Target Enzymes of Origanum majorana and Rosmarinus officinalis Essential Oils in Black Cutworm (Agrotis ipsilon): In Vitro and In Silico Studies

Simple Summary The black cutworm, Agrotis ipsilon (Hufnagel), poses a significant threat to various crops. Marjoram (Origanum majorana) and rosemary (Rosmarinus officinalis) essential oils (EOs) were investigated for their toxicity using in vitro and in silico methods. GC-MS analysis identified the main constituents, with O. majorana being more toxic than R. officinalis to A. ipsilon larvae. R. officinalis EO inhibited Na+/K+ pump activity consistently, while O. majorana showed varied effects. Both EOs influenced detoxification enzymes differently over time. Molecular docking indicated a strong binding affinity of the main EO constituents to target enzymes. These findings suggest the potential of EOs as insecticides in integrated pest management programs, particularly in organic agriculture. Abstract In this study, in vitro and in silico approaches were employed to assess the toxicity of marjoram (Origanum majorana) and rosemary (Rosmarinus officinalis) essential oils (EOs) to A. ipsilon larvae. The study determined the activities of ATPases in the larvae after treatment with the LC20 and LC70 of each EO. α-esterase and glutathione-S-transferase (GST) activities were also determined after treatment with LC10 and LC30 of each EO. Furthermore, molecular docking was employed to determine the binding affinity of terpinene-4-ol and α-pinene, the major constituents of O. majorana, and R. officinalis EOs, respectively, compared to the co-crystallized ligand of α-esterase, diethyl hydrogen phosphate (DPF). Toxicity assays revealed that O. majorana EO was more toxic than R. officinalis EO to the A. ipsilon larvae at 96 h post-treatment. However, the LC20 and LC70 of the latter significantly inhibited the activity of the Na+-K+ pump at almost all intervals. The same concentrations significantly inhibited the Mg2+/Ca2+-ATPase and Ca2+ pump at 96 h post-treatment. In contrast, O. majorana EO showed a variable effect on the Na+-K+ pump across different time intervals. On the other hand, LC10 and LC30 of both EOs showed varied effects on α-esterase and GST over time. Molecular docking revealed energy scores of −4.51 and −4.29 kcal/mol for terpinene-4-ol and α-pinene, respectively, compared to a score of −4.67 for PDF. Our study demonstrated the toxicity of the tested EOs to A. ipsilon, suggesting their potential efficacy as insecticides.


