Mechanistic insights into the toxicological effects of insecticides with distinct modes of action on Apis mellifera: An integrated in silico study

Mechanistic insights into the toxicological effects of insecticides with distinct modes of action on Apis mellifera: An integrated in silico study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mechanistic insights into the toxicological effects of insecticides with distinct modes of action on Apis mellifera: An integrated in silico study Serkan SUGEÇTİ This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8945595/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The extensive use of insecticides in modern agriculture raises concerns regarding their sublethal molecular effects on non-target pollinators such as the honeybee (Apis mellifera). This study employed an integrated in silico approach combining AMED profiling, molecular docking, and molecular dynamics (MD) simulations to investigate the interactions of chlorantraniliprole, imidacloprid, and thiamethoxam with two key honeybee proteins: glutathione S-transferase (GST) and odorant-binding protein (OBP). Docking analyses revealed selective binding patterns, with chlorantraniliprole showing the strongest affinity toward GST, suggesting a prominent interaction with detoxification pathways. Thiamethoxam exhibited the highest affinity for OBP, indicating potential interference with olfactory signaling, whereas imidacloprid demonstrated moderate binding to both proteins. MD simulation of the GST–chlorantraniliprole complex confirmed structural stability based on RMSD, RMSF, Rg, SASA, and hydrogen bond analyses. These findings provide mechanistic insights into how insecticides may simultaneously affect detoxification and sensory systems in A. mellifera . The integration of AMED prediction with docking and MD simulations offers a complementary computational framework for ecotoxicological risk assessment of agrochemicals. Apis mellifera molecular dynamics simulation AMED analysis glutathione S-transferase odorant-binding protein ecotoxicology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction The extensive use of insecticides in modern agriculture has raised serious concerns regarding their unintended environmental consequences, particularly their adverse effects on non-target organisms (Serrão et al. 2022 ). While insecticides are designed to control pest populations, they often persist in environmental matrices such as soil, water, and vegetation, leading to chronic exposure of beneficial insects and other wildlife. Pollinators, especially honeybees ( Apis mellifera ), are highly vulnerable to such exposure due to their foraging behavior and close interaction with treated crops (Liao et al. 2017 ). Numerous studies have demonstrated that insecticides can induce sublethal effects in bees, including impaired learning, disrupted navigation, altered foraging efficiency, and weakened immune responses, ultimately threatening colony health and ecosystem services essential for agricultural sustainability (Goulson 2013 ; Sanchez-Bayo and Goka 2014 ; Pisa et al. 2015 ). Chlorantraniliprole, imidacloprid, and thiamethoxam represent widely used insecticides belonging to different chemical classes and modes of action. Chlorantraniliprole is a diamide insecticide that selectively activates ryanodine receptors, causing uncontrolled calcium release and muscle paralysis in insects (Sun and Xu 2018 ; Du and Fu 2023 ). Imidacloprid and thiamethoxam are neonicotinoid insecticides that act as agonists of nicotinic acetylcholine receptors, leading to persistent neuronal excitation and eventual insect death (Jeschke et al. 2011 ; Thany 2023 ). Although these compounds are often regarded as selective toward target pests, increasing evidence indicates that they can exert significant sublethal and molecular effects on non-target insects, including honey bees, even at environmentally relevant concentrations (Blacquière et al. 2012 ). At the molecular level, detoxification and sensory perception pathways play crucial roles in mediating insect responses to xenobiotics. Glutathione S-transferases (GSTs) constitute a major family of phase II detoxification enzymes that catalyze the conjugation of reduced glutathione to electrophilic compounds, facilitating their detoxification and excretion (Kumar and Trivedi 2018 ; Potęga 2022 ). Elevated GST activity has frequently been associated with insecticide tolerance and adaptive stress responses in insects (Zhang et al. 2022 ). Odorant binding proteins (OBPs), on the other hand, are small soluble proteins located in the sensillar lymph of insect antennae and are essential for transporting hydrophobic odorant molecules to olfactory receptors (Ha and Smith 2022 ). Emerging evidence suggests that OBPs may also interact with exogenous chemicals such as insecticides, potentially interfering with olfactory signaling and behavioral processes critical for pollinator survival (Abendroth et al. 2023 ). In silico approaches, particularly molecular docking, have become valuable tools for investigating protein–ligand interactions at the atomic level (Sugeçti 2025a ). Molecular docking enables the prediction of binding affinities, interaction modes, and key amino acid residues involved in ligand recognition, offering mechanistic insights that complement experimental ecotoxicological studies (Waghmode et al. 2025 ). Given ethical, logistical, and economic constraints associated with in vivo testing on pollinators, in silico modeling provides an efficient and predictive framework for screening potential toxicological interactions of insecticides with honeybee proteins (Sugeçti 2025b ). Such computational approaches are increasingly recognized as essential components of integrative environmental risk assessment strategies. The present study aims to evaluate the potential molecular interactions of chlorantraniliprole, imidacloprid, and thiamethoxam with Apis mellifera glutathione S-transferase and odorant binding protein using molecular docking analyses. By comparing insecticides with distinct modes of action, this work seeks to elucidate possible molecular mechanisms underlying detoxification responses and sensory disruption in honeybees, thereby contributing to a deeper understanding of insecticide-related ecotoxicological risks to pollinators. 2. Materials and methods 2.1. Chemicals The chemical structures of chlorantraniliprole, imidacloprid, and thiamethoxam were obtained from the PubChem database of the National Center for Biotechnology Information (NCBI). The three-dimensional (3D) structures were downloaded in SDF format and subsequently prepared for molecular docking analysis. Prior to docking, ligand structures were energy-minimized to achieve stable conformations using the MMFF94 force field. All ligands were converted to the PDBQT format following the addition of polar hydrogen atoms and assignment of Gasteiger partial charges. 2.2. Protein structure preparation The three-dimensional structures of A. mellifera GST and OBP were retrieved from the Protein Data Bank (PDB). Selected protein structures were chosen based on resolution quality and biological relevance. Prior to docking, all water molecules, co-crystallized ligands, and heteroatoms were removed from the protein structures. Polar hydrogen atoms were added, and Kollman charges were assigned to prepare the proteins for docking analysis. Protein structures were subsequently saved in PDBQT format. 2.3. Molecular docking analysis Molecular docking simulations were performed using AutoDock Vina to evaluate the binding interactions between the selected insecticides and the target proteins (Trott and Olson 2010 ). Docking grids were defined to encompass the active or binding sites of GST and OBP based on known functional regions reported in the literature. Docking calculations were conducted using default exhaustiveness parameters, and the best-ranked binding poses were selected based on minimum binding free energy (kcal/mol). The predicted protein–ligand complexes were visualized and analyzed using Discovery Studio Visualizer. 