Insecticidal and Detoxification Enzyme Inhibition Activities of Essential Oils for the Control of Pulse Beetle, Callosobruchus maculatus (F.) and Callosobruchus chinensis (L.) (Coleoptera: Bruchidae)

Pulse beetle is the most harmful pest attacking stored grains and affecting quality and marketability. Continuous use of chemical-based pesticides against pulse beetle led to the development of insecticidal resistance; essential oils (EOs) can be an effective natural alternative against this pest. The main objective was to study the chemical composition of seven EOs viz., Acorus calamus, Hedychium spicatum, Lavandula angustifolia, Juniperus recurva, Juniperus communis, Cedrus deodara and Pinus wallichiana, their insecticidal and enzyme inhibition activities against pulse beetle. The primary compounds present in these EOs were cis-asarone, 1,8-cineole, linalyl isobutyrate, 2-β-pinene, camphene, α-dehydro-ar-himachalene and camphene. A. calamus oil showed promising fumigant toxicity to Callosobruchus maculatus and C. chinensis (LC50 = 1357.86 and 1379.54 µL/L, respectively). A combination of A. calamus + L. angustifolia was effective against C. maculatus and C. chinensis (LC50 = 108.58 and 92.18 µL/L, respectively). All the combinations of EOs showed synergistic activity. In the repellency study, A. calamus showed more repellence to C. maculatus and C. chinensis (RC50 = 53.98 and 118.91 µL/L, respectively). A. calamus and L. angustifolia oil at 2500, 5000 and 10,000 µL/L significantly inhibited the AChE and GST enzymes in C. maculatus and C. chinensis after 24 and 48 h.


Introduction
The global population is continuously rising which needs a huge amount of food to feed people, so the production of food grains creates a critical source of food for growing population. Among these, pulse crops are sustainable and high-quality protein sources [1]. However, increasing pest infestation causes stored grain loss, affecting food security worldwide [2]. During storage, pulse beetle can cause a 5-10% loss in temperate and a 20-30% loss in tropical regions [3]. If no control measures are employed, there will be a 50% loss of grains in store within 3-4 months [4]. At the global level, the pulses (green/black gram) are considered as major host of pulse beetle [5] and these pulses are rich source of proteins, carbohydrates, vitamins etc. [6]. Callosobruchus maculatus and Callosobruchus chinensis are the major pest of cowpea and French beans in Africa and Asia which contains rich source of proteins (20-25%) and carbohydrates (50-60%) [7,8]. The grubs of the C. maculatus and C. chinensis feed on grains affecting quantitative and qualitative damage to the stored grains [9]. Usually, fumigation with phosphine/aluminium phosphide and chemical pesticides are more effective for controlling pulse beetle where the grains are stored in small bins and big go downs. However, fumigation causes some minor side effects on consumer's health [10]. Also, the indiscriminate use of synthetic insecticides a Retention index value of compounds in the literature [22]. b Retention index value determined relative to n-alkanes (C9-C24) on the DB-5 GC column, * Percentage of compounds class in analyzed essential oil samples.

Percent Mortality of Essential Oils against C. maculatus
The fumigant toxicity of EOs, concentrations, and interaction effect (oils × concentrations) against C. maculatus after 72, 96 and 120 h were presented in Figure 1. The pooled mean percent mortality was found to be significantly different across the oils (F 6 The fumigant toxicity of different EOs and their combinations against C. chinensis was presented in Table 3. Among EOs, L. angustifolia showed more toxicity against C. chinensis (LC 50

C. chinensis
Toxicity of Different Essential Oils against C. chinensis The fumigant toxicity of different EOs and their combinations against C. chinensis was presented in Table 3. Among EOs, L. angustifolia showed more toxicity against C. chinensis (LC50 = 4316.34 µL/L) after 72 h of treatment, followed by C. deodara and J.