Introduction
Agrotis ipsilon, the black cutworm, is a globally recognized insect pest of significant agricultural concern.Its impact is notable, particularly in Egypt, where it has garnered substantial research interest due to the extensive damage it inflicts on a variety of such crucial vegetable and field crops as corn, cotton, soybeans, beans, potatoes, clover, and tomatoes [1].Historically, the management of this pest has been heavily reliant on the use of insecticides [2].However, the exclusive dependence on insecticides as a rapid solution for pest eradication poses such challenges as resistance, secondary pest outbreaks, and health and environmental issues [3,4].These challenges have sparked interest in exploring naturally occurring compounds as alternatives for pest management [5][6][7][8].Recent studies have provided evidence that plant EOs could serve as an effective tool in managing this destructive pest [9][10][11][12][13][14].This shift towards more natural pest control represents a significant advancement in our approach to sustainable agriculture.
Plant EOs are produced as secondary metabolites within the secretory structures of plant organs.Based on their synthesis, these metabolites are classified into two chemical groups (i) terpenoids, and (ii) phenylpropanoids [15].Both groups have exhibited enormous potential as acute or chronic insecticides [6], insect growth regulators [16], or antifeedants [17,18] against a variety of insect species.Such effects may be correlated with the magnitude of biochemical changes in the test species.For example, [19] revealed that Lavandula multifida EO affected the detoxification enzymes α-esterase and glutathione-Stransferase (GST) of A. ipsilon and Spodoptera littoralis at 96 h post-treatment.Study [20] also reported that Cymbopogon citratus EOs inhibited the carboxylesterases (CarEs) and GST enzymes of A. ipsilon larvae.Study [21] reported that Arisaema fargesii EOs significantly affected the α-esterase, β-esterase, p-nitrophenyl acetate (p-NPA) esterase, and GST of Aedes aegypti larvae.Furthermore, numerous studies have validated the inhibitory potential of EOs on insect cytochrome P450s, CarEs, and GSTs establishing these enzymes as prospective target sites in insects [4,22].Nevertheless, the precise mechanisms underlying the enzyme inhibition by EOs remain unclear [23].
Adenosine triphosphate-hydrolyzing enzymes (ATPases) are transmembrane proteins that play a key role in a varied array of cellular functions across all kingdoms of life [24].These dynamic proteins transport solutes across membranes and act as molecular motors that use the energy of ATP hydrolysis to conduct such mechanical works as ion pumping, cellular metabolism, muscle movement, protein trafficking, unfolding, replication, and transcription [25].These transmembrane proteins were reported to be target sites for insecticides such as DDT, chlorpyrifos, beta cypermethrin, abamectin, thiamethoxam, and diafenthiuron [26][27][28].In addition, some reports have revealed that some plant compounds have insecticidal effects due to their inhibition of ATPases [29,30].However, to our knowledge, little or no data are available on the effect of Origanum majorana and Rosmarinus officinalis EOs on the activities of ATPases such as Na + /K + -ATPase (Na + /K + -pump), Mg 2+ /Ca 2+ -ATPase, and Ca 2+ -ATPase (Ca 2+ pump) in A. ipsilon.
In the realm of drug discovery, in silico methods play a pivotal role by enabling the virtual screening of millions of compounds within a short timeframe [47].This approach significantly reduces the initial costs associated with compound identification and enhances the likelihood of identifying promising drug candidates.Currently, a diverse array of molecular modeling techniques exists to facilitate drug discovery.These methods are primarily categorized based on their structural and ligand-based approaches [48] and one of the widely used techniques is molecular docking, which predicts the molecular orientation of a ligand within the receptor.Subsequently, the replacement of the ligand in the receptor is estimated using a scoring function [49].
The current study investigated the chemical composition of O. majorana and R. officinalis EOs and their insecticidal activity on A. ipsilon larvae.The primary objective is to delve into the effects of O. majorana and R. officinalis EOs on the ATPases, namely Na + /K + -ATPase, Ca 2+ -ATPase, and Mg 2+ /Ca 2+ -ATPase, to elucidate whether these enzymes are a possible target site for these EOs.Furthermore, our research aims to gain a comprehensive understanding of the biochemical repercussions of these EOs on A. ipsilon with a specific focus on the modulation of detoxifying enzyme activity, namely α-esterase and GST.Additionally, we performed a molecular docking analysis to recognize the amino acids' interactions, the lengths of hydrogen bonds (Å), the affinity (Kcal/mol), and the docking energy score of the major constituents of O. majorana and R. officinalis Eos, i.e., terpinene-4-ol and α-pinene, against the active site of α-esterase enzyme, compared to the co-crystallized ligand of this enzyme, DPF (diethyl hydrogen phosphate).

Insect
A strain of Agrotis ipsilon was reared in the laboratory (26 ± 1 • C, 65 ± 5% RH, 16:8 [L:D] h) [1], away from any insecticide exposure.The newly hatched larvae were placed in a clean glass jar (1 L) covered with muslin and secured with a rubber band.They were daily fed fresh castor bean (Ricinus communis L.) leaves until they reached the third larval instar [50].To avoid excessive cannibalism, the larvae were individually placed in small plastic cups (7.0 cm in diameter, 3.5 cm in height) with fresh castor bean leaves at the beginning of the fourth instar [50].The pupae were kept surrounded with paper towels in glass jars until they matured.The adult moths were transferred to larger jars supplied with hung pieces of cotton moistened with 10% sugar solution [51] and covered with black muslin strips for egg deposition [6].The eggs were collected daily and transferred to new jars, and the neonates were fed castor bean leaves as described above.

Essential Oils Preparation and GC-Mass Analysis
The EOs of Origanum majorana and Rosmarinus officinalis were extracted from fresh leaves as described by [13].The chemical composition of the EOs was identified using a GC Ultra-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) (Table S1 in the Supplementary File).

Insect Susceptibility Assay
The EOs of O. majorana and R. officinalis were examined for their bioactivity on the 2nd instar larvae (24 h old) of A. ipsilon.The castor bean leaf dipping method was used with five concentrations of the EOs: 0.5, 1.0, 2.0, 4.0, and 8.0 mg mL −1 [52].Three replicates (10 larvae/replicate) were prepared for each concentration.Water stock solutions of EOs were mixed in cups with Tween-20 (0.05%), as an emulsifying agent.In the control cups, the EO was replaced with water.The experiment was kept under the same insect-rearing conditions.After 24 h of larval feeding, the live larvae were transferred to clean jars and the treated castor bean leaves were replaced with untreated ones.Mortalities of larvae were recorded at 24, 48, 72, and 96 h post-treatment.The sublethal and lethal concentrations (LC 10 , LC 20 , LC 30 , and LC 70 ) of the EOs at 96 h post-treatment were calculated using probit analysis [53].

