Introduction

Stored products meet heavy loss in both quality and quantity due to pest attack. Insect pests are major problem to stored products throughout the world. Globally, around 10 to 40 % of grain loss has been reported due to infestation by insects and other bioagents (Papachristos and Stamopoulos 2002). The cowpea weevil, Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), a cosmopolitan pest of legumes, is a common throughout the tropics and subtropics of the world (Jackai and Daoust 1986). Rice weevil, Sitophilus oryzae (L.), is one of the major pests in stored cereals and predominant of stored rice. India is considered to be the native land of S. oryzae (Zacher 1937).

Fumigation is one of the major chemical methods to control a stored product insect infestation in worldwide. Fumigation is the effective method for management of the stored grains because it is effective against all the life stages of pests, cost-effective, fast and leaves minimal residues (Graver 2004). Currently, phosphine and methyl bromide are used (Lee et al. 2004). Methyl bromide has been reported for its deleterious effect on the ozone layer (Anonymous 1993), and phosphine causes serious health problems to human beings (Garry et al. 1989). Phosphine also causes pesticide resistance in the target pests (Zettler et al. 1989; Bell and Wilson 1995; Chaudhry 1997).

Recently, there has been a growing interest in research concerning the possible use of plant extracts as alternative to synthetic insecticides. Plant products offer protection through repellent property and toxicity against the insect pests. Volatile compounds in plants are responsible for the repellent activity and fumigation toxicity. Plant products are safe to human beings and the environment. In the perspective, many plant products have been evaluated for their toxicity against stored product pests, exclusively in the form of essential oils (Regnault-Roger 1997; Negahban and Moharramipour 2007; Rajenderan and Sriranjiani 2008; Nattudurai et al. 2014). Essential oils are a complex mixture of monoterpenes, phenols and sesquiterpenes and are extracted through hydro or stem distillation of plant materials (Isman et al. 2007). Essential oils are particularly plentiful in some families of plant, namely Conifers, Umbelliferae and Rutaceae, and are often localized in specialized geographical structures (Regnault-Roger 1997).

Bioactivity of essential oils is directly related to their chemical composition, which can vary it chemical composition in the same species. Even in the same species, it varies due to differences in the plant part, location, time of the year and environmental factors (Angioni et al. 2006; Isman et al. 2007). Bioactivity of essential oils is also affected by interaction among their components. Even minor compounds can have produced effective activity due to additional action between the chemical compounds and synergism of antagonism (Sampson et al. 2005; Angioni et al. 2006; Bakkali et al. 2008). Essential oils that possess fumigation toxicity (Lee et al. 2004; Negahban and Moharramipour 2007; Zapata and Smagghe 2010; Nattudurai et al. 2014), repellent activity (Shaaya et al. 1997; Papachristos and Stamopoulos 2002; Nerio et al. 2009), oviposition deterrent, ovicidal activity and adult emergence (Regnault-Roger and Hamraoui 1994, 1995; Raja et al. 2001; Papachristos and Stamopoulos 2002; Nattudurai et al. 2012) in stored product pests were investigated.

Protein molecules play a vital function in many organisms including insects. Different types of protein molecules are involved in the digestion of food, growth and development of insects. Proteins contain vital molecules including enzymes that are involved not only in the ordinary physiological functions but also in the important task of detoxification of chemicals. The proteins show quantitative and qualitative variations in all the insects’ it affect the developmental stages (Kanost et al. 1990). Esterase, glutathione S-transferase and monooxygenase are present in the insect antennal sensilla for detoxifying the volatile allelochemical compounds. Neutralization of synthetic insecticides and resistance mechanisms by glutathione S-transferase in insects have been widely studied (Siegfried and Scharf 2001).