Percent Mortality of Different EOs against C. chinensis
The fumigant toxicity of EOs, concentrations, and interaction effect (oils × concentrations) against C. chinensis after 72, 96 and 120 h were presented in Figure 2. The pooled mean percent mortality was found to be significantly different across the oils (F 6   The interaction effect (oils × concentrations) showed significantly different against C chinensis after 72 (F24,174 = 1.96; p < 0.0001) and 96 h (F24,174 = 1.89; p < 0.05). Results showe that among oils, pooled mean mortality was higher in L. angustifolia (38.00 ± 25.00) afte The interaction effect (oils × concentrations) showed significantly different against C. chinensis after 72 (F 24,174 = 1.96; p < 0.0001) and 96 h (F 24,174 = 1.89; p < 0.05). Results showed that among oils, pooled mean mortality was higher in L. angustifolia (38.00 ± 25.00) after 72 h of treatment and was at par with C. deodara as compared to other oils. Similarly, pooled mean mortality was maximum in A. calamus (62.80 ± 23.72) after 96 h and was at par with L. angustifolia (58.80 ± 27.43) followed by C. deodara (49.20 ± 21.78) as compared to EOs. After 120 h, mortality was higher in L. angustifolia (76.80 ± 22.49), followed by A. calamus (68.00 ± 24.66) compared to the remaining oils. Among concentrations, pooled mean mortality was higher in 10,000 µL/L (55.43 ± 15.40, 75.43 ± 17.71 and 92.00 ± 8.33, respectively) after 72, 96 and 120 h as compared to other concentrations.

Repellent Activity of Essential Oils against Pulse Beetle
The repellent activity of A. calamus, H. spicatum, J. recurva, J. communis, L. angustifolia, P. wallichiana and C. deodara against two species of pulse beetle after 1, 2, 3, 4, 5 and 24 h was presented in Tables 4 and 5. The repellent activity of EOs against pulse beetle decreased over time. Results showed that A. calamus showed more repellence against C. maculatus and C. chinensis (RC 50

Ovipositional Inhibition (OI) Effect of Essential Oils against Pulse Beetle
The ovipositional inhibition (OI) of different EOs and concentrations against pulse beetle reflected in the percent OI after 24, 48 and 72 h were summarized from Tables S7-S12.

C. chinensis
The percent OI of different EOs and concentrations against C. chinensis and its interaction effect (oils × concentrations) after 24, 48 and 72 h were presented in Tables S10-S12. The pooled mean percent OI was found to be significantly different across the oils (F 6,174 = 7.45 and 3.77; p < 0.0001) after 24 and 72 h, concentrations (F 4,174 = 25.18, 20.16 and 36.46; p < 0.0001) after 24, 48 and 72 h of treatment. However, the mean percent OI was not differed significantly (p > 0.05) in the interaction (oils × concentrations). Among oils, the pooled mean OI was highest in P. wallichiana (34.84 ± 7.28) after 24 h, followed by J. communis and C. deodara (28.72 ± 12.05 and 27.96 ± 10.31%, respectively) as compared to other EOs. Similarly, after 48 h, pooled mean percent OI was maximum in P. wallichiana (32.04 ± 9.52) and was followed by A. calamus (27.56 ± 8.98) as compared to other EOs. In the case of 72 h, pooled mean percent OI was higher in P. wallichiana (32.64 ± 7.58) and was at par with H. spicatum (31.04 ± 7.44) followed by A. calamus (29.20 ± 10.61) as compared to remaining oils. Among concentrations, pooled mean percent OI was higher in 10,000 µL/L (36.03 ± 11.03, 34.05 ± 9.02 and 37.28 ± 6.45%, respectively) after 24, 48 and 72 h as compared to other concentrations.