Biochemical Assays 2.5.1. ATPase Assays Preparation of Insect's Homogenate
One thousand five hundred 2nd instar larvae of A. ipsilon in triplicate were treated with the LC 20 (0.25 and 1.85 mg mL −1 ) and LC 70 (0.49 and 4.41 mg mL −1 ) of O. majorana and Rosmarinus officinalis, respectively.Additionally, there were more than two hundred untreated larvae that were allowed to feed on untreated castor bean leaves.Forty-five live larvae were collected every 24, 48, 72, and 96 h post-treatment to determine the activity of Na + /K + -, Mg 2+ /Ca 2+ -, and Ca 2+ -ATPases in triplicate for each assay.They were weighed, rinsed, and homogenized in 10 mM Tris-HCl buffer (pH 7) containing 1 mM EDTA and 0.32 M Sucrose.The homogenate was then centrifuged at 4 • C at 2500× g for 10 min and the supernatant was centrifuged again at 22,000× g for 30 min.The supernatant was discarded, and the sediments were resuspended in Tris-HCl buffer (pH 7.4) containing 1 mM EDTA [29,54].The samples were kept frozen at −20 • C until used for ATPase assays.
The activity of Na + /K + -ATPase was determined using three groups of reaction systems containing 50 mM Tris-HCl buffer (pH 7), 5 mM MgCl 2 , and sample enzyme source according to the method described by [29].Reaction system 1 contained 150 mM NaCl, and 20 mM KCl; reaction system 2 contained 1 mM ouabain in 50 mM Tris-HCl buffer (pH 7); and reaction system 3 was considered as control (contained 50 mM Tris-HCl buffer (pH 7), 5 mM MgCl 2 , and sample enzyme source).These reaction systems were repeated three times (1 mL per system).All reaction systems were incubated for 5 min at 37 • C, and then 1.5 mM of adenosine triphosphate (ATP) was added.Reaction systems were incubated again for 15 min at 37 • C before reaction systems 1 and 2 were terminated by adding 15% of ice-cold trichloroacetic acid (TCA).Reaction systems were mixed by inversion, and then the phosphorous stain (1% ammonium molybdate tetrahydrate in 0.5 N sulfuric acid) and 1% freshly prepared ascorbic acid were added.The absorbance was read at 625 nm (Jenway-7205UV/Vis Spectrophotometer) after the systems were incubated for 30 min at 25 • C. Mg 2+ /Ca 2+ -and Ca 2+ -ATPases Mg 2+ /Ca 2+ -ATPase and Ca 2+ -ATPase activities were assayed using two groups of reaction systems according to the method of [55] with some modifications [28].In the Mg 2+ /Ca 2+ -ATPase assay determination, the two reaction systems (1 mL per system, with three replicates) contained 50 mM Tris-HCl buffer (pH 7), 1 mM MgCl 2 , 0.1 mM CaCl 2 , 10 mM KCl, 1 mM ouabain, and sample enzyme source.In the Ca 2+ -ATPase assay determination, the two reaction systems (1 mL per system, with three replicates) contained 50 mM Tris-HCl buffer (pH 7.4), 5 mM CaCl 2 , and 1 mM ouabain.Reaction system 1 in both determinations contained 1.5 mM ATP substrate while reaction system 2 contained 15% ice-cold trichloroacetic acid and 1.5 mM ATP substrate.Before adding the ATP substrate in both determinations, the two reaction systems were placed in a dry bath incubator at 37 • C. Reaction system 1 in both determinations was terminated by adding 15% of ice-cold trichloroacetic acid after 5 min.The phosphorous stain was added to the two reaction systems in both determinations before reading the absorbance at 625 nm.
ATPases were determined as described by [56,57] and the absorbance level was compared to a standard absorbance curve for known inorganic phosphate concentrations.

Detoxification Enzymes Assay α-Esterase Activity Assay
Three hundred 2nd instar larvae of A. ipsilon in triplicate were treated with the LC 10 (131 and 399 mg/L) and LC 30 (242 and 818 mg/L) of O. majorana and Rosmarinus officinalis, respectively.At 24, 48, 72, and 96 h, fifteen live larvae of the treatment or control groups were weighed, rinsed with distilled water, and homogenized in 40 mM potassium phosphate buffer containing 1 mM EDTA at pH 7. The homogenates were then centrifuged for 10 min at 14,000× g.The activity of α-esterase was determined in the supernatants according to [58] with small modifications [59].The absorbance levels were compared to a standard curve of absorbance obtained from known concentrations of α-naphthol (50 mM methanolic stock solution).The specific activities of α-esterase were reported as µ moles of α-naphthol formed min −1 mg −1 protein.