Atalantia monophylla (L.) belongs to the family Rutaceae and is commonly called as wild lime. Various parts of A. monophylla have been used in folk medicine, such as for rheumatoid pain and glandular swelling (Sankaranarayanan et al. 2010). The root is believed to be antispasmodic (Kirtikar and Basu 1999), whereas a decoction of the leaves is often applied for itching and other skin complaints (Panda 2004). Hexane, chloroform and ethyl acetate extracts of A. monophylla showed antifeedant, larvicidal, pupicidal and ovicidal activities against Helicoverpa armigera and Spodoptera litura (Baskar et al. 2009, 2012, 2015a, Baskar and Ignacimuthu 2012). Methanolic extract of the leaves of A. monophylla was evaluated for mosquitocidal activity (Sivagnaname and Kalyanasundaram 2004). Ethanol extract of the leaves of A. monophylla was evaluated for antifungal and antioxidant activities (Reddy et al. 2010).

Toxic effects and repellent activity of A. monophylla essential oil have not been studied against C. maculatus and S. oryzae. Hence, the present study was undertaken to investigate the fumigant toxicity, repellent activity, developmental toxicity and biochemical changes of C. maculatus and S. oryzae by the treatment of A. monophylla oil.

Materials and methods

Insect culture

C. maculatus and S. oryzae were reared and maintained continuously several generations on cowpea and whole rice respectively, at the Entomology Research Institute insectary at 29 ± 1 °C; 60–65 % R.H.; and 11:13 h light and dark. Adult insects (1–7 days old) were used the further studies.

Plant collection and preparation

Fresh leaves of A. monophylla were collected from Poondy hills, Thiruvallur district, Tamil Nadu, India in December 2012. Essential oil was extracted by using Clevenger-type apparatus. About 50 g of fresh leaves was taken in a 1-L flask along with 500 mL of distilled water and distilled 4 h. Anhydrous sodium sulphate was used to remove water from the extracted essential oil and were stored at 4 °C until use.

Gas chromatography mass spectrometry

Gas chromatography-mass spectrometry (GC-MS) analysis was performed with a SHIMADZU-QP2010 with helium as a carrier gas with a linear velocity flow on a Resteck-624 ms column (30 m × 0.32 mm id, 1.8 μm film thickness). Column flow rate was 1.491 mL/min. The oven was programmed to rise to 45 °C (4 min) isotherm and then to 175 leads to 240 °C at a rate of 10 and 25 °C/min respectively. Injector and detector temperatures were 140 °C. The identification of the constituents was performed by computer library identification of the constituents was performed by computer library.

Fumigation toxicity

The toxicity of A. monophylla essential oil on C. maculatus and S. oryzae adults were tested by filter paper dip method as described by Negahban et al. (2007) at laboratory conditions (29 ± °C; 60–65 % R.H.). Whatman no. 1 filter paper discs (2 cm diameter) were impregnated with different concentrations (20, 40, 80, 120 and 160 μL/L) of essential oils separately and were attached to the under surface of the screw caps of glass vials (volume 50 mL) separately. The cap was screwed tightly on the vial after the release of ten adult beetles (5–7 days old) along with little amount of cowpea and wheat as food for C. maculatus and S. oryzae respectively. For comparison, a set of control, without essential oil, was maintained. Each treatment and control was replicated ten times. Mortality was recorded after 4, 8, 12 and 24 h from the commencement of exposure. When no leg or antennal movements were observed, insects were considered as dead. Percentage insect mortality was calculated using the Abbott’s formula (Abbott 1925).

Repellent activity

The repellent activity was studied at five different concentrations viz., 5, 10, 15, 20 and 25 μL using a Y-tube glass olfactometer. The stem of the Y-tube was 20 cm long, and each of the two arms was also 20 cm long. The inner diameters of the stem tube and arms were 2.5 cm. About 5 μL of volatile oil was applied on a piece of filter paper (2 cm × 3 cm) and placed inside one arm tube near the opening. In the other arm of the Y-tube, a blank filter paper strip without volatile oil (control) was placed. An air current was created by an aerator through the arms, and the air passed out through the stem of the Y-tube. The rate of airflow was adjusted as 1.25 L/min near each arm. Twenty beetles were released into the olfactometer through the opening of the stem tube. The number of insects that moved into the control side and essential oils treated side was recorded at the end of every 1 h period, and the entire experiment lasted in 3 h. The experiment was replicated five times for each insect species. All the concentrations of the oil were tested by same method. Percentage of repellency was calculated by the using formula of Nerio et al. (2009);