2.4. In silico prediction of AMED-related physicochemical and toxicokinetic properties In silico AMED-related physicochemical and toxicokinetic properties of chlorantraniliprole, imidacloprid, and thiamethoxam were predicted using SwissADME ( https://www.swissadme.ch/index.php ) and pkCSM online platforms. These analyses provided comparative insights into absorption, distribution, metabolic interaction potential, and ecotoxicological risk, supporting the interpretation of molecular docking results. 2.5. Molecular Dynamics (MD) Simulations All-atom molecular dynamics (MD) simulations were performed using the GROMACS 2023.3 simulation package to investigate the structural stability and dynamic behavior of the glutathione S-transferase (GST)–chlorantraniliprole complex. The protein and ligand were described using the OPLS-AA force field, while the TIP3P water model was employed for explicit solvation. The complex was placed in a triclinic simulation box with a minimum distance of 10 Å between the solute and the box boundaries, and the system was neutralized by the addition of appropriate counterions. Energy minimization was carried out using the steepest descent algorithm until the maximum force fell below 1000 kJ·mol⁻¹·nm⁻¹ to remove unfavorable steric contacts. The system was then equilibrated in two phases: a 100 ps NVT ensemble followed by a 100 ps NPT ensemble, during which position restraints were applied to the protein backbone atoms. Temperature was maintained at 300 K using the velocity-rescaling (V-rescale) thermostat, while pressure was controlled at 1 bar using the Parrinello–Rahman barostat with isotropic coupling. Long-range electrostatic interactions were calculated using the Particle Mesh Ewald (PME) method with a real-space cutoff of 1.0 nm. Van der Waals interactions were treated using a force-switch scheme between 0.9 and 1.0 nm. All bonds involving hydrogen atoms were constrained using the LINCS algorithm, allowing an integration time step of 2 fs. Following equilibration, a 30 ns production MD simulation was performed under the NPT ensemble without position restraints. Periodic boundary conditions were applied in all three spatial dimensions. Trajectory coordinates were saved every 2 ps for subsequent analyses. Structural stability and conformational dynamics of the complex were evaluated using root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and hydrogen bond analyses based on the production trajectory. 3. Results Molecular docking simulations were performed to evaluate the binding affinities and interaction profiles of chlorantraniliprole, imidacloprid, and thiamethoxam with A. mellifera GST and OBP. The predicted binding free energy values (ΔG, kcal/mol) and key molecular interactions are summarized in Table 1 . Table 1 The docking results of insecticides on GST and Odorant binding protein of A. mellifera Insecticides Protein ΔG (kcal/mol) Interactions Amino Acid Chlorantraniliprole GST -7.3 Carbon hydrogen bonds SER34 Pi-sigma LEU58 Pi-sulfur MET134 Pi-Pi T-shaped TRY138 Pi-alkyl TRY138; PHE229; PHE142 Alkyl MET134; PRO35; LEU58; LEU31; LEU232; ILE141 Odorant binding protein -7.8 Pi-sigma VAL108; Pi-Pi T-shaped TRP116; PHE117 Pi-alkyl TRP116; PHE117; ALA82; ALA112; LEU78; VAL85; LEU74; ARG81; ARG81; Alkyl ALA105; MET86; VAL85; MET70; VAL108; LEU73; LEU53; LEU58 Imidacloprid GST -6.5 Conventional hydrogen bonds SER34; TYR130 Alkyl MET228; LEU232 Odorant binding protein -7.4 Pi-sulfur MET70; Pi-alkyl LEU74; VAL85; ALA82; VAL108 Alkyl LEU74; ALA82; LEU78; MET86 Thiamethoxam GST -5.8 Conventional hydrogen bonds SER34; TYR130 Carbon hydrogen bonds SER34 Pi-Pi stacked PHE229 Pi-alkyl LEU232 Alkyl MET228; LEU232 Odorant binding protein -11.8 Pi-Pi stacked TRP116 Pi-alkyl ALA105; MET49; TRP116; LEU53 Alkyl MET49 3.1. Interactions with glutathione S-transferase Among the tested insecticides, chlorantraniliprole exhibited the strongest binding affinity toward GST with a ΔG value of -7.3 kcal/mol (Table 1 ). The binding was stabilized by multiple non-covalent interactions, including carbon hydrogen bonding with SER34, π–σ interaction with LEU58, π–sulfur interaction with MET134, and a π–π T-shaped interaction involving TYR138. Additionally, several hydrophobic π–alkyl and alkyl interactions were observed with residues such as PHE229, PHE142, PRO35, LEU31, LEU58, LEU232, and ILE141, indicating a stable accommodation of chlorantraniliprole within the GST binding pocket (Fig. 1 ). Imidacloprid showed a moderate binding affinity to GST (ΔG = -6.5 kcal/mol) (Table 1 ). Its interaction profile was characterized primarily by conventional hydrogen bonds with SER34 and TYR130, accompanied by hydrophobic alkyl interactions involving MET228 and LEU232. Compared to chlorantraniliprole, imidacloprid formed fewer stabilizing interactions within the GST active site (Fig. 2 ). Thiamethoxam displayed the weakest binding affinity toward GST among the tested insecticides (ΔG = -5.8 kcal/mol) (Table 1 ). The interaction pattern included conventional and carbon hydrogen bonds with SER34, a π–π stacking interaction with PHE229, and hydrophobic π–alkyl and alkyl contacts with LEU232 and MET228. Overall, the lower binding energy and reduced interaction diversity suggest a comparatively weaker association of thiamethoxam with GST (Fig. 3 ). 3.2. Interactions with odorant binding protein Docking results indicated that all three insecticides interacted favorably with OBP, with binding affinities generally stronger than those observed for GST. Chlorantraniliprole exhibited a binding free energy of -7.8 kcal/mol and formed multiple hydrophobic interactions within the OBP binding cavity (Table 1 ). These included π–σ interaction with VAL108, π–π T-shaped interactions with TRP116 and PHE117, and extensive π–alkyl and alkyl contacts involving residues such as ALA82, ALA112, LEU78, LEU74, VAL85, MET70, MET86, LEU53, and LEU58 (Fig. 4 ). Imidacloprid demonstrated a comparable binding affinity to OBP (ΔG = − .4 kcal/mol). The interaction profile was dominated by hydrophobic contacts, including π–sulfur interaction with MET70 and π–alkyl interactions with LEU74, VAL85, ALA82, and VAL108. Additional alkyl interactions with LEU78 and MET86 further contributed to ligand stabilization within the OBP binding pocket (Fig. 5 ). Thiamethoxam showed the strongest binding affinity to OBP with a ΔG value of -11.8 kcal/mol. This interaction was primarily stabilized by a π–π stacking interaction with TRP116, along with multiple π–alkyl and alkyl interactions involving ALA105, MET49, LEU53, and TRP116. The markedly lower binding free energy suggests a high affinity of thiamethoxam for OBP compared to both GST and the other insecticides tested (Fig. 6 ). 3.3. Molecular Dynamics Simulation Analysis of GST–Chlorantraniliprole Complex The structural stability and dynamic behavior of the GST–chlorantraniliprole complex were evaluated through a 30 ns all-atom molecular dynamics simulation using multiple structural descriptors, including RMSD, RMSF, radius of gyration (Rg), solvent-accessible surface area (SASA), and hydrogen bond analysis. Root Mean Square Deviation (RMSD) The RMSD profile of the protein backbone revealed an initial increase during the early phase of the simulation, followed by stabilization after approximately 6–8 ns (Fig. 8 ). Throughout the remaining simulation time, RMSD values fluctuated within a narrow range (~ 0.16–0.20 nm), indicating that the GST–chlorantraniliprole complex reached a stable conformational state. The absence of large deviations suggests that ligand binding did not induce significant structural destabilization of the protein, supporting the overall stability of the complex during the simulation. Root Mean Square Fluctuation (RMSF) Residue-wise flexibility of the protein was assessed using RMSF analysis (Fig. 9 ). Most residues exhibited low fluctuations (< 0.15 nm), indicating a rigid and stable protein backbone. Higher fluctuations were mainly observed at the N- and C-terminal regions and in selected loop regions, which is typical for solvent-exposed and flexible segments of proteins. Importantly, residues associated with the ligand-binding region showed relatively low RMSF values, suggesting that chlorantraniliprole binding contributes to local stabilization of the active site. Radius of Gyration (Rg) The radius of