Glutathione-S-Transferase Activity Assay
The GST enzyme activity was assayed using 1-chloro-2,4 dinitrobenzene (CDNB) as a substrate, based on the method of [60].Three hundred 2nd instar larvae of A. ipsilon in triplicate were treated with the LC 10 (131 and 399 mg/L) and LC 30 (242 and 818 mg/L) of O. majorana and R. officinalis, respectively.At 24, 48, 72, and 96 h, fifteen live larvae of the treatment or control groups were weighed, rinsed with distilled water, and homogenized in 100 mM potassium phosphate buffer containing 1 mM EDTA at pH 6.5.The homogenates were then centrifuged at 10,000× g for 10 min.The GST activity was determined in the supernatant as described by [60].The GST-specific activities were expressed as nmols min −1 mg −1 protein.Three replicates from the treated and untreated (control) groups were used for all enzyme assays and correction for non-enzymatic conjugation in the samples was made.

Protein Assay
Protein concentration (mg protein/mL homogenates) was determined according to [61] and was used for standardization.

Molecular Docking
The structure of terpinene-4-ol and α-pinene, the major constituents of O. majorana and Rosmarinus officinalis, respectively, were created in the PDB file format using the Gaussian 09 software outputs.α-esterase crystal structure (PDB ID: 4FNM) was downloaded from the protein data bank (http://www.rcsb.org/,accessed on 26 June 2024).The molecular docking studies were performed using the MOE 2015 software.The co-crystallized ligand was re-docked in its original enzyme structure using the default parameters.

Statistical Analysis
The data underwent assessment to ensure that they met the assumptions required for parametric tests.Continuous variables were evaluated for normality using both the Shapiro-Wilk and Kolmogorov-Smirnov tests.The lethal and sublethal concentrations of the EOs were estimated by probit analysis [53] utilizing the Log Dose Probit line ® software (https://www.ehabsoft.com/ldpline/,accessed on 26 June 2024).Effects of EO treatments, and time post-treatment on the ATPase and detoxification enzymes (α-esterase, and GST) were subjected to ANOVA (Type II) using the generalized linear method (GLM) procedure, followed by Tukey's multiple comparisons test to compare the significance level between every two groups (p ≤ 0.05).This analysis and data representation as figures were conducted using GraphPad Prism, version 9.3.1 (GraphPad Software LLC, San Diego, CA, USA).The EO treatments and times post-treatment were considered fixed factors while the enzyme activity was treated as a random factor.Within the same day, the means of enzyme activities were separated using Duncan's multiple range test (p ≤ 0.05) (SPSS, V. 19.0,IBM Corporation, New York, NY, USA).

Toxicity of the Tested EOs to A. ipsilon Larvae
The insecticidal activity of O. majorana and R. officinalis EOs on A. ipsilon larvae was evaluated using the leaf dipping technique and data are shown in Table 1.The LC10, LC20, LC30, and LC70 (mg mL -1 ) were 0.13, 0.25, 0.39, and 1.85 mg mL −1 for O. majorana and 0.24, 0.49, 0.82, and 4.41 mg mL -1 for R. officinalis, respectively.Table 1.Larvicidal activity (in mg mL −1 ) of Origanum majorana and Rosmarinus officinalis essential oils (EOs) against A. ipsilon 2nd instar larvae at 96 h post-treatment.

ATPase Assays
Effects of the EOs at LC20 and LC70 at 24, 48, 72, and 96 h post-treatment on the Na + /K + -, Mg 2+ /Ca 2+ -, and Ca 2+ -ATPases of the A. ipsilon 2nd instar larvae were studied (Figure 2).Two-way ANOVA (GLM) and Tukey's multiple comparisons test were utilized to compare the treatments with the control.Additionally, both tested EOs were compared with each other at different time intervals (Table 2).

Toxicity of the Tested EOs to A. ipsilon Larvae
The insecticidal activity of O. majorana and R. officinalis EOs on A. ipsilon larvae was evaluated using the leaf dipping technique and data are shown in Table 1.The LC 10 , LC 20, LC 30 , and LC 70 (mg mL −1 ) were 0.13, 0.25, 0.39, and 1.85 mg mL −1 for O. majorana and 0.24, 0.49, 0.82, and 4.41 mg mL −1 for R. officinalis, respectively.

