Chemical Composition, Insecticidal, Persistence and Detoxification Enzyme Inhibition Activities of Essential Oil of Artemisia maritima against the Pulse Beetle

Pulse beetle is the major pests of pulses that cause significant loss to grains leads to unfit for consumption and marketing. Indiscriminate use of synthetic pesticides for the control of pulse beetle (Callosobruchus chinensis and Callosobruchus maculatus) led to insect resistance, pesticide residues on grains which affect consumer’s health and environment. Essential oils (EOs) are good alternatives to synthetics due to their safety to the environment and consumers’ health. The main objective of the present study was to explore the chemical composition, fumigant, repellency, ovipositional deterrence, persistence, and detoxification enzyme inhibition of Artemisia maritima essential oil against pulse beetle. Results showed that primary components of the EO were 1,8-Cineole and bornyl acetate. EO showed promising fumigant toxicity to C. chinensis and C. maculatus (LC50 = 1.17 and 0.56 mg/L, respectively) after 48 h. In the repellent assay, EO at 8 mg/L showed 92–96% repellence after 1 h. In ovipositional deterrence assay, EO showed more ovipositional deterrence against C. chinensis (OD50 = 3.30 mg/L) than C. maculatus (OD50 = 4.01 mg/L). Higher concentrations of oil (8 and 6 mg/L) in C. maculatus showed significant inhibition of the glutathione-S-transferase enzyme (7.14 and 5.61 n mol/min/mL, respectively).


Introduction
Infestation of bruchids and lepidopterans pests causes significant damage to grains and their products in storage. More than 500 species of the stored grains and cereal products are often infested by more than 600 species of coleopterans [1] causing 20-30% loss, affecting nutritional value and germination [2]. Bruchids, Callosobruchus chinensis and C. maculatus (Coleoptera: Bruchidae), are primary pests of pulses and cause 50% loss in storage after three to four months [3,4]. The grub's bore into grains, feed internal contents, affecting nutritional quality [5]. In severe infestation, seeds become completely hollow and unsuitable for marketing [6]. The control of stored grain pests generally depends on synthetic insecticides, including fumigants [5,7]. The use of synthetic insecticides resulted in several negative effects in the environment, natural enemies, human health, and resistance development in insects. Now the focus is shifted to search of potent insecticide from natural origin. Plant essential oils (EOs), its monoterpenoids and sesquiterpenoids are known to possess significant insecticidal activities against stored grain pests [8][9][10] The EOs are extracted from different parts (leaves/flowers/seeds/bark) of aromatic and medicinal plants. Nearly 10% of EOs used in aromatic, flavor and fragrance industries [11].

Fumigant and Persistence Toxicity of A. maritima Oil against Pulse Beetle
Fumigant toxicity and persistence of A. maritima oil against pulse beetle were presented in Tables 2-4. The oil showed more promising toxicity against C. maculatus (LC 50 = 1.91 and 0.56 mg/L) after 24, and 48 h of treatment respectively as compared to C. chinensis (LC 50 = 2.06 and 1.17 mg/L) ( Table 2). Based on persistence study, A. maritima oil showed significantly more promising in protecting the grains/seeds (Table 3) against C. chinensis (82% mortality) up to 10 days of storage (F 3,19 = 55.30; p < 0.0001) as compared to C. maculatus (44% mortality) (F 3,19 = 33.47; p < 0.0001). A. maritima oil showed moderate residual toxicity of 10-20 days only, later toxicity was gradually decreased and became 8 and 0% mortality, against C. chinensis and C. maculatus, respectively after 40 days of storage. A. maritima oil also showed residual toxicity with the lethal time taken to kill 50% of test insects was 4.49 days and 9.33 days for C. chinensis and C. maculatus, respectively ( Table 4). The seeds used for the persistence study also showed 100% germination within 24 h.