gyration remained relatively constant throughout the simulation, fluctuating around an average value of approximately 2.24–2.27 nm (Fig. 10 ). This consistent Rg profile indicates that the overall compactness of the GST structure was preserved during the simulation. No significant expansion or collapse of the protein structure was observed, further confirming the conformational stability of the GST–chlorantraniliprole complex. Solvent-Accessible Surface Area (SASA) SASA analysis showed moderate fluctuations around an average value of ~ 215–225 nm² during the simulation period (Fig. 11 ). These variations reflect minor conformational rearrangements at the protein surface but do not indicate large-scale unfolding events. The relatively stable SASA profile suggests that the global exposure of the protein to the solvent remained largely unchanged upon ligand binding. Hydrogen Bond Analysis The number of hydrogen bonds within the system displayed stable behavior over the 30 ns simulation, with values fluctuating within a consistent range (Fig. 12 ). Although minor decreases were observed toward the later stages of the simulation, the overall hydrogen bond network remained well maintained. This indicates sustained intermolecular and intramolecular interactions contributing to the structural integrity of the protein–ligand complex. 3.4. In silico AMED profiling of chlorantraniliprole, imidacloprid, and thiamethoxam SwissADME-based AMED predictions revealed marked physicochemical differences among the tested insecticides (Table 2 ). Chlorantraniliprole exhibited the highest molecular weight (483.15 g/mol) and lipophilicity (XlogP3 = 4.80), along with relatively low polarity (TPSA = 88.91 Ų), suggesting a strong tendency for hydrophobic interactions and limited aqueous solubility. In contrast, imidacloprid showed substantially lower lipophilicity (XlogP3 = 0.57) and high predicted solubility, whereas thiamethoxam displayed intermediate lipophilicity but higher polarity (TPSA = 115.02 Ų). All compounds were predicted to exhibit high gastrointestinal absorption and lack blood–brain barrier permeability. Table 2 In silico AMED properties of the tested insecticides Parameter Chlorantraniliprole Imidacloprid Thiamethoxam Molecular weight (g/mol) 483.15 255.66 291.71 LogP (XlogP3) 4.80 0.57 1.52 Topological polar surface area (TPSA, Ų) 88.91 86.34 115.02 H-bond donors 2 1 0 H-bond acceptors 4 4 5 GI absorption High High High BBB permeability No No No P-gp substrate No No No CYP inhibition (general) Yes (CYP2C19, CYP2C9, CYP3A4) No Yes (CYP1A2) ESOL solubility class Moderately soluble Very soluble Soluble Silicos-IT solubility class Poorly soluble Soluble Soluble Bioavailability score 0.55 0.55 0.55 4. Discussion The molecular docking analyses presented in this study provide detailed insights into the potential molecular interactions of three major insecticides such as chlorantraniliprole, imidacloprid, and thiamethoxam with GST and OBP of A. mellifera . These findings support and extend recent evidence that sublethal exposure to modern insecticides can perturb both detoxification and sensory systems in honeybees, even at environmentally relevant concentrations (Li et al. 2024a ). Chlorantraniliprole showed the highest binding affinity toward GST, suggesting its strong interaction with phase II detoxification enzymes. GSTs play an essential role in conjugating electrophilic xenobiotics with glutathione, facilitating their elimination from the organism (Higgins and Hayes 2011 ). Previous studies have reported that exposure to chlorantraniliprole can alter antioxidant enzyme activities in A. mellifera and related species (Li et al. 2024b ). Transcriptomic analyses further demonstrated upregulation of GST and related detoxification genes in insects following chlorantraniliprole exposure (Wang et al. 2018 ; Xu et al. 2022 ). The predominance of hydrophobic and π–π interactions observed in the present study indicates a stable accommodation of chlorantraniliprole within the GST catalytic site, consistent with its capacity to trigger enzymatic detoxification pathways. These findings collectively suggest that chlorantraniliprole may be efficiently metabolized by honeybee GSTs, potentially contributing to adaptive tolerance mechanisms. In contrast, thiamethoxam exhibited the strongest binding affinity to OBP, implying possible interference with olfactory perception mechanisms. OBPs are vital in transporting odorant molecules through the sensillar lymph to odorant receptors, thereby enabling floral recognition and foraging communication. Neonicotinoids have been shown to reduce antennal sensitivity and alter odor learning in bees (Mitchell and Alexandre 2021). The current docking results revealing extensive π–π stacking and hydrophobic interactions between thiamethoxam and key OBP residues (e.g., TRP116, LEU53, and MET49), provide a structural basis for such behavioral impairments. Similar molecular interactions have been reported in Aphis craccivora for neonicotinoids, suggesting that OBP binding may represent a conserved mechanism of chemosensory disruption (Abdelmoteleb et al. 2023 ) Imidacloprid displayed intermediate affinity for both GST and OBP, suggesting potential dual molecular targets. This broader binding profile may underlie its diverse sublethal effects, including impaired learning, reduced foraging, and increased oxidative stress observed in Apis species (Peng et al. 2023 ). Furthermore, imidacloprid exposure has been linked to the upregulation of detoxification enzymes such as UDP-glucosyltransferases and GSTs, which are known to contribute to resistance in pests (Chen et al. 2019 ; Peng et al. 2023 ). The ability of imidacloprid to engage with both metabolic and olfactory proteins could thus potentiate cumulative physiological stress, even at sublethal exposure levels. It was determined that the structure and mechanism of action of the insecticide determined the different protein binding behaviors in honeybees. These findings are in accordance with in vivo results demonstrating that exposure to insecticides induces enzyme activity changes and behavioral alterations (Alfuhaid et al. 2025 ). From an ecotoxicological standpoint, these protein–ligand interaction patterns provide molecular evidence that sublethal insecticide exposure may simultaneously challenge detoxification capacity and sensory integrity, leading to synergistic physiological consequences. The predicted AMED profiles were highly consistent with the docking-based interaction patterns observed in this study. The pronounced lipophilicity and moderate polarity of chlorantraniliprole favor stable accommodation within the hydrophobic active site of GST, supporting its strong binding affinity and extensive hydrophobic interactions (Chen et al. 2026 ). Conversely, thiamethoxam exhibited higher polarity and moderate lipophilicity, physicochemical characteristics that are compatible with efficient interaction within the OBP cavity, where ligand stabilization is largely driven by π–π stacking and hydrophobic contacts rather than membrane partitioning. Imidacloprid displayed comparatively low lipophilicity and high solubility, consistent with its moderate binding affinities toward both GST and OBP (Li et al. 2025 ). Collectively, these findings indicate that physicochemical and toxicokinetic properties critically shape protein-binding behavior and sublethal molecular interference mechanisms in A. mellifera . Future research should integrate molecular docking with transcriptomic, enzymatic, and behavioral studies to validate these computational predictions under biological conditions. Additionally, comparative docking analyses across pollinator taxa such as A. cerana and Bombus terrestris could elucidate interspecific variations in susceptibility, informing pollinator-safe pesticide development. Collectively, this study demonstrates that in silico modeling can serve as a valuable component of mechanistic risk assessment frameworks, bridging molecular mechanisms with ecological outcomes in pollinator toxicology. Declarations Disclosure statement The authors declare no competing interests. Data availability statement The datasets generated during the current study are available from the corresponding author on reasonable request. 