$$ \mathrm{Percent}\ \mathrm{repellency}=\frac{C-T}{C+T}\times 100 $$

Fecundity

Ten pairs of freshly emerged C. maculatus adults were introduced in 220 mL of airtight Petri dish (2 cm diameter) along with 20 g of cowpea as food. Filter paper discs (Whatman no. 1; 2 cm diameter) impregnated with 4.10 μL/L (LC10), 7.74 μL/L (LC20) and 43.28 μL/L (LC30) air sub-lethal concentration of A. monophylla were placed inside the individual Petri dish. After 24 h of treatment, the adults were transferred to an untreated Petri dish and the number of eggs laid was counted up to 2 days. Fecundity per female was calculated.

Ovicidal activity

Ten pairs of C. maculatus adults were taken in a 220 mL of Petri dish (2 cm diameter) and 20 g of cowpea as food. After 2 days, all the adults were removed. The eggs were counted and treated with sub-lethal concentrations of 4.10 μL/L (LC10), 7.74 μL/L (LC20) and 43.28 μL/L (LC30) air concentration in airtight 220 mL of Petri dish by fumigation method. After 5 days, hatched and unhatched eggs were counted and calculated the percent ovicidal activity.

$$ \mathrm{Ovicidal}\ \mathrm{activity}=\frac{\mathrm{Egg}\ \mathrm{hatched}\ \mathrm{in}\ \mathrm{control}-\mathrm{Egg}\ \mathrm{hatched}\ \mathrm{in}\ \mathrm{treatment}}{\mathrm{Egg}\ \mathrm{hatched}\ \mathrm{in}\ \mathrm{control}}\times 100 $$

Treatment for biochemical studies

For biochemical studies, the selected insects were treated with A. monophylla sub-lethal concentrations of 4.10 μL/L (LC10), 7.74 μL/L (LC20) and 43.28 μL/L (LC30) air for C. maculatus and 5.41 μL/L (LC10), 16.27 μL/L (LC20) and 52.98 μL/L (LC30) air for S. oryzae. The fumigation treatment was given for a period of 24 h. The live insects of each species were used for the estimation of total protein, esterase, acetylcholine esterase and glutathione S-transferase. For each concentration, five replications with 30 mg of insects/replications were used.

Estimation of protein

The total protein in the whole body extracts of C. maculatus and S. oryzae was estimated according to Bradford’s method using bovine serum albumin as the standard (Bradford 1976).

Preparation of Bradford reagent

Twenty-five milligrammes of coomassive brilliant blue G250 was dissolved with 12.5 mL of ethanol and added 25 mL of orthophosphoric acid mixed well and make up to 250 mL with double distilled water. The reagent was filtered in Whatman no. 1 filter paper and stored at 4 °C in amber colour bottle.

The insect pests were homogenized with 350 μL of phosphate buffer pH 7.2, and samples were centrifuged at 14,000×g for 10 min at 4 °C. Ten microlitres of supernatant was mixed with 90 μL of phosphate buffer and added 3 mL of Bradford reagent. Incubated for 20 min. The absorbance was read at 595 nm.

Estimation of esterase

The esterase was determined according to Asperen (1962) method. In this experiment, α-naphtlyacetate (α-NA) was used as substrates. The whole insects were homogenized with 1000 μL of 0.1 M phosphate buffer (pH 7) containing Triton X—100 at the ratio of 0.01 %, then the homogenized solution was centrifuged at 10,000×g for 10 min at 4 °C. The supernatant was transferred to new microfuge tube and was diluted with phosphate buffer. This solution reacted with the substrate, and by using dye indicator (Fast Blue RR salt) (1 mM), a coloured solution was formed and the absorbance was read at 630 nm.

Estimation of acetylcholinesterase

Enzyme source preparation

Sub-lethal concentrations exposed insects (30 mg) were homogenized with 2 mL of phosphate buffer at 0.1 M (pH 8). The un-centrifuged homogenate was used as enzyme source.