Ovipositional Deterrence of A. maritima Oil against Pulse Beetle
Results on ovipositional deterrence of A. maritima oil against C. chinensis and C. maculatus was presented in Table 6 and Tables S2 and S3. A. maritima oil was showed promising deterrence against C. chinensis (OD 50 = 2.3, 3.09 and 3.30 mg/L) after 24, 48, and 72 h of treatment, respectively (Table 6) as compared to C. maculatus (OD 50 = 2.89, 3.36 and 4.01 mg/L). With respect to percent deterrence against C. chinensis, the A. maritima oil at higher concentration (12 mg/L) reported 100% deterrence against C. chinensis at 24 h of treatment (F 4,24 = 16.08; p < 0.0001) and was at par with other concentrations (78.2 to 94.38% deterrence) except 2 and 1 mg/L. Similarly, 48 and 72 h after treatment, A. maritima oil at 12 mg/L showed higher deterrence (98.28-100%) and was at par with 8 mg/L (92.34 to 93.28% deterrence) and was followed by 4 and 2 mg/L which were at par as compared to lower concentration. Similarly, for C. maculatus, A. maritima oil at 8 mg/L showed 100% deterrence after 24 h against C. maculatus (F 4,24 = 30.38; p < 0.0001) and was at par with 4 mg/L (86.54%) followed by 2 mg/L (70%) as compared to lower concentrations. At 48 h after treatment same trend was observed as that of 24 h. At 72 h, A. maritima oil at 8 mg/L showed significantly higher deterrence (81.78%) (F 4,24 = 41.78; p < 0.0001) and was at par with all other concentrations except lower concentration.

Detoxification Enzyme Inhibition of A. maritima Oil against Pulse Beetle
Detoxifying enzyme (AChE and GST) activities of A. maritima oil against C. chinensis and C. maculatus after 12 h of treatment is presented (Table S4 and Figure 1). Significant differences were not observed (p > 0.001) among the concentrations of oil (4 to 10 mg/L) in inhibiting the enzyme GST and AChE in C. chinensis. Similarly, higher concentrations of A. maritima oil at 6 and 8 mg/L significantly inhibited the GST (7.14 and 5.61 n mol/min/mL) (F 4,14 = 14.6; p < 0.003) and AChE (mU/mg) (F 4,14 = 8.14; p < 0.003) in C. maculatus and were at par as compared to lower concentrations and control.

Detoxification Enzyme Inhibition of A. maritima Oil against Pulse Beetle
Detoxifying enzyme (AChE and GST) activities of A. maritima oil against C. chinensis and C. maculatus after 12 h of treatment is presented (Table S4 and Figure 1). Significant differences were not observed (p > 0.001) among the concentrations of oil (4 to 10 mg/L) in inhibiting the enzyme GST and AChE in C. chinensis. Similarly, higher concentrations of A. maritima oil at 6 and 8 mg/L significantly inhibited the GST (7.14 and 5.61 n mol/min/mL) (F4, 14 = 14.6; p < 0.003) and AChE (mU/mg) (F4, 14 = 8.14; p < 0.003) in C. maculatus and were at par as compared to lower concentrations and control.

Discussion
The chemical composition, insecticidal, and enzyme inhibition activities of A. maritima oil against C. chinensis and C. maculatus are discussed. Present results revealed that
The insecticidal activities of A. maritima oil against targeted insects in the present study may be due to the presence of major compounds including 1,8-cineole, bornyl acetate, myrcene, and sabinene. Current results also confirmed with the earlier studies in which insecticidal activities of oils are due to monoterpenoids and sesquiterpenoids which are volatile and rather lipophilic compounds that can penetrate insect cuticle and interfere with their physiological functions [45][46][47]. Du to th volatile nature of EOs, they act as a fumigant and kill the stored-grain insects by asphyxiation. The insecticidal activities also depend on nature and type of components, application dose/concentrations [48][49][50].
The chemical constituents present in the EOs and their blends, inhibit the GST activity [58][59][60]. In the current studies, A. maritima oil was not significantly inhibiting GST/AChE enzyme in C. chinensis and AChE in C. maculatus. However, higher concentrations of A. maritima oil showed inhibition of GST enzyme in C. maculatus and these results confirm the findings of previous work in which A. brachyloba oil significantly inhibited the GST in T. castaneum after 24 h but the same oil also inhibit the AChE after 60 h of treatment [61]. In a similar study, eucalyptol and caryophyllene oxide isolated from EO of A. lavandulaefolia showed inhibition of GST and CarE in the larvae of Plutella xylostella after 24 h [26]. Based on insecticidal activities, A. maritima oil showed promising fumigant toxicity, repellence, ovipositional deterrence, and enzyme inhibition (GST and AChE) activities against C. chinensis and C. maculatus.

Plant Material
The plant material was collected from Keylong (Lahaul & Spiti District, H.P, India) (latitude 32.571 • N and longitude 77.041 • E) of Himachal Pradesh during September 2019. The specimens are authenticated by the Taxonomist, and a voucher specimen (PLP 17794) was deposited in the herbarium.