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Ecotoxicol Environ Saf 230:113145. https://doi.org/10.1016/j.ecoenv.2021.113145 Zhang Y, Yang B, Yu N et al (2022) Insecticide resistance associated overexpression of two sigma GST genes assists Nilaparvata lugens to remedy oxidative stress from feeding on resistant rice variety. Pestic Biochem Physiol 188:105230. https://doi.org/10.1016/j.pestbp.2022.105230 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8945595","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":611155120,"identity":"6f5e8b56-7396-45b5-9211-06a145258219","order_by":0,"name":"Serkan SUGEÇTİ","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIiWNgGAWjYHACZiS2gU0CmE4oIFbLAYO0BAY2kBYDorUwHIZoYcCjRb6997HBzx0M9gbnFx/8/KHgfB6/fHfihwcGDPL8YgewajE4c9w4sfcMQ+KGG8+SJQ4Y3C6WbOPdLAF0mOHM2QnYtUikMR/gbQM6/sYZM6BfbiduOMa7AaQlweA2di3y858xH/zbBnTYjfPfgFrOgbRs/oFPC8MNNuZkoC2MG873sAG1HABp2YbXFoMzaczGsm0SiTNvsBlLnDFITpzZlrvNIsFAAqdf5NuPMUu+bbOx5zt/+OGHij92if3MZzff/FFhI88vjcNhECABRAnoIgQB/wEiFI2CUTAKRsGIBABhUGCpvtohIgAAAABJRU5ErkJggg==","orcid":"","institution":"Zonguldak Bülent Ecevit University","correspondingAuthor":true,"prefix":"","firstName":"Serkan","middleName":"","lastName":"SUGEÇTİ","suffix":""}],"badges":[],"createdAt":"2026-02-23 09:54:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8945595/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8945595/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105377676,"identity":"efc4bafd-86c4-4347-803f-96a6169351fc","added_by":"auto","created_at":"2026-03-25 10:33:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":340546,"visible":true,"origin":"","legend":"\u003cp\u003eThe Molecular docking results of chlorantraniliprole on the GST in \u003cem\u003eA. mellifera\u003c/em\u003e. (a) Docking result of chlorantraniliprole and GST. (b) 2D interactions of the chlorantraniliprole with amino acids in the active site of the GST.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8945595/v1/78b9804ffaefd67eef190e6e.png"},{"id":105377670,"identity":"dbfdecf0-053b-4f4e-ab8f-01c8cb3777b5","added_by":"auto","created_at":"2026-03-25 10:33:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":470209,"visible":true,"origin":"","legend":"\u003cp\u003eThe Molecular docking results of chlorantraniliprole on the \u003cem\u003eA. mellifera\u003c/em\u003eodorant binding protein. (a) Docking result of chlorantraniliprole and \u003cem\u003eA. mellifera\u003c/em\u003e odorant binding protein. (b) 2D interactions of the chlorantraniliprole with amino acids in the active site of the \u003cem\u003eA. mellifera\u003c/em\u003eodorant binding protein.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8945595/v1/4881f76408c93ba5de0d8738.png"},{"id":105565355,"identity":"8a672f96-960d-40cb-9077-27e91a55c378","added_by":"auto","created_at":"2026-03-27 12:53:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":279387,"visible":true,"origin":"","legend":"\u003cp\u003eThe Molecular docking results of Imidacloprid on the \u003cem\u003eA. mellifera\u003c/em\u003e GST. (a) Docking result of Imidacloprid and \u003cem\u003eA. mellifera\u003c/em\u003e GST. (b) 2D interactions of the Imidacloprid with amino acids in the active site of the \u003cem\u003eA. mellifera\u003c/em\u003eGST.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8945595/v1/45c8df298bf4fe84216bc343.png"},{"id":105565453,"identity":"c678ed1f-2655-410c-85fa-832a2643cb2c","added_by":"auto","created_at":"2026-03-27 12:53:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":345688,"visible":true,"origin":"","legend":"\u003cp\u003eThe Molecular docking results of Imidacloprid on the \u003cem\u003eA. mellifera\u003c/em\u003e odorant binding protein. (a) Docking result of Imidacloprid and \u003cem\u003eA. mellifera\u003c/em\u003eodorant binding protein. (b) 2D interactions of the Imidacloprid with amino acids in the active site of the \u003cem\u003eA. mellifera\u003c/em\u003e odorant binding protein.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8945595/v1/7b54f7ba204828042ef58548.png"},{"id":105565677,"identity":"455c7993-a528-415b-96b0-cd01ac02fc13","added_by":"auto","created_at":"2026-03-27 12:54:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":334822,"visible":true,"origin":"","legend":"\u003cp\u003eThe Molecular docking results of Thiamethoxam on the \u003cem\u003eA. mellifera\u003c/em\u003e GST. (a) Docking result of Thiamethoxam and \u003cem\u003eA. mellifera\u003c/em\u003e GST. (b) 2D interactions of the Thiamethoxam with amino acids in the active site of the \u003cem\u003eA. mellifera\u003c/em\u003eGST.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8945595/v1/80a587333a394afd5a5f0e2c.png"},{"id":105377673,"identity":"1f8822a0-4073-4cba-95ec-51a9c4eb9507","added_by":"auto","created_at":"2026-03-25 10:33:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":309801,"visible":true,"origin":"","legend":"\u003cp\u003eThe Molecular docking results of Thiamethoxam on the \u003cem\u003eA. mellifera\u003c/em\u003e odorant binding protein. (a) Docking result of Thiamethoxam and \u003cem\u003eA. mellifera\u003c/em\u003eodorant binding protein. (b) 2D interactions of the Thiamethoxam with amino acids in the active site of the \u003cem\u003eA. mellifera\u003c/em\u003e odorant binding protein.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8945595/v1/57de691303be9e7567d5c29b.png"},{"id":105377677,"identity":"3f4a11e7-07fc-4f9a-8744-cc657c05cde4","added_by":"auto","created_at":"2026-03-25 10:33:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":409363,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 8. \u003c/strong\u003eTime evolution of the root mean square deviation (RMSD) of the GST backbone atoms during the 30 ns molecular dynamics simulation of the GST–chlorantraniliprole complex. The RMSD profile indicates that the system reaches equilibrium early in the simulation and remains structurally stable throughout the production run.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8945595/v1/329fe3023df192791cf98584.png"},{"id":105565422,"identity":"2bc1b9b3-620b-4b47-9c18-add78faa8f08","added_by":"auto","created_at":"2026-03-27 12:53:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":434859,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 9. \u003c/strong\u003eRoot mean square fluctuation (RMSF) of Cα atoms of GST residues over the 30 ns MD simulation. The fluctuation pattern reveals flexible loop regions, while the majority of secondary structure elements remain relatively rigid, indicating overall structural stability of the protein upon ligand binding.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8945595/v1/97a7ba6a8657092a17704842.png"},{"id":105566075,"identity":"fbdaa34a-e288-4226-a884-a6d890b645c9","added_by":"auto","created_at":"2026-03-27 12:55:14","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":422992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 10. \u003c/strong\u003eRadius of gyration (Rg) of the GST protein as a function of simulation time during the 30 ns MD simulation. The nearly constant Rg values suggest that the protein maintains its compactness and does not undergo significant unfolding events in the presence of chlorantraniliprole.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8945595/v1/a5ee5b23e0cd933fb5ede6cb.png"},{"id":105377678,"identity":"596dc280-b022-423a-b032-3e0273a0bb26","added_by":"auto","created_at":"2026-03-25 10:33:17","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":421065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 11. \u003c/strong\u003eTime-dependent solvent-accessible surface area (SASA) of the GST protein during the 30 ns MD simulation. The stable SASA profile indicates consistent exposure of the protein surface to the solvent, supporting the structural stability of the GST–chlorantraniliprole complex.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8945595/v1/abcbd8f004edeb489b1d1a83.png"},{"id":105377680,"identity":"b1755c0c-29af-4fc9-a30e-b06f0eeeb4a4","added_by":"auto","created_at":"2026-03-25 10:33:17","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":452371,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 12. \u003c/strong\u003eTotal number of hydrogen bonds in the simulated system as a function of time over the 30 ns MD simulation. The relatively stable hydrogen bond count reflects a well-equilibrated system and stable intermolecular interactions within the solvated GST–chlorantraniliprole complex.