Detoxification Enzyme Activities of A. calamus and L. angustifolia against Pulse Beetle
Detoxification enzyme inhibition activities of A. calamus and L. angustifolia against C. maculatus and C. chinensis after 24 and 48 h of treatment were presented in Figures 3a-d and 4a-   at 10,000 µL/L showed higher inhibition (2.39 ± 0.70 nmol/min/mL) and was at par with 5000 µL/L (5.91 ± 1.71 nmol/min/mL), as compared to other concentrations.

Discussion
The chemical composition of selected EOs, insecticidal activities (fumigant toxicity, synergistic, repellence, ovipositional activities) and their effect on detoxification enzyme inhibition in C. chinensis and C. maculatus are discussed. EOs are volatile essences and aetheroleum found in aromatic plants as a mixture of compounds produced by secondary metabolites [23]. The EOs extracted by steam/water distillation which has a lower density than water [24,25]. The composition of EO is very complex, and the individual components have valuable applications in agriculture, cosmetics, human health, and the environment. EO has been discovered to be an effective complement to synthetic compounds used in the chemical industry [26]. EOs containing monoterpenes, sesquiterpenes, and terpenoids [27] play a significant role in pesticide activities. In the present study, the chemical composition of seven EOs viz., A. calamus, H. spicatum, J. recurva, J. communis, L. angustifolia, P. wallichiana, and C. deodara were studied and presented. In the present study, the major constituents are cis-asarone in the EO of A. calamus; 1,8-cineole in H. spicatum; α-dehydro-ar-himachalene and α-himachalene in C. deodara; camphene in J. communis and P. wallichiana; 2-β-pinene in J. recurva; linalyl isobutyrate and linalool in L. angustifolia. The constituents of EOs play a significant role in insecticidal activities against pests. In this study, the percent composition of 1,8-cineole is higher in H. spicatum than in EO of Artemisia maritima and M. piperita [9,21]. Similarly, the composition of 1,8-cineole is lesser (12.2-23.15%) in H. spicatum oil [28][29][30] than in the current results. The composition of asarone in the present study is higher than in earlier reports, which observed comparatively less (9.5-50.09%) [31,32]. Earlier studies showed that the composition of linalool in L. angustifolia in previous studies was also lesser (19.71, 28.06, and 31.17%) [33][34][35] than present study (33.30%). In the recent study, the composition of camphene was higher (46.21%) in P. wallichiana as compared to the previous (0.9-1%) studies [36,37]. The composition of α-himachalene (18.11%) in C. deodara was higher in the present study than in previous studies [38]. The variation in the chemical components of EOs may be due to geographical, seasonal, and climatic conditions, genetic/hereditary, chemotype, and nutrition of the plants [39].
Insecticidal activities of the EO depend upon the presence of significant constituents, mode of application, concentration, stage, and type of insect [40,41]. In the present study, A. calamus showed the highest fumigant toxicity against C. maculatus after 96 h, compared to the previous study (LC 50 = 3043.94 µL/L) [42]. In a similar study, A. calamus showed fumigant toxicity against C. maculatus [43] and C. chinensis [44,45]. The EO of A. calamus was less effective against Trogaderma granarium [46] than in present studies. H. spicatum is the second-best oil that showed fumigant toxicity against C. maculatus (LC 50 = 2177.08 and 806.92 µL/L) and C. chinensis (LC 50 = 4875.82 and 1585.15 µL/L) after 96, and 120 h of treatment and these results are comparable with that of Plutella xylostella [47]. Similarly, the EOs of other species viz., H. forrestii, H. elatum, H. bousigonianum, H. flavum, H. thysiforme showed promising mortality against Stephanitis pyrioides [48] and H. gardnerianum against Artemia salina [49]. In the present findings, the EO of L. angustifolia was also found effective against C. chinensis and C. maculatus (LC 50 = 1715.57-1876.15 µL/L) after 96 h. These results agree with previous studies of L. angustifolia against Ryzopertha dominica, Tribolium castaneum and L. dentata against C. maculatus [50][51][52].
In the current study, EOs of C. deodara, J. recurva and A. calamus at higher concentrations showed significant ovipositional inhibition (37.8-42.6%) against both species of pulse beetle, and these findings agree with the earlier reports in which the EOs of C. tangerina, C. limonium, C. paradisi, C. aurantifolia and C. sinensis [56] and Eugenia caryophyllus and Illicium verum [57] showed promising ovipositional inhibition against C. maculatus as compared to the present results.
AChE is categorised as the enzymes that catalyse the hydrolysis of acetylcholine (ACh), a neurotransmitter converted into acetic acid and choline [58]. GST enzyme detoxifies various insecticides, including organochlorines, pyrethroids, organophosphates, and carbamates [59]. GST activity is mainly inhibited by the chemical compounds present in the EOs [60,61]. In the present study, EOs of A. calamus and L. angustifolia were significantly inhibiting the AChE and GST activity against C. maculatus and C. chinensis. The earlier reports confirmed the current results, which reported that the EO of A. maritima inhibited the GST activity in C. maculatus [21]. Similarly, EOs of A. monosperma, A. judaica, C. aurantifolia, C. viminals, C. lemon and Origanum vulgare also inhibited the AChE and adenosine triphosphatases (ATPases) activity against S. oryzae [62].