ATPase Assays
Effects of the EOs at LC 20 and LC 70 at 24, 48, 72, and 96 h post-treatment on the Na + /K + -, Mg 2+ /Ca 2+ -, and Ca 2+ -ATPases of the A. ipsilon 2nd instar larvae were studied (Figure 2).Two-way ANOVA (GLM) and Tukey's multiple comparisons test were utilized to compare the treatments with the control.Additionally, both tested EOs were compared with each other at different time intervals (Table 2).
Na + /K + -ATPase At LC 20 , a significant difference was found among treatments (F (2,6) = 290.4,p ≤ 0.05) at different time intervals (F (3,8) = 17.02, p ≤ 0.05), and among the interaction between treatments and time (F (6,18) = 28.14, p ≤ 0.05) in the specific activity of Na + /K + -ATPases.Similar results were found for LC 70 of the same EO, and the corresponding values were (F (2,6) = 41.83,p ≤ 0.05) for treatments, (F (3,18) = 58.35,p ≤ 0.05) for time, and (F (6,18) = 7.030, p ≤ 0.05) for the interaction between treatments and time.On the other hand, at LC 20 Tukey's multiple comparisons displayed a significant (p ≤ 0.05) difference between the control and O. majorana, the control and R. officinalis, and O. majorana versus R. officinalis at all time intervals, except for the control and R. officinalis at 96 h post-treatment.Similarly, at LC 70 of both EOs, Tukey's multiple comparisons indicated a significant difference (p ≤ 0.05) between the control and O. majorana, the control and R. officinalis, and O. majorana versus R. officinalis at all time intervals, except for the control and R. officinalis at 24 and 72 h post-treatment.Overall, O. majorana showed a contrasting effect to Na + /K + -ATPase, as the LC 20 significantly inhibited the Na+-K+ pump activity at 48 and 72 h after-treatment although it activated it at 24 and 96 h after-treatment.In addition, the LC 70 of the same EO inhibited this activity at 24 and 72 h post-treatment but activated it at 24 h post-treatment.However, R. officinalis suppressed the Na + /K + pump across all time intervals. 1SS = sums of squares, 2 MS = mean source/square, 3 p-values below 0.05 are displayed in bold. 4Times post-treatment, 5 Control group (untreated).
On the other hand, at LC 20 , Tukey's test displayed a significant difference (p ≤ 0.05) between the control and O. majorana at all time intervals.Additionally, distinctions were noted between the control and R. officinalis at 24 and 96 h, as well as between O. majorana and R. officinalis at 48 and 72 h.Similarly, at LC 70 of both EOs, Tukey's test revealed significant (p ≤ 0.05) differences between the control and O. majorana at 24, 72, and 96 h.Moreover, distinctions were observed between the control and R. officinalis at 24 and 96 h, and between O. majorana and R. officinalis at 24 and 72 h post-treatment.
Overall, similar to the Na+-K+ pump, O. majorana exhibited the same trend between activation and inhibition of Mg 2+/ Ca 2+ -ATPase at different time intervals in response to LC 20 and LC 70 .However, both tested concentrations of R. officinalis, LC 20 and LC 70 , significantly inhibited the activity of Ca 2+ Mg 2+ -ATPase at 96 h post-treatment.
On the other hand, at LC 20 , Tukey's test displayed a significant (p ≤ 0.05) difference between the control and O. majorana at 24 and 72 h, and between the control and R. officinalis at 24, 72, and 96 h.Additionally, a distinction was observed between O. majorana and R. officinalis at 96 h.Similarly, at LC 70 of both EOs, Tukey's test uncovered significant (p ≤ 0.05) differences between the control and O. majorana at 48, 72, and 96 h, the control and R. officinalis at 24, 72, and 96 h, and O. majorana and R. officinalis at 24, 48, and 72 h post-treatment.
Overall, the Ca 2+ -ATPases were significantly inhibited at 96 h post-treatment with LC 70 of both O. majorana and R. officinalis and with LC 20 of R. officinalis only.

Detoxification Enzymes Assay
The effects of LC 20 and LC 70 of the EOs on carboxylesterase and GST activities in the A. ipsilon 2nd instar larvae at different time intervals (24,48,72, and 96 h) post-treatments are presented in Figure 3. Two-way ANOVA and Tukey's test were used to compare the treatments with the control.Additionally, both tested EOs were compared with each other at different time intervals (Table 3).

α-Esterase
The LC 10 of O. majorana EO significantly inhibited the α-esterase activity at 96 h post-treatment.Additionally, the LC 30 of the same EO significantly increased the activity of α-esterase at 96 h post-treatment, while it inhibited the activity of α-esterase at 72 h post-treatment.
The LC 10 of R. officinalis significantly increased the activity of α-esterase at 48 h posttreatment.In addition, the LC 30 of the same EO significantly inhibited the activity of α-esterase at 48, 72, and 96 h post-treatment.