Extraction of A. maritima Oil
The whole plant (aerial parts) material of A. maritima was dried under shade for 10 days and chopped into small pieces. About 10 kg plant material was used for extraction of oil by hydro-distillation in Clevenger apparatus as per the method followed [16]. The yield obtained was 3.4 mL and was kept below 4 • C until further use.

Gas Chromatography Analysis
The composition of EO 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.d., 0.25 µm film thickness) with a flame ionization detector (FID) [16,62]. The GC oven temperature programmed at 70 • C (initial temperature) held for 4 min and 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, 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.

Test Insect
C. chinensis and C. maculatus were maintained on green gram seeds in plastic jars under controlled conditions (27-28 • C and 60 ± 5% humidity) in the Entomology laboratory, Agrotechnology Division, CSIR-IHBT, Palampur for >50 generations. The newly emerged adults (2-3 days old) were used for the study.

Fumigant Toxicity of A. maritima Oil against Pulse Beetle
Five different concentrations of A. maritima oil for C. chinensis (10,8,6,4, and 2 mg/L) and C. maculatus (8, 4, 2, 1, and 0.5 mg/L) were prepared based on preliminary evaluation for dose-response bioassay against pulse beetle adults. The fumigant toxicity assay was studied on glass desiccators (2.5 L capacity). Five grams of green gram were taken in a glass Petri dish and kept at the bottom of the desiccators in which 10 adults are released. In another Petri dish, Whatman No. 9 filter paper was placed and kept at the top of the desiccators. Five concentrations of A. maritima oil were applied on the filter paper separately by using a micropipette and then the lid of the desiccators was closed to make it airtight. The desiccators were kept in the controlled laboratory conditions for recording the mortality at 24 h intervals. There are five treatments/concentrations, and each treatment was replicated thrice.

Persistence of A. maritima Oil against Pulse Beetle
The persistence of A. maritima oil was carried out as per the standard method followed by Nenaah [8]. Higher concentrations (4 and 8 mg/L) of oil were used against C. chinensis and C. maculatus, respectively. Briefly, sterilized green gram seeds were treated with two concentrations of EO and seeds were vigorously hand-shaken for 20-30 s for thorough coating. After evaporation of the solvent, the treated grains were packed in jute sacks (20-30 cm) and stored in dark conditions. Samples of treated seeds (20 g) were withdrawn after 10, 20, 30, and 40 days of treatment and then 10 adults (1 day old) are released in the Petri-dish (9 cm diameter). The same no. was also used for the control treated with 0.05% of Triton-X 100 LR water. Each treatment was replicated five times. The insects were exposed to treated seeds were continued for 48 h and then mortality was recorded. For germination study, the treated green gram seeds with A. maritima oil were placed on moistened cotton in a Petri dish and incubated under laboratory conditions. Observations on the number of seeds germinated were recorded after 24 and 48 h.

Repellent Activity of A. maritima Oil against Pulse Beetle
Repellent activity of A. maritima oil was tested against C. chinensis and C. maculatus as per the method followed by Eccles et al. [63]. Briefly, five concentrations (8, 6, 4, 2, 1 mg/L) were prepared from stock solutions. The Whatman No. 9 filter paper (diameter 9 cm) was cut and marked with a pencil into two halves and each labeled as treated (T) and untreated (UT). Filter papers were transferred to Petri plates (diameter 9 cm) and treated with required concentrations of EOs and then allowed to air dry for 15 min. Ten adults (3-4 days old) were released in the center of the filter paper containing ten grains, and the plates were sealed with parafilm to prevent the escape of adults. The dispersal of the beetles on each side of the filter paper was recorded after treatment. Observation on repellency was recorded at 1, 2, 3, 4, and 5 h after treatment. In this study, there were five treatments, and each treatment was replicated five times. About 250 insects were used in different treatments (5 treatments × 50 insects = 250 insects). The Percent repellency (PR) [64] was calculated based on the formula: PR = [(Nc − Nt)/(Nc + Nt)] × 100. Where Nc = number of insects on control half of filter paper after required exposure interval; Nt = number of insects on treated half of filter paper after required exposure interval.
The Repellent Index (RI) [65] was calculated based on the formula; RI = 2G/G+P. Where G = number of adults on the treated side and P = number of adults on the untreated side. The repellent index of EOs is considered as repellent, attractant, or indifferent based on the mean value of RI and its respective standard deviation (SD). If the mean RI is higher than 1 + SD, the oil is an attractant, while if the mean RI is less than 1-SD, the oil is repellent, and for the mean RI in between 1 − SD and 1 + SD, the oil is indifferent.