The activity of acetylcholinesterase (AChE) was measured according to the method described by Ellman et al. (1961). Colour reagent containing 0.25 mM Dithiobis, 100 mM NaCl and 20 mM MgCl2 was prepared in 50 mM Tris-HCl, pH 8. Acetylcholine iodide (33 mM) was used as substrate. The kinetics of the enzyme reaction was monitored continuously for 5 min in double beam spectrometer Bio spec-1601 (Shimadzu), adjusted at 405 nm and 25 °C. The specific activity was evaluated by the increase in absorbance after adding 20 μL enzyme stock. Absorbance values were converted to units of concentration using a molar extinction coefficient of 13,300 M–1 cm−1 for acetylthiocholine iodide. The specific activity was expressed as 1 μmol of acetylcholine iodide hydrolysed/min/mL/mg protein at 25 °C and pH 8.

Estimation of glutathione S-transferase

The glutathione S-transferase was determined according to method of Habig et al. (1974). 1-Chloro-2, 4-dinitrobenzene (CDNB) (20 mM) was used as the substrate. Initially, insect pests were homogenized in 20 μL distilled water, then the homogenized solution was centrifuged at 12,500×g for 10 min at 4 °C. Fifteen microlitres of supernatant was mixed with 135 μL of phosphate buffer (pH = 7), 50 μL of CDNB and 100 μL of GST. The absorbance was read at 340 nm.

Statistical analysis

The significances of treatments of biochemical changes and enzyme inhibition were found out by students ‘t’ test and effective treatment was separated by Tukey’s multiple range test by one-way ANOVA. Differences between means were considered significant at p ≤ 0.05. All statistical analyses were done by using SPSS 20.5 version.

Result

Yield of the oil

The yield of essential oil from 50 g leaves of A. monophylla was 200 μL. The extraction process was repeated till sufficient quantity of the oil was obtained for the experiments.

Chemical composition of A. monophylla oil

The chemical compositions of A. monophylla are shown in the Table 1. Totally, forty components were detected in the essential oil of A. monophylla. The major components were eugenol (19.76 %), sabinene (19.57 %), 1,2-dimethoxy-4-(2-methoxyethenyl) benzene (9.84 %), beta-asarone (7.02) and methyl eugenol (5.52 %).

Table 1 Composition of essential oil of Atalantia monophylla as analysed by GC-MS
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Fumigation toxicity

The fumigant action of essential oil of A. monophylla was tested on adults of C. maculatus and S. oryzae. Mortality of C. maculatus and S. oryzae was dependent upon the concentration and exposure period. Considerable difference in mortality of the insect was observed in different concentrations and experimental periods (Table 2). All the concentrations of A. monophylla oil were more toxic against C. maculatus than S. oryzae. At the highest concentration, 160 μL/L produced mortality of 70.22 % (C. maculates) and 65.44 % of (S. oryzae) at 24-h exposure period; 160 μL/L concentration exhibited more than 50 % mortality of C. maculatus and S. oryzae within 12-h exposure period. The lowest concentration, 20 μL/L produced mortality of 23.78 % of C. maculatus and 20.44 % of S. oryzae. The A. monophylla exhibited LC50 value of 101.69 μL/L air for C. maculatus and 113.67 μL/L air for S. oryzae (Table 2).

Table 2 Insecticidal activity of Atalantia monophylla against Callosobruchus maculatus and Sitophilus oryzae
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Repellent activity

A. monophylla essential oil showed strong repellent activity against C. maculatus and S. oryzae (Table 3). The repellent effect was more on C. maculatus than S. oryzae. In the higher concentration, 25 μL, A. monophylla showed 85.24 % (C. maculates) and 75.24 % (S. oryzae) repellent activities. The middle concentration at 15 μL was repelled more than 50 % of C. maculatus but S. oryzae required about 20 μL of essential oil. At the onset, the insects were agitated and slowly move towards treatment or control side in Y-tube olfactometer. The repellent activity was directly propositional to the concentration and exposure period. With increasing the concentration and exposure period, the repellent activity was also increased.