Glutathione-S-Transferase (GST)
Overall, the LC 10 of O. majorana EO significantly increased the GST activity at 24 h post-treatment but inhibited the activity of GST at 72 and 96 h post-treatment.Additionally, the LC 30 of the same EO significantly increased the activity of GST at 24 h post-treatment, while it significantly inhibited the activity of GST at 96 h post-treatment.
The LC 10 of R. officinalis significantly inhibited the activity of GST at 72 and 96 h post-treatment.In addition, the LC 30 of the same EO significantly increased the activity of GST at 48 and 72 h post-treatment but inhibited the activity of GST at 96 h post-treatment.

Molecular Docking
Molecular docking was performed for terpinene-4-ol and α-pinene, the major constituents of O. majorana and R. officinalis EOs, respectively, against the active site of α-esterase enzyme (PDB ID: 4FNM).The binding and interactions with the significant amino acids were carried out by docking studies.The docking process was validated using the co-crystallized ligand DPF (diethyl hydrogen phosphate).Interactions of amino acids, lengths of hydrogen bonds (A • ), affinity (Kcal/mol), and docking energy score are shown in Table 4.

Discussion
Egypt's fertile agricultural terrain provides an ideal environment for cultivating aromatic plants like rosemary and marjoram [62].These perennials offer economic benefits as they regrow after harvest, thereby eliminating the need for labor-intensive annual replanting [63].Egypt's climate further supports its growth across diverse regions [64,65].Leveraging the plentiful availability of these plants in Egypt to produce these EOs and their application in pest management aligns with the principles of sustainable agriculture, providing a safe and effective solution.
Furthermore, the EOs derived from these plants are multifunctional as they possess medicinal properties [66,67] and are used as flavorings [68,69].In comparison to chemical insecticides, these EOs present a safer alternative.Our research highlights the potent insecticidal properties of marjoram and rosemary EOs against Agrotis ipsilon larvae, a notably invasive and harmful pest.
The potential insecticidal activity of the EOs from O. majorana (marjoram) and R. officinalis (rosemary) against various species has been investigated by a plethora of studies and pilot tests [31,[78][79][80].Our research corroborated these findings, demonstrating significant insecticidal activity of these EOs against A. ipsilon larvae.Notably, while O. majorana oil exhibited higher toxicity than R. officinalis against the 2nd instar larvae of A. ipsilon, the latter showed considerable toxicity, compared to the control.
The existence or absence of the phosphate group distinguishes four types of ATPases: P, V, F, and ABC ATPases [81].The P-type is found in all living cells, regulating the transport of ions across membranes.It transports ions like protons, sodium, potassium, calcium, and heavy metals across diverse biological membrane systems [82].Different types of P-type ATPases were found to be the target site of insect management tools like pesticides, i.e., chlorinated hydrocarbons and pyrethroids [83][84][85][86] or their bio-alternative, i.e., plant EOs [87,88].
In the current study, we determined the specific activity of three types of P-type AT-Pases, Na + /K + -ATPase, Mg 2+ /Ca 2+ -ATPase, and Ca 2+ -ATPase, at different time intervals post-treatment with lethal and sub-lethal concentrations of O. majorana and R. officinalis.This was based on the hypothesis that these ATPases might affect the tested EO toxicity to A. ipsilon larvae.Surprisingly, significant effects of these ATPases were observed for different concentrations of the tested EOs.Additionally, an interaction between time and treatment was also recorded.
O. majorana showed a contrasting effect on the Na + /K + -ATPase or Na + -K + pumps.This contrasting effect between activation and inhibition might indicate that the Na + -K + pump is not the main target site of this EO.On the contrary, R. officinalis inhibited the Na + -K + pump activity at both tested concentrations, LC 20 and LC 70 , at almost all-time intervals post-treatment, except for at 96 h post-treatment of the LC 20 .
The Na + -K + pump exists in the plasma membrane of almost all animal cells, and functions as an antiporter, actively pumping Na + out of the cell and pumping K + in, thereby maintaining the cell's equilibrium and the cell's ability to generate electrical impulses [89,90].The main difference between this pump and voltage-gated sodium channels (VGSCs) lies in their function and mechanism of action.The former is an active pump that uses energy to maintain ion gradients, while the latter are passive channels that respond to changes in membrane potential to generate action potentials.Their roles in the action of pyrethroids also differ, with the VGSCs being the primary target of these insecticides [91].
The suppression of this pump observed in the current study following exposure to R. officinalis across all time intervals suggests that this pump is a potential target for this EO.This suppression might elevate the concentration of Na + ions, triggering heightened neuronal excitation, and ultimately causing a knockdown effect and death.In harmony with this, a dose-response and time-course study conducted by [92] reported that the Na + -K + pump inhibition is related to the knockdown effect of the decalesides, a novel category of bio-insecticides.However, an electrophysiology study, using the patch clamp technique, is needed to confirm this hypothesized mode of action of R. officinalis.
The Mg 2+ /Ca 2+ -ATPase, which shows the same mechanism as the Na + /K + -ATPase, can pump calcium ions against a concentration gradient.The calcium gradients made by this enzyme are critical to muscle relaxation [93].In the current work, O. majorana showed the same trend, as shown in the Na + -K + pump, between activation and inhibition of Mg 2+ /Ca 2+ -ATPase at different time intervals in response to LC 20 and LC 70 .The LC 20 of this EO inhibited the activity of the Mg 2+ /Ca 2+ -ATPase at 96 h post-treatment.This might indicate that the Mg 2+ /Ca 2+ -ATPase target site is one of the target sites of O. majorana but not the major one.