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8945595/v1/2cc5887f81a300bb7f5b0d24.png"},{"id":108237147,"identity":"dd208d4b-0704-4676-8c82-d237b80d8a6e","added_by":"auto","created_at":"2026-04-30 19:10:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4527643,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8945595/v1/cc271fea-6397-4e32-8b66-30c219798eed.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanistic insights into the toxicological effects of insecticides with distinct modes of action on Apis mellifera: An integrated in silico study","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe extensive use of insecticides in modern agriculture has raised serious concerns regarding their unintended environmental consequences, particularly their adverse effects on non-target organisms (Serr\u0026atilde;o et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). While insecticides are designed to control pest populations, they often persist in environmental matrices such as soil, water, and vegetation, leading to chronic exposure of beneficial insects and other wildlife. Pollinators, especially honeybees (\u003cem\u003eApis mellifera\u003c/em\u003e), are highly vulnerable to such exposure due to their foraging behavior and close interaction with treated crops (Liao et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Numerous studies have demonstrated that insecticides can induce sublethal effects in bees, including impaired learning, disrupted navigation, altered foraging efficiency, and weakened immune responses, ultimately threatening colony health and ecosystem services essential for agricultural sustainability (Goulson \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sanchez-Bayo and Goka \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Pisa et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChlorantraniliprole, imidacloprid, and thiamethoxam represent widely used insecticides belonging to different chemical classes and modes of action. Chlorantraniliprole is a diamide insecticide that selectively activates ryanodine receptors, causing uncontrolled calcium release and muscle paralysis in insects (Sun and Xu \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Du and Fu \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Imidacloprid and thiamethoxam are neonicotinoid insecticides that act as agonists of nicotinic acetylcholine receptors, leading to persistent neuronal excitation and eventual insect death (Jeschke et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Thany \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although these compounds are often regarded as selective toward target pests, increasing evidence indicates that they can exert significant sublethal and molecular effects on non-target insects, including honey bees, even at environmentally relevant concentrations (Blacqui\u0026egrave;re et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAt the molecular level, detoxification and sensory perception pathways play crucial roles in mediating insect responses to xenobiotics. Glutathione S-transferases (GSTs) constitute a major family of phase II detoxification enzymes that catalyze the conjugation of reduced glutathione to electrophilic compounds, facilitating their detoxification and excretion (Kumar and Trivedi \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Potęga \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Elevated GST activity has frequently been associated with insecticide tolerance and adaptive stress responses in insects (Zhang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Odorant binding proteins (OBPs), on the other hand, are small soluble proteins located in the sensillar lymph of insect antennae and are essential for transporting hydrophobic odorant molecules to olfactory receptors (Ha and Smith \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Emerging evidence suggests that OBPs may also interact with exogenous chemicals such as insecticides, potentially interfering with olfactory signaling and behavioral processes critical for pollinator survival (Abendroth et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn silico approaches, particularly molecular docking, have become valuable tools for investigating protein\u0026ndash;ligand interactions at the atomic level (Suge\u0026ccedil;ti \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e). Molecular docking enables the prediction of binding affinities, interaction modes, and key amino acid residues involved in ligand recognition, offering mechanistic insights that complement experimental ecotoxicological studies (Waghmode et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Given ethical, logistical, and economic constraints associated with in vivo testing on pollinators, in silico modeling provides an efficient and predictive framework for screening potential toxicological interactions of insecticides with honeybee proteins (Suge\u0026ccedil;ti \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e). Such computational approaches are increasingly recognized as essential components of integrative environmental risk assessment strategies.\u003c/p\u003e \u003cp\u003eThe present study aims to evaluate the potential molecular interactions of chlorantraniliprole, imidacloprid, and thiamethoxam with Apis mellifera glutathione S-transferase and odorant binding protein using molecular docking analyses. By comparing insecticides with distinct modes of action, this work seeks to elucidate possible molecular mechanisms underlying detoxification responses and sensory disruption in honeybees, thereby contributing to a deeper understanding of insecticide-related ecotoxicological risks to pollinators.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals\u003c/h2\u003e \u003cp\u003eThe chemical structures of chlorantraniliprole, imidacloprid, and thiamethoxam were obtained from the PubChem database of the National Center for Biotechnology Information (NCBI). The three-dimensional (3D) structures were downloaded in SDF format and subsequently prepared for molecular docking analysis. Prior to docking, ligand structures were energy-minimized to achieve stable conformations using the MMFF94 force field. All ligands were converted to the PDBQT format following the addition of polar hydrogen atoms and assignment of Gasteiger partial charges.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Protein structure preparation\u003c/h2\u003e \u003cp\u003eThe three-dimensional structures of \u003cem\u003eA. mellifera\u003c/em\u003e GST and OBP were retrieved from the Protein Data Bank (PDB). Selected protein structures were chosen based on resolution quality and biological relevance. Prior to docking, all water molecules, co-crystallized ligands, and heteroatoms were removed from the protein structures. Polar hydrogen atoms were added, and Kollman charges were assigned to prepare the proteins for docking analysis. Protein structures were subsequently saved in PDBQT format.