Test Insect
C. maculatus and C. chinensis obtained from Council of Scientific and Industrial Research-Central Food Technological Research Institute (CSIR-CFTRI), Mysore, Karnataka, India, for further rearing in the Entomology laboratory, CSIR-Institute of Himalayan Bioresource Technology (IHBT), Palampur, H.P, India under controlled conditions at 25 ± 2 • C temperature, 60 ± 5% relative humidity. The adults were fed on the uninfected dried green gram (Vigna radiata (L.)) seeds in plastic jars and covered with a black muslin cloth. The adults were inspected for growth regularly (20-30 days intervals). The newly emerged adults were then transferred to 1 L plastic jars containing uninfested seeds for mating and egg-laying to ensure sufficient adults. The adults (1-4 days old) were used for bioassay and other experiments. The dead adults removed after their adult period competed, either by sieving the grains or handpicking, depending on the number of adults.

Gas Chromatography (GC) Analysis
The composition of EOs determined by gas chromatography (GC) on a Shimadzu GC 2010 equipped with DB-5 (J &W Scientific, Folsom, CA, USA) fused silica capillary column (30 m × 0.25 mm, i.e., 0.25 µm film thickness) with a flame ionisation detector (FID) [21,47]. The GC oven temperature was programmed at 70 • C (initial temperature), held for 4 min, then increased at a rate of 4 • C/min to 220 • C and held for 5 min. The injector temperature was 240 • C, the detector temperature was 260 • C, and the samples were injected in split mode. The carrier gas was nitrogen at a column flow rate of 1.05 mL/min (100 kPa). The sample's retention indices (RI) were determined based on homologous n-alkane hydrocarbons under the same conditions.

GC-MS Analysis and Identification
The gas chromatography-mass spectrometry (GC-MS) analysis of EOs carried out using a Shimadzu QP 2010 using a DB-5 (J&W Scientific, Folsom, CA, USA) capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness) [9]. The GC oven temperature was 70 • C for 4 min and then increased to 220 • C at 4 • C /min and held for 5 min. The injector temperature was 240 • C, the interface temperature was 250 • C, the mass acquisition range was 800-50 amu, and the ionisation energy was 70 eV. The carrier gas used was Helium. Compounds were identified using a library search of the National Institute of Standards and Technology (NIST) database [63], as well as by comparing their RI and mass spectral frame pattern with those reported in the literature [22].