On the contrary, both tested concentrations of R. officinalis, LC 20 and LC 70 , significantly inhibited the activity of Mg 2+ /Ca 2+ -ATPase at 96 h post-treatment, the time at which we recorded the highest mortality rate in A. ipsilon larvae in the concentration gradient bioassay.This inhibition of the Mg 2+ /Ca 2+ -ATPase leads to inhibition in muscle relaxation, which might be a response to the long-lasting inhibition in the Na + -K + pump due to treatment with the two tested concentrations of R. officinalis recorded in the current work.
Ca 2+ -ATPase, or the Ca 2+ pump, exists in the sarcoplasmic reticulum membrane of skeletal muscle cells and is responsible for about 90% of the organelle membrane protein.This pump serves as an intracellular store of Ca 2+ and accounts for moving Ca 2+ from the cytosol to the sarcoplasmic reticulum [24].As reported by [94], the regulation of Ca 2+ inner or outer nerve membranes is chiefly performed by Mg 2+ /Ca 2+ -ATPase and Ca 2+ -ATPase.In the current work, the Ca 2+ pump was significantly inhibited at 96 h post-treatment with LC 70 of both O. majorana and R. officinalis and with LC 20 of R. officinalis only.This finding supports our hypothesis that Mg 2+ /Ca 2+ -ATPase and Ca 2+ -ATPase are target sites for O. marjoram and might be a major target for R. officinalis.
The activities of α-esterase, mixed-function oxidase, and Glutathione S-transferase (GST) have been the subject of extensive research, particularly on insects' exposure to xenobiotics [95].These detoxification enzymes serve as potent biological indicators due to their sensitivity in signaling exposure to chemicals [96].However, their activity after xenobiotic exposure has been reported to show a varied level of activity between activation and inhibition in different insects after exposure to various chemicals [28].Hence, the activity of these detoxifying enzymes does not directly reveal the target site of a compound.Instead, it provides insights into how the insect's enzymatic system interacts with these xenobiotic substances, essentially shedding light on the compound's mode of action.In our research, we assessed the α-esterase and GST activities as an indicator of the detoxification process of the EOs in A. ipsilon larvae.While cytochrome P450s are undoubtedly important in detoxification processes, our previous findings [13] suggested that GST and esterases may play a more direct role in mediating the effects of our tested EOs.
The data revealed that the two tested EOs, O. majorana and R. officinalis, exhibited a varied level of activation and inhibition of the detoxification enzymes, α-esterase and GST, over time post-treatment.The increased activity of detoxification enzymes following xenobiotic treatment may represent a direct adaptive response aimed at neutralizing toxic compounds and detoxifying insecticides, thereby enhancing survival rates [97].Furthermore, Ref. [98] suggested that the induction of detoxification enzymes is associated with increased tolerance of insects to insecticides.
However, in the current work, the most common effect of both tested EOs on αesterase and GST was inhibition.Insects utilize such detoxification enzymes as α-esterase and GST to metabolize the secondary metabolites of plants, thereby protecting themselves from oxidative damage [99].This suggests that the mortality of A. ipsilon larvae following exposure to R. officinalis and O. majorana EOs might be a consequence of the reduced activity of α-esterase and GST.Our findings suggest that α-esterase and GST play a part in the detoxification process of O. majorana and R. officinalis in A. ipsilon larvae.However, further research, at the molecular level, could provide more precise insights.
Our current results align with those of [100], who observed varied levels in the activity of AChE, carboxylesterase, and GST in the cereal weevil, Sitophilus zeamais, in response to Melaleuca alternifolia EO over time post-treatment.However, the varied level recorded in their study in the EO-treated insects was consistently lower than that recorded in the untreated insects at all time intervals, presenting a contrasting result with ours.This variation between our study and theirs could be attributed to the different concentrations used; they used a lethal concentration (LC 50 ), while we used sublethal ones (LC 10 and LC 30 ).Additionally, they employed a fumigation method to treat their insects, whereas we used the leaf dipping technique.This latter point suggests a potential future comparative study testing the same EOs using different bioassay methods to identify the most efficient bioassay method for these EOs against A. ipsilon larvae.
Furthermore, the inhibition trend of detoxification enzymes by the tested EOs in the current work highlights the potential of using these EOs to enhance the toxicity of synthetic insecticides as previously suggested by [101].In accordance with this suggestion, [102] reported that various plant EOs have been shown to inhibit detoxification enzymes such as GST and cytochrome P450 monooxygenases of Aedes aegypti and Anopheles gambiae, both pyrethroid-susceptible and pyrethroid-resistant strains, increasing the efficacy of pyrethroids against the resistant populations of these pests.Additionally, lemongrass EO was found to synergize the toxic effect of certain insecticides on Bemisia tabaci adults [103].These findings suggest that EOs could be valuable synergistic agents in conventional insecticides.Future research should focus on exploring the precise mechanisms of enzyme inhibition by these EOs, synergistic effects in various pest management scenarios, and ensuring environmental safety.Large-scale field trials are also recommended to assess the practicality and long-term benefits of integrating EOs with traditional insecticides in pest control programs across urban, public health, and agricultural settings.
The molecular docking results indicated that terpinene-4-ol and α-pinene, the major constituents of O. majorana and R. officinalis EOs, respectively, exhibited favorable energy scores (S); −4.51 and −4.29 Kcal/mol, respectively, which closely resemble the score of the diethyl hydrogen phosphate (DPF) ligand (−4.67).This alignment with the toxicity assay data further supports the observation that O. majorana is more toxic than R. officinalis to A. ipsilon larvae.Additionally, the molecular docking results reinforce our earlier hypothesis, initially derived from the biochemical analysis of detoxification enzymes, regarding the involvement of the detoxification enzyme α-esterase in the response of A. ipsilon larvae to both tested EOs.