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Molecular docking analysis\u003c/h2\u003e \u003cp\u003eMolecular docking simulations were performed using AutoDock Vina to evaluate the binding interactions between the selected insecticides and the target proteins (Trott and Olson \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Docking grids were defined to encompass the active or binding sites of GST and OBP based on known functional regions reported in the literature. Docking calculations were conducted using default exhaustiveness parameters, and the best-ranked binding poses were selected based on minimum binding free energy (kcal/mol). The predicted protein\u0026ndash;ligand complexes were visualized and analyzed using Discovery Studio Visualizer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. In silico prediction of AMED-related physicochemical and toxicokinetic properties\u003c/h2\u003e \u003cp\u003eIn silico AMED-related physicochemical and toxicokinetic properties of chlorantraniliprole, imidacloprid, and thiamethoxam were predicted using SwissADME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.swissadme.ch/index.php\u003c/span\u003e\u003cspan address=\"https://www.swissadme.ch/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and pkCSM online platforms. These analyses provided comparative insights into absorption, distribution, metabolic interaction potential, and ecotoxicological risk, supporting the interpretation of molecular docking results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Molecular Dynamics (MD) Simulations\u003c/h2\u003e \u003cp\u003eAll-atom molecular dynamics (MD) simulations were performed using the GROMACS 2023.3 simulation package to investigate the structural stability and dynamic behavior of the glutathione S-transferase (GST)\u0026ndash;chlorantraniliprole complex. The protein and ligand were described using the OPLS-AA force field, while the TIP3P water model was employed for explicit solvation. The complex was placed in a triclinic simulation box with a minimum distance of 10 \u0026Aring; between the solute and the box boundaries, and the system was neutralized by the addition of appropriate counterions.\u003c/p\u003e \u003cp\u003eEnergy minimization was carried out using the steepest descent algorithm until the maximum force fell below 1000 kJ\u0026middot;mol⁻\u0026sup1;\u0026middot;nm⁻\u0026sup1; to remove unfavorable steric contacts. The system was then equilibrated in two phases: a 100 ps NVT ensemble followed by a 100 ps NPT ensemble, during which position restraints were applied to the protein backbone atoms. Temperature was maintained at 300 K using the velocity-rescaling (V-rescale) thermostat, while pressure was controlled at 1 bar using the Parrinello\u0026ndash;Rahman barostat with isotropic coupling.\u003c/p\u003e \u003cp\u003eLong-range electrostatic interactions were calculated using the Particle Mesh Ewald (PME) method with a real-space cutoff of 1.0 nm. Van der Waals interactions were treated using a force-switch scheme between 0.9 and 1.0 nm. All bonds involving hydrogen atoms were constrained using the LINCS algorithm, allowing an integration time step of 2 fs.\u003c/p\u003e \u003cp\u003eFollowing equilibration, a 30 ns production MD simulation was performed under the NPT ensemble without position restraints. Periodic boundary conditions were applied in all three spatial dimensions. Trajectory coordinates were saved every 2 ps for subsequent analyses. Structural stability and conformational dynamics of the complex were evaluated using root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and hydrogen bond analyses based on the production trajectory.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eMolecular docking simulations were performed to evaluate the binding affinities and interaction profiles of chlorantraniliprole, imidacloprid, and thiamethoxam with \u003cem\u003eA. mellifera\u003c/em\u003e GST and OBP. The predicted binding free energy values (ΔG, kcal/mol) and key molecular interactions are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe docking results of insecticides on GST and Odorant binding protein of \u003cem\u003eA. mellifera\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInsecticides\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eΔG (kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInteractions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmino Acid\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"9\" rowspan=\"10\"\u003e \u003cp\u003eChlorantraniliprole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eGST\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e-7.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCarbon hydrogen bonds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSER34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePi-sigma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLEU58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePi-sulfur\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMET134\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePi-Pi T-shaped\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTRY138\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePi-alkyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTRY138; PHE229; PHE142\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlkyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMET134; PRO35; LEU58; LEU31; LEU232; ILE141\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eOdorant binding protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e-7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePi-sigma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVAL108;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePi-Pi T-shaped\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTRP116; PHE117\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePi-alkyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTRP116; PHE117; ALA82; ALA112; LEU78; VAL85; LEU74; ARG81; ARG81;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlkyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eALA105; MET86; VAL85; MET70; VAL108; LEU73; LEU53; LEU58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eImidacloprid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGST\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e-6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConventional hydrogen bonds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSER34; TYR130\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlkyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMET228; LEU232\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eOdorant binding protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e-7.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePi-sulfur\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMET70;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePi-alkyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLEU74; VAL85; ALA82; VAL108\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlkyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLEU74; ALA82; LEU78; MET86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e \u003cp\u003eThiamethoxam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eGST\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e-5.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConventional hydrogen bonds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSER34; TYR130\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCarbon hydrogen bonds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSER34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePi-Pi stacked\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePHE229\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePi-alkyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLEU232\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlkyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMET228; LEU232\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eOdorant binding protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e-11.