Ovipositional Deterrent Activity of A. maritima Oil against C. chinensis and C. maculatus
The ovipositional deterrent of A. maritima oil against C. chinensis and C. maculatus was studied as per the method followed by Eccles et al. [63]. Briefly, five concentrations (12,8,4, 2, 1 mg/L) for C. chinensis and C. maculatus (8, 6, 4, 2, 1 mg/L) were prepared from stock solutions by mixing EOs in acetone. Seeds (30 no./plate) dipped in different concentrations for 10s, then removed and placed on filter paper to air dry for 15 min. Treated seeds were placed in a Petri plate (diameter 9 cm) and then ten adults (5 male and 5 female) of one day old were released. Petri plates were sealed with parafilm to prevent the escape of the adults. For the control, seeds were treated with acetone only. There were five treatments, and each treatment was replicated five times. The number of eggs laid on seeds of green gram was observed from 24 to 72 h. The percentage of oviposition inhibition was calculated by using the formula [45].  Detoxification enzyme (Glutathione-S-Transferase and Acetylcholinesterae) inhibition activities were performed as per the standard methods [60,66,67]. Four different concentrations of A. maritima oil (4, 6, 8, and 10 mg/L for C. chinensis and 2, 4, 6, and 8 mg/L for C. maculatus) were chosen for detoxification enzyme inhibition activity based on fumigant toxicity assay described in Section 4.5. The adults who survived after 12 h of treatment (7-8 adults weighing 20 mg/concentration) were collected for enzyme assay. The adults in each test concentration were transferred to a centrifuge tube and homogenized in 0.1 M 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 (Tarsons Micro Pestle). The homogenate was transferred immediately under ice bath conditions and then centrifuged at 12,000 rpm and 4 • C for 30 min. The supernatant was taken into a new centrifuge tube for protein estimation by Bradford assay [68] for all the concentrations before proceeding for 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 [68] 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 with respect to lower concentrations for the AChE assay.

AChE 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, 900 µL of assay buffer). The AChE activity was spectrophotometrically measured at 410 nm in a microplate reader (Biotek SYNERGY H1 Microplate Spectrophotometer). The enzyme activities were expressed as micromolar per milligram protein per minute (µmol/min/mg). For the determination of AChE, the Acetylcholinesterase Assay Kit was procured from Abcam, UK.

GST Assay
The reaction contains 1000 µL of the solution, in which 75 µL of Assay buffer, 10 µL of the homogenized sample, 10 µL of glutathione were added. To initiate the reactions, 5 µL CDNB was added to each well in triplicates to microplate at room temperature. To measure the enzyme kinetics, a 96 well microplate was loaded with reaction solution and shaken for 10 s. After 60 s of lag time, the absorbance was read at 340 nm and the homogenates were read for 20 min at 37 • C with a microplate reader. The GST activity was determined from the extinction coefficient of 0.0096 µM −1 cm −1 for CDNB. The enzyme activities were expressed as micromolar per milligram protein per minute (µmol/min/mg protein). For the determination of GST and AChE 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, ovipositional deterrent, and persistence of A. maritima oil was compiled. Lethal concentration (LC 50 ), lethal time (LT 50 ), and ovipositional deterrence (OD 50 ) values were calculated by Probit analysis [69] using SPSS software v. 16.0. The data on repellency, ovipositional deterrence, persistence, and enzyme inhibition were subjected to one-way ANOVA by SPSS software, and means were compared by Tukey's post hoc test to know the significant differences between treatments. The assumptions of normality and homogeneity of variance test for different parameters/variables and no data transformations were required.

Conclusions
A. maritima oil showed promising fumigant toxicity (LC 50 = 0.56 to 1.17 mg/L) against pulse beetle after 48 h of treatment. Higher concentrations of oil (6 and 8 mg/L) significantly inhibited the GST enzyme in C. maculatus. However, the EO of A. maritima may be recommended for the control of pulse beetle particularly grains stored in bins based on a safe waiting period, persistence studies, and economics.
Supplementary Materials: The following are available online. Table S1: Repellent index of A. maritima oil against C. chinensis and C. maculatus. Table S2: Ovipositional inhibition of A. maritima oil against C. chinensis. Table S3: Ovipositional inhibition (deterrence) of A. maritima oil against C. maculatus. Table S4: Enzyme inhibition activity of A. maritima oil in C. chinensis and C. maculatus adults.