Ovicidal and Physiological Effects of Essential Oils Extracted from Six Medicinal Plants on the Elm Leaf Beetle, Xanthogaleruca luteola (Mull.)

Plant essential oils may serve as safe alternatives to detrimental synthetic pesticides due to relatively lower side effects on the environment and non-targeted organisms. The current study was conducted to investigate the ovicidal toxicity and physiological disruptions of six medicinal plant essential oils, including Artemisia annua L., Lavandula angustifolia Mill., Origanum vulgare L., Rosmarinus officinalis Spenn., Satureja hortensis L., and Thymus vulgaris L., on elm leaf beetle Xanthogaleruca luteola (Mull.). The LC50 (Lethal Concentration to kill 50% of tested insects) values of 122.8, 287.5, 152.8, 180.6, 315.9, and 1366.2 ppm were recorded for T. vulgaris, L. angustifolia, A. annua, S. hortensis, R. officinalis, and O. vulgare, respectively, 72 h after treatment of 3-day-old eggs of the pest. Significant decreases in the amounts of glucose, protein, and triglyceride macromolecules were also observed after treatment. The application of essential oils derived from T. vulgaris, A. annua, and S. hortensis at 400 ppm revealed 100% ovicidal activity. Accordingly, tested essential oils, particularly the essential oil of T. vulgaris, have been promising potential as biorational insecticides in the management of X. luteola.


Introduction
The elm leaf beetle, Xanthogaleruca luteola Müller (Coleoptera: Chrysomelidae), is a defoliating pest of elm trees (Ulmus spp.) widely distributed in Europe, North Africa, Asia, Australia, and temperate areas in North and South America [1]. Elm leaf beetle adults hibernate overwinter in bark fissures and woodpiles and emerge in spring to feed on elm foliage for 1-2 weeks before commencement of egg-laying. Adults feed through leaves, more often in a shot hole pattern. The eggs of elm leaf beetle are bright yellow and laid in clusters, averaging 14-20 eggs per cluster, that are mainly located on the underside of a leaf. Both larvae and adults of X. luteola feed on the emergent leaves. The larvae of the elm leaf beetle can feed on elm leaves for 2-4 weeks, undergo three instars, and cause more damage than adults by skeletonizing the leaf surface [2]. Severe infestations by elm leaf beetle larvae can cause physiological stress, defoliation of trees, and enhance the susceptibility of host plants to other pests and diseases [3]. Since elm trees are widely planted in cities and suburbs, the excessive use of chemical pesticides to protect them from the population was mixed with 1000 mL distilled water and kept for 24 h. Samples were subjected to 2 h hydrodistillation in a Clevenger-type apparatus. This process was repeated several times to receive the required amount of aqueous/oil distillate (about 3 mL). The oil phase was isolated from the obtained solution by dehydration over anhydrous sodium sulfate (Na 2 SO 4 ) (Merck Co., Darmstadt, Germany). Extracted essential oils were stored in standing 1.5 mL microtubes covered with aluminum foil and kept at 4 • C. Table 1. List of plants used for exploring the ovicidal effect of their essential oils on elm leaf beetle eggs.

Assessment of the Ovicidal Effect of Essential Oils
To assess the direct ovicidal effect of essential oils on elm leaf beetle eggs, 3-dayold eggs were randomly collected from the mass-reared colonies and exposed to EOs concentrations diluted in distilled water+Tween-80 by egg dipping method. Experiments were performed with 3 replications; 5 eggs-masses (12-15 eggs) per replication were exposed to EOs for 30 sec and left in Petri dishes (10 cm) to hatch. Untreated controls were exposed to only distilled water+Tween-80 for 30 secs. Egg mortality was determined for individual eggs within each egg mass per each treatment using a stereomicroscope. Percent ovicidal activity was assessed after 72 h exposure to EOs by the following formula [29]: Ovicidal Percentage = Number of eggs hatched in control − Number of eggs hatched in treatment Number of egg hatched in control × 100

Preparation of Eggs Homogenates for Biochemical Assay
After 72 h of treatment of X. luteola eggs with EOs at the concentrations of 100, 200, 300, and 400 ppm, about 10 g unhatched eggs from each treatment and control groups were weighed and homogenized by a glass pestle in 0.5 mL of distilled water. The homogenate was centrifuged at 13,000× g at 4 • C for 10 min. The homogenized egg supernatant was transferred into a 1.5 mL Eppendorf ® tube and stored at −20 • C. Biochemical assays were conducted with 3 replicates.