Table 3 Percent repellent activity of Atalantia monophylla essential oil against Callosobruchus maculatus and Sitophilus oryzae (mean ± SE)
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Fecundity

The fecundity was reduced compared to control in all the treatments (Table 4). Results showed the number of eggs laid by female in control 75.20, LC10 = 28.29, LC20 = 23.23 and LC30 = 10.11 eggs/insect at 48 h. Compared to control in the treatments of LC10 and LC30, 62.38 and 85.56 % of the fecundity were reduced. In all the treatments, fecundity was reduced and highly significant when compared to control. On the other hand, LC10 was not significantly deviated the reduction of fecundity compared to LC20.

Table 4 Fecundity, ovicidal activity and adult emergence in Callosobruchus maculatus at sub-lethal concentrations
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Ovicidal activity and adult emergence

The essential oil of A. monophylla was found toxic to the C. maculatus eggs. The ovicidal activity of A. monophylla was 37.62, 44.04 and 100 % in sub-lethal concentrations of LC10, LC20 and LC30 respectively in C. maculatus. In control, 10.64 % ovicidal was observed. The essential oil of A. monophylla was interfered with the growth and development of the young stages, which leads to reduction of adult emergence (Table 4). In control, 89.36 % adults were emerged. In the treatment of LC10 and LC20 concentrations, 62.38 and 55.96 % of adult emergence occurred. No adult was emerged in LC30 concentration. So, LC30 concentration (43.28 μL/L) was the most effective treatment for inhibition of C. maculatus. Table 4 shows ovicidal activity and reduction of the adult emergence in all the treatments.

Total protein in C. maculatus and S. oryzae

The sub-lethal doses of A. monophylla were inhibited the total protein content of C. maculatus and S. oryzae in a concentration-defended manners. The LC30 concentration significantly reduced the total protein level (19.39 μg/g) when compared to control (30.13 μg/g) (Table 5). The total protein content was reduced significantly (35.66 %) in the LC30 concentration compared to control in C. maculatus.

Table 5 Impact of Sub-lethal concentrations of Atalantia monophylla essential oil on total protein, esterase, acetylcholinesterase and glutathione S-transferase of Callosobruchus maculatus
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The total protein content was 12.14 μg/g in LC30 concentration; it was a highly significant reduction of the protein content when compared to control. The total protein reduction in S. oryzae was calculated as 40.90 % in the LC30 concentration (52.98 μL/L) (Table 6). Therefore, A. monophylla oil was more effective on S. oryzae than C. maculatus.

Table 6 Impact of sub-lethal concentrations of Atalantia monophylla essential oil on total protein, esterase, acetylcholinesterase and glutathione S-transferase of Sitophilus oryzae
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Impact of A. monophylla oil on total esterase activity of C. maculatus and S. oryzae

Total esterase activity was decreased in all the treatments of A. monophylla against C. maculatus and S. oryzae. The LC30 concentration exhibited decreased esterase activity (53.54 %) when compared to control of C. maculatus (Table 5). The total esterase activity in S. oryzae was decreased by the A. monophylla essential oil treatment. The LC30 concentration exhibited 38.29 % reduction of esterase activity in test insects (Table 6).

Impact of A. monophylla oil on acetylcholinesterase activity of C. maculatus and S. oryzae

The treatment exhibited decreased acetylcholinesterase activity 45.21–10.96 % in the concentration of LC10 and LC30 on C. maculatus (Table 5). The treatment showed highly significant reduction in acetylcholinesterase activity in LC30 concentration when compared to control. The total acetylcholinesterase activity in S. oryzae was decreased by the A. monophylla essential oil treatment. The decrease in the acetylcholinesterase activity was 9.18 and 23.47 % at LC10 and LC20 concentrations respectively. The LC30 concentration reduced the acetylcholinesterase activity 44.90 % of the test insects (Table 6). The LC30 treatment had highly significant reduction in the acetylcholinesterase enzyme activity in C. maculatus and S. oryzae compared to control.

Glutathione S-transferase activity in C. maculatus and S. oryzae

The glutathione S-transferase activity of C. maculatus was decreased by the treatment of A. monophylla when compared to control (Table 5). The GST activity in S. oryzae was decreased by the A. monophylla essential oil treatment. The decrease in the GST activity was 9.23 and 22.31 % at LC10 and LC20 respectively. At the 43.08 % of GST, activity was decreased at concentration of LC30 of A. monophylla essential oil (Table 6). In the treatment of LC30 concentration, this exhibited decreased GST activity of test insects when compared to control.