Conclusions
This study indicates that the Na+-K+ pump may be a primary target for Rosmarinus officinalis.Additionally, the Mg 2+ /Ca 2+ -ATPase and Ca 2+ pumps may also be targeted by this EO.However, while Origanum marjoram may influence these pumps, they might not be its primary targets.Furthermore, the findings regarding detoxification enzymes suggest that α-esterase and GST play roles in the detoxification process of these EOs in A. ipsilon larvae.Overall, O. majorana and R. officinalis EOs can be promising insecticides in organic farming and IPM programs for A. ipsilon management.Additionally, in silico studies could serve as a powerful tool to validate and enhance the understanding of the toxicity and biochemical data.To conclude, our research provided good insights; however, a more granular understanding of our results can be achieved through subsequent research employing advanced molecular and electrophysiological methods.
Author Contributions: F.S.A. and M.A.M.M. were responsible for conceptualization, methodology, software, validation, formal analysis, project administration, writing-original draft preparation, writing-review and editing, visualization, supervision, and project administration.W.S.H. helped with methodology, writing-original draft preparation, writing-review and editing, and visualization.Finally, N.A.A. contributed to the formal analysis, supervision, and resources.All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Figure 1 .
Figure 1.Chemical structure of the main bioactive compounds of Origanum majorana and Rosmarinus officinalis EOs.

Figure 1 .
Figure 1.Chemical structure of the main bioactive compounds of Origanum majorana and Rosmarinus officinalis EOs.

Figure 4 .
Figure 4. Diagrams of 2D and 3D interactions of the major bioactive components of Origanum majorana (terpinene-4-ol) and Rosmarinus officinalis (α-pinene) and diethyl hydrogen phosphate (DPF) in the active site of α-esterase (PDB ID: 4FNM).Hydrogen bonds are displayed in cyan and H-pi-bonds in magenta.

Table 2 .
Two-way ANOVA (GLM procedure) and Tukey's multiple comparisons test for the effects of LC 20 and LC 70 of Origanum majorana and Rosmarinus officinalis EOs at different time intervals post-treatments on the Na + /K + -, Mg 2+ /Ca + -, and Ca 2+ -ATPases of A. ipsilon 2nd instar larvae.

Table 3 .
Two-way ANOVA (GLM procedure) and Tukey's multiple comparisons test for the effects of LC 10 and LC 30 of Origanum majorana and Rosmarinus officinalis on the detoxification enzymes (α-esterase (Est.) and GST) of A. ipsilon 2nd instar larvae at different time intervals post-treatment.

Table 3 .
Two-way ANOVA (GLM procedure) and Tukey's multiple comparisons test for the effects of LC10 and LC30 of Origanum majorana and Rosmarinus officinalis on the detoxification enzymes (αesterase (Est.) and GST) of A. ipsilon 2nd instar larvae at different time intervals post-treatment.