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePi-Pi stacked\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTRP116\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePi-alkyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eALA105; MET49; TRP116; LEU53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAlkyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMET49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Interactions with glutathione S-transferase\u003c/h2\u003e \u003cp\u003eAmong the tested insecticides, chlorantraniliprole exhibited the strongest binding affinity toward GST with a ΔG value of -7.3 kcal/mol (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The binding was stabilized by multiple non-covalent interactions, including carbon hydrogen bonding with SER34, π\u0026ndash;σ interaction with LEU58, π\u0026ndash;sulfur interaction with MET134, and a π\u0026ndash;π T-shaped interaction involving TYR138. Additionally, several hydrophobic π\u0026ndash;alkyl and alkyl interactions were observed with residues such as PHE229, PHE142, PRO35, LEU31, LEU58, LEU232, and ILE141, indicating a stable accommodation of chlorantraniliprole within the GST binding pocket (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImidacloprid showed a moderate binding affinity to GST (ΔG = -6.5 kcal/mol) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Its interaction profile was characterized primarily by conventional hydrogen bonds with SER34 and TYR130, accompanied by hydrophobic alkyl interactions involving MET228 and LEU232. Compared to chlorantraniliprole, imidacloprid formed fewer stabilizing interactions within the GST active site (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThiamethoxam displayed the weakest binding affinity toward GST among the tested insecticides (ΔG = -5.8 kcal/mol) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The interaction pattern included conventional and carbon hydrogen bonds with SER34, a π\u0026ndash;π stacking interaction with PHE229, and hydrophobic π\u0026ndash;alkyl and alkyl contacts with LEU232 and MET228. Overall, the lower binding energy and reduced interaction diversity suggest a comparatively weaker association of thiamethoxam with GST (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Interactions with odorant binding protein\u003c/h2\u003e \u003cp\u003eDocking results indicated that all three insecticides interacted favorably with OBP, with binding affinities generally stronger than those observed for GST. Chlorantraniliprole exhibited a binding free energy of -7.8 kcal/mol and formed multiple hydrophobic interactions within the OBP binding cavity (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These included π\u0026ndash;σ interaction with VAL108, π\u0026ndash;π T-shaped interactions with TRP116 and PHE117, and extensive π\u0026ndash;alkyl and alkyl contacts involving residues such as ALA82, ALA112, LEU78, LEU74, VAL85, MET70, MET86, LEU53, and LEU58 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImidacloprid demonstrated a comparable binding affinity to OBP (ΔG\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;.4 kcal/mol). The interaction profile was dominated by hydrophobic contacts, including π\u0026ndash;sulfur interaction with MET70 and π\u0026ndash;alkyl interactions with LEU74, VAL85, ALA82, and VAL108. Additional alkyl interactions with LEU78 and MET86 further contributed to ligand stabilization within the OBP binding pocket (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThiamethoxam showed the strongest binding affinity to OBP with a ΔG value of -11.8 kcal/mol. This interaction was primarily stabilized by a π\u0026ndash;π stacking interaction with TRP116, along with multiple π\u0026ndash;alkyl and alkyl interactions involving ALA105, MET49, LEU53, and TRP116. The markedly lower binding free energy suggests a high affinity of thiamethoxam for OBP compared to both GST and the other insecticides tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Molecular Dynamics Simulation Analysis of GST\u0026ndash;Chlorantraniliprole Complex\u003c/h2\u003e \u003cp\u003eThe structural stability and dynamic behavior of the GST\u0026ndash;chlorantraniliprole complex were evaluated through a 30 ns all-atom molecular dynamics simulation using multiple structural descriptors, including RMSD, RMSF, radius of gyration (Rg), solvent-accessible surface area (SASA), and hydrogen bond analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRoot Mean Square Deviation (RMSD)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe RMSD profile of the protein backbone revealed an initial increase during the early phase of the simulation, followed by stabilization after approximately 6\u0026ndash;8 ns (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Throughout the remaining simulation time, RMSD values fluctuated within a narrow range (~\u0026thinsp;0.16\u0026ndash;0.20 nm), indicating that the GST\u0026ndash;chlorantraniliprole complex reached a stable conformational state. The absence of large deviations suggests that ligand binding did not induce significant structural destabilization of the protein, supporting the overall stability of the complex during the simulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRoot Mean Square Fluctuation (RMSF)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eResidue-wise flexibility of the protein was assessed using RMSF analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Most residues exhibited low fluctuations (\u0026lt;\u0026thinsp;0.15 nm), indicating a rigid and stable protein backbone. Higher fluctuations were mainly observed at the N- and C-terminal regions and in selected loop regions, which is typical for solvent-exposed and flexible segments of proteins. Importantly, residues associated with the ligand-binding region showed relatively low RMSF values, suggesting that chlorantraniliprole binding contributes to local stabilization of the active site.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRadius of Gyration (Rg)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe radius of gyration remained relatively constant throughout the simulation, fluctuating around an average value of approximately 2.24\u0026ndash;2.27 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003e). This consistent Rg profile indicates that the overall compactness of the GST structure was preserved during the simulation. No significant expansion or collapse of the protein structure was observed, further confirming the conformational stability of the GST\u0026ndash;chlorantraniliprole complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSolvent-Accessible Surface Area (SASA)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSASA analysis showed moderate fluctuations around an average value of ~\u0026thinsp;215\u0026ndash;225 nm\u0026sup2; during the simulation period (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e11\u003c/span\u003e). These variations reflect minor conformational rearrangements at the protein surface but do not indicate large-scale unfolding events. The relatively stable SASA profile suggests that the global exposure of the protein to the solvent remained largely unchanged upon ligand binding.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHydrogen Bond Analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe number of hydrogen bonds within the system displayed stable behavior over the 30 ns simulation, with values fluctuating within a consistent range (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e12\u003c/span\u003e). Although minor decreases were observed toward the later stages of the simulation, the overall hydrogen bond network remained well maintained. This indicates sustained intermolecular and intramolecular interactions contributing to the structural integrity of the protein\u0026ndash;ligand complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4. In silico AMED profiling of chlorantraniliprole, imidacloprid, and thiamethoxam\u003c/h2\u003e \u003cp\u003eSwissADME-based AMED predictions revealed marked physicochemical differences among the tested insecticides (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Chlorantraniliprole exhibited the highest molecular weight (483.15 g/mol) and lipophilicity (XlogP3\u0026thinsp;=\u0026thinsp;4.80), along with relatively low polarity (TPSA\u0026thinsp;=\u0026thinsp;88.91 \u0026Aring;\u0026sup2;), suggesting a strong tendency for hydrophobic interactions and limited aqueous solubility. In contrast, imidacloprid showed substantially lower lipophilicity (XlogP3\u0026thinsp;=\u0026thinsp;0.57) and high predicted solubility, whereas thiamethoxam displayed intermediate lipophilicity but higher polarity (TPSA\u0026thinsp;=\u0026thinsp;115.02 \u0026Aring;\u0026sup2;). All compounds were predicted to exhibit high gastrointestinal absorption and lack blood\u0026ndash;brain barrier permeability.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIn silico AMED properties of the tested insecticides\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChlorantraniliprole\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eImidacloprid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThiamethoxam\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMolecular weight (g/mol)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e483.