Discussion

A large number of essential oils were extracted from different plant species. Fumigation is a successful method for eradication of stored product pest from food products. Fumigant toxicity of many plant essential oils against stored product insects has been reported by many investigators (Lee et al. 2004; Negahban et al. 2007; Nattudurai et al. 2012). In this study, essential oil of A. monophylla had more fumigation toxicity and repellency against C. maculatus and S. oryzae. Our report was correlated with previous investigations of Mahmoudv et al. (2011) that reported that Mentha pulegium essential oil was more toxic to C. maculatus. Toddalia asiatica essential oil showed strong fumigation toxicity and repellency against C. maculatus, S. oryzae and Tribolium castaneum (Nattudurai et al. 2014). Fumigation toxicity of Marjoram hortensis essential oil showed 100 % mortality to Phthorimaea operculella adult at 0.1–0.2 mL or 100–200 μL/L after 24 h exposure (El-Aziz 2011). The essential oil of Lantana camera showed fumigation toxicity and its lethal concentrations LC50: 187.9 μL L−1 for male and 282.7 μL L−1 for female C. maculatus (Zandi-Sohani et al. 2012). Fumigation toxicity and repellent activity of essential oil of A. monophylla against C. maculatus and S. oryzae are reported for the first time in the present study. The fumigant toxicity of three essential oils, Pelargonium graveolens, Ocimum basilicum and Foeniculum vulgare, were investigated against S. oryzae and C. maculatus (Adel et al. 2015) repellent activity against S. oryzae and C. maculatus (Seada et al. 2016).

The fumigation and repellent activity of essential oil are due to the presence of volatile compounds especially monoterpenes. The strong insecticidal and repellent activities of A. monophylla might be due to the presence of some major components such as eugenol (19.76 %), sabinene (19.57 %), 1,2-dimethoxy-4-(2-methoxyethenyl) benzene (9.84 %), beta-asarone (7.02 %) and methyl eugenol (5.52 %). Some previous reports were available for constitutes of A. monophylla. Manimaran et al. (2002) reported that the major compounds were methyl eugenol (36.46 %), sabinene (24.89 %) and elemicin (24.61 %) in A. monophylla essential oil. Das and Swamy (2013) have reported that the major compounds were asarone (28.82 %), sabinene (13.19 %), eugenol methyl ether (12.71 %), 1,2-dimethoxy-4(2-methoxyethenyl) benzene (11.63 %) and β-pinene (5.3 %) in A. monophylla. Similar compounds have been identified in the present study, even though there is substantial variation in the identified compounds as well as in their concentration.

Similarly, six compounds, including eugenol, methyl eugenol, methyl isoeugenol, elemicin, myristicin and safrole isolated from Myristica fragrans, showed potent contact toxicity against Lasioderma serricorne adults. Methyl eugenol, safrole and myristicin are also the main compounds; the toxic properties of the essential oil is due to the synergistic effects of its diverse major and minor components (Du et al. 2014). The difference in chemical constitution may be probably due to different environmental and genetic factors, different chemotypes and the nutritional status of the plants as well as other factors that can influence the oil composition (Ozcan and Chalchat 2002). In the present study, the compounds of eugenol and Sabinene were major concentrations in A. monophylla. The effective fumigation and repellent activity were due to the major compounds. Previously, eugenol, as a monoterpene, has already been reported as a fumigant against C. maculatus and Sitophilus zaemais and most effective repellent against C. maculatus (Reis et al. 2016). Similarly, linalool, camphor, camphene and sabinene hydrate exhibited 100 % fumigant toxicity (Kim et al. 2016).