Protein Assay
The protein concentration of the homogenized egg supernatant treated with EOs was determined via Biuret's method [30] using a total protein assay kit (Biochem Co., Tehran, Iran).

Determination of Glucose
Glucose concentration of the homogenized egg supernatant, treated with EOs was determined by automated enzymatic assays using a glucose assay kit (Biochem Co., Tehran, Iran) as described by Siegert [31] with some modifications: reagent A and reagent B were mixed (4:1) followed by addition of samples. Glucose levels were estimated by measuring reduction in absorption at 492 nm in an ELISA reader (Awareness, Temecula, CA, USA).

Determination of Triglyceride
Triglyceride concentration of the homogenized egg supernatant, treated with EOs, was determined by a triglyceride kit (Biochem Co., Tehran, Iran) according to protocol described by Fossati and Prencipe [32]. The reducing absorption rate for triglyceride analysis was read at 545 nm in an ELISA reader (Awareness, Temecula, CA, USA).

Statistical Analysis
Control mortality was rare, and where corrections for mortality were necessary, these were accomplished using Abbott's formula [33]. Egg mortality and lethal concentrations of EOs were determined 72 h after treatment, and data were analyzed by POLO-PC software to calculate LC 50 values as the effective concentrations to follow the biochemical experiments. Relative potency (RP) for each mixture was determined based on O. vulgare (lowest toxicity) and calculated using the following formula: RP = LC 50 of O. vulgare essential oil/LC 50 of individual essential oil. Raw data obtained from the biochemical assay and ovicidal bioassay were subjected to a one-way statistical analysis of variance test (ANOVA) for significant differences in the measured parameters. The Tukey-Kramer test at a 5% significance level was considered for comparison of means using SAS statistical software.

Determination of Triglyceride
Triglyceride concentration of the homogenized egg supernatant, treated with EOs, was determined by a triglyceride kit (Biochem Co., Tehran, Iran) according to protocol described by Fossati and Prencipe [32]. The reducing absorption rate for triglyceride analysis was read at 545 nm in an ELISA reader (Awareness, Temecula, CA, USA).

Statistical Analysis
Control mortality was rare, and where corrections for mortality were necessary, these were accomplished using Abbott's formula [33]. Egg mortality and lethal concentrations of EOs were determined 72 h after treatment, and data were analyzed by POLO-PC software to calculate LC50 values as the effective concentrations to follow the biochemical experiments. Relative potency (RP) for each mixture was determined based on O. vulgare (lowest toxicity) and calculated using the following formula: RP = LC50 of O. vulgare essential oil/LC50 of individual essential oil. Raw data obtained from the biochemical assay and ovicidal bioassay were subjected to a one-way statistical analysis of variance test (ANOVA) for significant differences in the measured parameters. The Tukey-Kramer test at a 5% significance level was considered for comparison of means using SAS statistical software.
Concentration-dependent toxicity was determined for all EOs. Untreated insects in control groups indicated mortality ranging from 5-10%, which were corrected through Abbott formula. All doses of EOs revealed ovicidal activity on X. luteola by the maximum mortality of 100 ± 00, 92.76 ± 3.17, 100 ± 00, 100 ± 00, 74.61 ± 2.31, and 21 The LC 50 values were estimated for each EO. T. vulgaris essentials showed the highest ovicidal toxicity with the lowest LC 50 of 122.8 ppm, indicating the highest relative potency ( Table 2). The LC 50 value estimated for A. annua EO was 152.8 ppm, which was much lower than the LC 50 value of Satureja hortensis EO (180.6 ppm), indicating a better efficacy and ovicidal activity followed by T. vulgaris (Table 2).
The LC50 values were estimated for each EO. T. vulgaris essentials showed the highest ovicidal toxicity with the lowest LC50 of 122.8 ppm, indicating the highest relative potency ( Table 2). The LC50 value estimated for A. annua EO was 152.8 ppm, which was much lower than the LC50 value of Satureja hortensis EO (180.6 ppm), indicating a better efficacy and ovicidal activity followed by T. vulgaris (Table 2).