Fumigant Toxicity of Essential Oils and Their Combinations against the Pulse Beetle
Five different test concentrations (625 to 10,000 µL/L) of EOs viz., A. calamus, H. spicatum, J. recurva, J. communis, L. angustifolia, P. wallichiana, and C. deodara and their combinations (1:1 ratio) were taken for bioassay against the adults of pulse beetle for the synergistic activity. The experiments were carried out in 15 mL glass vials. Whatman No. 9 filter paper was inserted in the inner portion of the vial cap, and EO was released into the filter papers. The vials were kept in controlled laboratory conditions to record the adult mortality at different intervals. There were five treatments per EO, and each was replicated five times. Aluminium phosphide (0.5-0.9 mg/100 g grain) was also tested as a positive control against adults of C. maculatus. Observations on mortality were recorded from 24 to 120 h after treatment for EOs and their combinations/binary mixtures to calculate LC 50 values and the co-toxicity coefficient for binary mixtures.

Repellent Activity of Essential Oils against the Pulse Beetle
The repellency of EOs against pulse beetle was studied as per the suggested methodology [21,64]. Five concentrations (62.5 to 1000 µL/L) were prepared, and each concentration/treatment was replicated five times. Whatman No. 9 filter paper (diameter 9 cm) was cut and marked with a pencil into two halves, each labelled as untreated (UT) and treated (T). Filter papers were transferred to Petri plates (diameter 9 cm), treated with required concentrations of EOs, and then allowed to air dry for 5 min. Ten adults (2-3 days old) were released in the centre of the Petri plate, and the Petri plates were sealed with parafilm to prevent the escape of adults. Observations on repellency were recorded after 1, 2, 3, 4, 5, and 24 h of treatment to calculate RC 50 values.

Ovipositional Deterrent Activity of Essential Oils against the Pulse Beetle
Ovipositional deterrence of EOs against pulse beetle was studied as per the suggested methodology [64]. There were five concentrations (625 to 10,000 µL/L). Five concentrations were made by mixing EOs in Tween 80. Seeds (20 no./plate) were dipped in different concentrations for 10 s, then removed and placed on filter paper to air dry for 10-15 min. Treated seeds were placed in a Petri plate (diameter 9 cm), and then ten adults of one-day old (5 male and 5 female) were released. Petri plates were sealed with parafilm to prevent the escape of the adults. For the control, seeds were treated with Tween 80 only. Each treatment was replicated five times. The number of eggs laid on seeds of green gram was observed from 24 to 72 h. Percent oviposition inhibition was calculated by using the formula [65].

OI = [(NC − NT)/NC] ×100
(2) where NT = No. of eggs in untreated and NT = No. of eggs laid in treated. Detoxification enzymes, i.e., Acetylcholinesterase (AChE) and Glutathione-S-Transferase (GST) inhibition activities, were completed as per the standard methods [21]. Four different concentrations of A. calamus and L. angustifolia EO (10,000, 5000, 2500, and 1250 µL/L) were taken for both C. maculatus and C. chinensis for detoxification enzyme inhibition activity. The adults alive after 24 and 48 h (5-8 adults weighing 20 mg/concentration) were collected for enzyme assay. The adults in each test concentration were transferred to a centrifuge tube and homogenized phosphate buffer (pH 7.4) in a ratio of 1:9. The weight of an adult (mg): the volume of buffer (mL) was kept in a ratio of 1:9. The adults were then homogenized with a homogenizer in a micro pestle mortar. The homogenate was transferred immediately under ice bath conditions and then centrifuged at 4 • C and 12,000 rpm for 30 min. The supernatant was taken into a new centrifuge tube for protein estimation by Bradford assay [66] for all the concentrations before proceeding with enzyme assays. The same assay was repeated thrice for separate homogenates, and then average values were taken for protein estimation.

Protein Estimation
Protein estimation was done using the Bradford method [66] by adding 2 µL of homogenate, 38 µL of MilliQ to 160 µL of Bradford reagent in triplicates. After incubation of the mixture for 15 min at room temperature, the absorbance was measured at 595 nm. Absorbance was converted into protein concentrations, and dilutions were made concerning lower concentrations for the AChE assay.