Our study also showed that A. monophylla oil significantly inhibited the oviposition of C. maculatus on cowpea seeds compared to control. Comparison of our results with earlier investigators (Huang et al. 2000) demonstrates different responses of the egg stage and active stages of stored product insects to the Elettaria cardamomum essential oil. Essential oil of E. cardamomum had oviposition deterrence in C. maculatus (Abbasipour et al. 2011). Millingtonia hortensis L. had significant oviposition deterrent activity against P. operculella and also adverse effect on egg hatchability of at lower dose 0.05 mL (El-Aziz 2011). The oil vapours diffused into eggs and affected the physiological and biochemical processes associated with embryonic development (Raja et al. 2001). In this experiment, LC30 concentration inhibited 100 % adult emergence of C. maculatus. Similarly, Eucalyptus and camphor oils significantly inhibited the adult emergence of T. castaneum (Nattudurai et al. 2012).

Proteins are the most important biochemical components that are important for the insect growth and development. Our results showed that the amount of protein decreased in the treated C. maculatus and S. oryzae. Similarly, previous reports were confirmed by our results; hexane extract of Capparis decidua was reduced the 44.19 % protein in T. castaneum (Upadhyay et al. 2011a), and aqueous extract of Cassia alata significantly reduced 68.50 % of total protein in C. maculatus (Upadhyay et al. 2011b). Baskar et al. (2015b, a) reported that eighth fraction from hexane extract of Couroupita guianensis reduced the protein concentrations of S. litura. The essential oil of Artemisia annua significantly reduced total protein of H. armigera when compared to control (Mahboubkar et al. 2015). This phenomenon could be due to the breakdown of proteins into their respective amino acids that could help to provide energy for the insect to survive. Similarly, protein and nucleic acid synthesis may also block at cellular level and catabolism get increased which results into low availability of proteins and nucleic acid (Pant and Gupta 1979; Shakoori and Saleem 1989).

In the present study, all the sub-lethal concentrations of A. monophylla oil were significantly decreased detoxifying enzymes concentration, such as esterase, acetylcholinesterase and glutathione S-transferase activities on C. maculatus and S. oryzae. Similarly, Baskar et al. (2014) reported that pectolinaringenin, a flavonoid from Clerodendrum phlomidis, inhibited the glutathione S-transferase and esterase activities of H. armigera and Earias vittella. Esterase is one of the detoxifying enzymes in many insects against ester bonds of the insecticides. This decreased activity of esterase occurred by biopesticide may increase the insect susceptibility to insecticides having an ester bond (Mukanganyama et al. 2003). So far, esterases are known to be involved in the detoxification of the major groups of insecticides (Pethuan et al. 2007). El-Aziz and El-Sayed (2009) reported that the activities of α, β-esterase and GST activities of Tribolium confusum were significantly reduction in LD50 dose of basil, garlic and sesame essential oils. The extract binds to the active site of the enzyme and therefore prevents detoxification role of the enzyme. Detoxification enzymes in insects are generally demonstrated as the enzymatic defence against foreign compounds and play a significant role in maintaining their normal physiological functions (Mukanganyama et al. 2003).

In our experiments, acetylcholinesterase activity was decreased significantly. Similarly, hexane extract of C. decidua had higher inhibitory activity of AChE in T. castaneum (Upadhyay et al. 2011a). Acetylcholinesterase plays an important role in neurotransmission at cholinergic synapses by catalysing the hydrolysis of the neurotransmitter acetylcholine. It is well known that AChE alteration is one of the main resistance mechanisms in many insect pests (Wang et al. 2004). Several essential oils from aromatic plants, monoterpenes and natural products act as AChE inhibitors (Shaaya and Rafaeli 2007; Lopez and Pascual-Villalobos 2010).

In the present study, GST was significantly decreased in all sub-lethal treatments against C. maculatus and S. oryzae. Previously, some investigations were carried out with plant product against insect pest detoxifying enzyme GST. Kolawole et al. (2009) has reported that crude ethanolic extracts of Tithonia diversifolia, Cyperus rotundus and Hyptis suaveolens have insecticidal activity against C. maculatus, and the plant extracts have potentially inhibited the GST activity.

Conclusion

Essential oil showed that good insecticidal, repellent and ovicidal activities, reduced the adult emergence and fecundity of the test insects. The mortality of insects may due to the reduction of protein content and reduction of GST, esterase and ACh activity. It may be considered as a stored pest control agent alternative to the chemical insecticides in large-scale usage in warehouse and container.