Total Glucose of X. luteola Eggs Treated
Profiling the glucose level in eggs per each treatment revealed a higher impact of O. vulgare in reducing the total glucose content even at its lower concentrations (

Total Glucose of X. luteola Eggs Treated
Profiling the glucose level in eggs per each treatment revealed a higher impact of O. vulgare in reducing the total glucose content even at its lower concentrations ( Figure 3) (F = 15.97; df = 5, 6; p < 0.05).

Total Triglyceride of X. luteola Eggs Treated
Profiling total triglyceride content of X. luteola eggs after exposing with A. annua EO at 400 ppm revealed higher reductions by significant differences ranging from 1.68 ± 0.09 mg/dL to 0.92 ± 0.05 mg/dL compared to triglycerides level assessed for control (F = 31.42; df = 5, 6; p < 0.001) (Figure 4). This is suggesting that the higher concentrations of A. annua were more effective than other applied concentrations of EOs in reducing the lipid contents of the X. luteola egg cells because there were no significant differences among the used concentrations. Agronomy 2021, 11, x FOR PEER REVIEW 6 of 11

Total Triglyceride of X. luteola Eggs Treated
Profiling total triglyceride content of X. luteola eggs after exposing with A. annua EO at 400 ppm revealed higher reductions by significant differences ranging from 1.68 ± 0.09 mg/dl to 0.92 ± 0.05 mg/dl compared to triglycerides level assessed for control (F = 31.42; df = 5, 6; p < 0.001) (Figure 4). This is suggesting that the higher concentrations of A. annua were more effective than other applied concentrations of EOs in reducing the lipid contents of the X. luteola egg cells because there were no significant differences among the used concentrations.

Discussion
The use of plant essential oils to control pests is gaining popularity, as they may provide an eco-friendly alternative to current synthetic insecticides in the control of elm leaf

Total Triglyceride of X. luteola Eggs Treated
Profiling total triglyceride content of X. luteola eggs after exposing with A. annua EO at 400 ppm revealed higher reductions by significant differences ranging from 1.68 ± 0.09 mg/dl to 0.92 ± 0.05 mg/dl compared to triglycerides level assessed for control (F = 31.42; df = 5, 6; p < 0.001) (Figure 4). This is suggesting that the higher concentrations of A. annua were more effective than other applied concentrations of EOs in reducing the lipid contents of the X. luteola egg cells because there were no significant differences among the used concentrations.

Discussion
The use of plant essential oils to control pests is gaining popularity, as they may provide an eco-friendly alternative to current synthetic insecticides in the control of elm leaf