Acetylcholinesterase Assay
The diluted 25 µL homogenates in triplicates were incubated for 30 min at room temperature with 25 µL of the reaction mixture (50 µL of DTNB, 50 µL of Acetothiocholine, and 900 µL of assay buffer). The AChE activity was spectrophotometrically measured at 410 nm in a microplate reader (Biotek, Synergy H 1 Hybrid Multi-Mode reader) and represented as milliunits per milligram of protein (mU/mg). For the determination of AChE, the Acetylcholinesterase Assay Kit was procured from Abcam, UK.

Glutathione-S-Transferase Assay
The reaction contains 100 µL of the solution, in which 75 µL of assay buffer, 10 µL of the homogenised sample, and 10 µL of glutathione were added. To start the reactions, 5 µL CDNB was added to each well in triplicates to the microplate at room temperature. These reaction mixtures were incubated at RT in a 96-well microplate. The enzyme kinetics were measured at the absorbance of 340 nm at 37 • C for 20 min in a microplate reader with continuous mixing for 10 s after 60 s of lag time. The extinction coefficient of 0.0096 µM −1 for CDNB was used to calculate the Glutathione-S-transferase activity and represented as nanomolar per minute per millilitre of a sample (nmol/min/mL). To determine the GST enzyme, the Glutathione-S-transferase Assay Kit was procured from Cayman Chemical, 1180 E, Ellsworth Road, Ann Arbor, MI, USA.

Statistical Analysis
The data on fumigant toxicity, synergistic activities, ovipositional inhibition, and repellence of different EOs was compiled. Lethal concentration (LC 50 ) and repellent concentration (RC 50 ) values were calculated by Probit analysis [67] using SPSS software v. 16.0. The data on fumigant toxicity and ovipositional inhibition were subjected to multivari-ate analysis, and enzyme inhibition was subjected to one-way ANOVA by SPSS software. Means were compared by Tukey's post hoc test to know the significant differences between treatments.

Conclusions
A. calamus, L. angustifolia and H. spicatum oil alone showed promising fumigant toxicity against both C. maculatus and C. chinensis. Among combinations, A. calamus oil with L. angustifolia and C. deodara against C. chinensis (LC 50 = 92.18-118.54 µL/L) and A. calamus with L. angustifolia and P. wallichiana against C. maculatus (LC 50 = 204.01-312.23 µL/L) showed promising toxicity/synergistic. A. calamus and H. spicatum also showed promising repellence to both species of pulse beetle. The insecticidal activities of promising EOs may be due to the presence of cis-asarone (85.37%), 1,8-cineole (28.31%) and 2-β-pinene (39.18%) are the major constituents. All the concentrations of A. calamus and L. angustifolia significantly inhibited the GST and AChE in both species of pulse beetle, so these enzymes may be the target site of action for tested oils in pulse beetle. Therefore, these promising EOs may be recommended to control pulse beetle where the grains are stored in bins and big godowns (staked grains) subject to large-scale trials.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules28020492/s1, The chemical composition and ovipositional deterrent activities of seven EOs are represented in the supplementary tables: Table S1, Chemical composition of essential oil of H. spicatum. Table S2, Chemical composition of essential oil of C. deodara. Table S3, Chemical composition of essential oil of J. communis. Table S4, Chemical composition of essential oil of J. recurva. Table S5, Chemical composition of essential oil of L. angustifolia. Table S6, Chemical composition of essential oil of P. wallichiana. Table S7, Ovipositional inhibition effect of different oils against adults of C. maculatus. Table S8, Ovipositional inhibition effect of different oils against adults of C. maculatus. Table S9, Ovipositional inhibition effect of different oils against adults of C. maculatus. Table S10, Ovipositional inhibition effect of different oils against adults of C. chinensis. Table S11, Ovipositional inhibition effect of different oils against adults of C. chinensis. Table S12, Ovipositional inhibition effect of different oils against adults of C. chinensis.