including limonene inhibiting AChE activity have been reported as constituents of A. marmelos essential oils and probably might attribute AChE inhibitory activities to the oils under investigation. The rapid action of essential oil against insect pest is indicative to their neurotoxic mode of action interfering with neuromodulator octopamine (Kostyukovsky et al., 2002) or with GABA-gated chloride channels (Priestley et al., 2003). The mode of action of this essential oil is yet to be confirmed but it appears that death of the adults, larvae may be due to the suffocation and inhibition of different biosynthetic processes of the insect metabolism (Don-Pedro, 1989). Therefore, one can conclude that the potent essential oil might be useful for management of stored grain insect pest.
CONCLUSION
In conclusion, A. marmelos oil may be used as botanical insecticide against different stored grain insect pests causing infestation in stored wheat and pulses. On the basis of results of present study, it can be concluded that the insecticidal nature of A. marmelos against T. castaneum might be due to its AChE inhibitory activities. Moreover, because of the use in traditional medicine in cure of different human diseases, the A. marmelos oil may be used as semiochemicals mediating phytopesticide to protect stored food commodities in developing countries, for which some farmers may not have easy access to chemical insecticides.