Discussion
The use of plant essential oils to control pests is gaining popularity, as they may provide an eco-friendly alternative to current synthetic insecticides in the control of elm leaf beetle [1,34,35]. Different plant EOs including A. annua, Artemisia sieberi Besser, L. angustifolia, Melia azedarach L., Pteridium aquillinum L., and T. vulgaris have previously been studied for their deterrent, toxic, and developmental inhibitory activities against X. luteola at the larval stage. The EOs derived from A. annua, A. sieberi [1,36], T. vulgaris, L. angustifolia [34], and R. officinallis [37] have shown repellence, toxicity, biochemical, behavioral, and physiological properties against X. luteola. However, the present study offers the first evidence of the ovicidal effect of A. annua, L. angustifolia, O. vulgare, R. officinalis, S. hortensis, and T. vulgaris EOs against X. luteola eggs. All applied EOs exhibited a degree of ovicidal activity against X. luteola, in which ovicidal activity of T. vulgaris, A. annua, and S. hortensis EOs was promising. Within this study, the difference observed in the level of ovicidal activities for each EO is presumably related to the difference in the accumulation level of active substances in each plant, such as tannic acid, which has embryotoxic effects on insects' eggs and larval specifically at high concentration levels [38]. It has been reported that the phenolic compounds of EOs affect the movement and vital systems of the fetus, and they have inhibitory effects on gas exchange inside the egg, which leads to hardening of the crust and direct influence on protoplasm, causing the death of the fetus inside the egg [38]. The ovicidal effects of thyme and lavender against Tetranychus urticae Koch were previously investigated, indicating that lavender oil was the most protective against this spider mite [39]. Bedini [24] reported the oviposition deterrence and toxicity effect of L. angustifolia on adults of common green bottle fly (Lucilia sericata Meigen). In general, many plant EOs have been reported to have an ovicidal effect and high potential to be developed into oviposition deterrent, ovicidal, and adulticidal agents for controlling populations of insects [40][41][42].
After the treatment of elm leaf beetle eggs with EOs, total protein, glucose, and triglyceride levels declined. Total protein, glucose, and triglyceride are the three required storage macromolecules closely linked with many metabolic pathways. Reduction in the amounts of these macromolecules as a result of insecticidal toxicity can influence several physiological functions such as energy demand, tissue repair, reproduction, etc. Plant EOs can coagulate the cytoplasm and crashed lipids and proteins [43]. Damage to the cell wall and membrane can lead to the leakage of macromolecules and lysis [44]. The result shows that the amount of total protein decreased in the eggs after exposure to different concentrations of EOs. This phenomenon could be due to the breakdown of proteins into free amino acids, which are transferred into Kreb's cycle [45]. The declines that were observed in the amounts of triglyceride and glucose caused by insecticide could also be attributed to an interruption in the absorption system [46].
The results of recent studies on the chemical profile of A. annua, L. angustifolia, O. vulgare, R. officinalis, S. hortensis, and T. vulgaris essential oils are reviewed and summarized in Table 3. Terpenes including monoterpene hydrocarbons such as α-pinene and β-pinene, oxygenated monoterpenes such as 1,8-cineole and thymol, and sesquiterpene hydrocarbons such as β-selinene were the main components of all studied essential oils except S. hortensis. Phenylpropanoid compounds estragole and eugenol were the main components of S. hortensis essential oil. Table 3. Main components of the essential oils of Artemisia annua, Lavandula angustifolia, Origanum vulgare, Rosmarinus officinalis, Satureja hortensis, and Thymus vulgaris.

Conclusions
Due to their volatile nature and lower residual characteristic of plant EOs, there is a much lower level of risk from EOs to the environment and pollinator insects comparing to current synthetic pesticides [58]. However, some EOs, such as thyme, have an impact on the movement and wax production by pollinator insects such as the western honeybee (Apis mellifera L.) [59]. Therefore, some EOs, such as thyme, should be applied at suitable times (large infestations) and with the concentrations recommended to reduce the inhibitory impacts on the hive. The reactivity of plant EOs depends upon the condition of their growing environment and their composition, which includes different concentrations of aldehydes, phenolics, terpenes, and other natural compounds and the orientation of their functional groups [60]. Further investigations must be undertaken to find the optimal dosage of T. vulgaris, A. annua, and S. hortensis for controlling target insects with the least mortal impacts on non-target organisms and beneficial insects such as hives. This requires further efforts to explore the molecular mechanisms of these essential oils and the effects of their individual chemical compounds on other insects and organisms. The result obtained from the present study indicates that the EOs of T. vulgaris, L. angustifolia, A. annua, S. hortensis, R. officinalis, and O. vulgare have ovicidal effects on elm leaf beetle X. luteola and an irreversible effect on some key biochemical compounds in this pest. It is expected that plant biorational pesticides will find their greatest commercial application in urban pest control. The ovicidal effects of essential oils reveal that these plants may be a good and safe natural toxicant that warrants further investigation.