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e255.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e291.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLogP (XlogP3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTopological polar surface area (TPSA, \u0026Aring;\u0026sup2;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e115.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH-bond donors\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH-bond acceptors\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGI absorption\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBBB permeability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP-gp substrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCYP inhibition (general)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes (CYP2C19, CYP2C9, CYP3A4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes (CYP1A2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eESOL solubility class\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eModerately soluble\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVery soluble\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoluble\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSilicos-IT solubility class\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePoorly soluble\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSoluble\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSoluble\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBioavailability score\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe molecular docking analyses presented in this study provide detailed insights into the potential molecular interactions of three major insecticides such as chlorantraniliprole, imidacloprid, and thiamethoxam with GST and OBP of \u003cem\u003eA. mellifera\u003c/em\u003e. These findings support and extend recent evidence that sublethal exposure to modern insecticides can perturb both detoxification and sensory systems in honeybees, even at environmentally relevant concentrations (Li et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChlorantraniliprole showed the highest binding affinity toward GST, suggesting its strong interaction with phase II detoxification enzymes. GSTs play an essential role in conjugating electrophilic xenobiotics with glutathione, facilitating their elimination from the organism (Higgins and Hayes \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Previous studies have reported that exposure to chlorantraniliprole can alter antioxidant enzyme activities in \u003cem\u003eA. mellifera\u003c/em\u003e and related species (Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). Transcriptomic analyses further demonstrated upregulation of GST and related detoxification genes in insects following chlorantraniliprole exposure (Wang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The predominance of hydrophobic and π\u0026ndash;π interactions observed in the present study indicates a stable accommodation of chlorantraniliprole within the GST catalytic site, consistent with its capacity to trigger enzymatic detoxification pathways. These findings collectively suggest that chlorantraniliprole may be efficiently metabolized by honeybee GSTs, potentially contributing to adaptive tolerance mechanisms.\u003c/p\u003e \u003cp\u003eIn contrast, thiamethoxam exhibited the strongest binding affinity to OBP, implying possible interference with olfactory perception mechanisms. OBPs are vital in transporting odorant molecules through the sensillar lymph to odorant receptors, thereby enabling floral recognition and foraging communication. Neonicotinoids have been shown to reduce antennal sensitivity and alter odor learning in bees (Mitchell and Alexandre 2021). The current docking results revealing extensive π\u0026ndash;π stacking and hydrophobic interactions between thiamethoxam and key OBP residues (e.g., TRP116, LEU53, and MET49), provide a structural basis for such behavioral impairments. Similar molecular interactions have been reported in \u003cem\u003eAphis craccivora\u003c/em\u003e for neonicotinoids, suggesting that OBP binding may represent a conserved mechanism of chemosensory disruption (Abdelmoteleb et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eImidacloprid displayed intermediate affinity for both GST and OBP, suggesting potential dual molecular targets. This broader binding profile may underlie its diverse sublethal effects, including impaired learning, reduced foraging, and increased oxidative stress observed in \u003cem\u003eApis\u003c/em\u003e species (Peng et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, imidacloprid exposure has been linked to the upregulation of detoxification enzymes such as UDP-glucosyltransferases and GSTs, which are known to contribute to resistance in pests (Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Peng et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The ability of imidacloprid to engage with both metabolic and olfactory proteins could thus potentiate cumulative physiological stress, even at sublethal exposure levels.\u003c/p\u003e \u003cp\u003eIt was determined that the structure and mechanism of action of the insecticide determined the different protein binding behaviors in honeybees. These findings are in accordance with \u003cem\u003ein vivo\u003c/em\u003e results demonstrating that exposure to insecticides induces enzyme activity changes and behavioral alterations (Alfuhaid et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). From an ecotoxicological standpoint, these protein\u0026ndash;ligand interaction patterns provide molecular evidence that sublethal insecticide exposure may simultaneously challenge detoxification capacity and sensory integrity, leading to synergistic physiological consequences.\u003c/p\u003e \u003cp\u003eThe predicted AMED profiles were highly consistent with the docking-based interaction patterns observed in this study. The pronounced lipophilicity and moderate polarity of chlorantraniliprole favor stable accommodation within the hydrophobic active site of GST, supporting its strong binding affinity and extensive hydrophobic interactions (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). Conversely, thiamethoxam exhibited higher polarity and moderate lipophilicity, physicochemical characteristics that are compatible with efficient interaction within the OBP cavity, where ligand stabilization is largely driven by π\u0026ndash;π stacking and hydrophobic contacts rather than membrane partitioning. Imidacloprid displayed comparatively low lipophilicity and high solubility, consistent with its moderate binding affinities toward both GST and OBP (Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Collectively, these findings indicate that physicochemical and toxicokinetic properties critically shape protein-binding behavior and sublethal molecular interference mechanisms in \u003cem\u003eA. mellifera\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eFuture research should integrate molecular docking with transcriptomic, enzymatic, and behavioral studies to validate these computational predictions under biological conditions. Additionally, comparative docking analyses across pollinator taxa such as \u003cem\u003eA. cerana\u003c/em\u003e and \u003cem\u003eBombus terrestris\u003c/em\u003e could elucidate interspecific variations in susceptibility, informing pollinator-safe pesticide development. Collectively, this study demonstrates that \u003cem\u003ein silico\u003c/em\u003e modeling can serve as a valuable component of mechanistic risk assessment frameworks, bridging molecular mechanisms with ecological outcomes in pollinator toxicology.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdelmoteleb M, Mohamed A, Genidy N, Abdel-Haleem D (2023) Computational and Toxicological Evaluation of Thiamethoxam as Nicotinic Acetylcholine Receptor Modulator Against Cowpea Aphid, Aphis craccivora Koch. 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Pestic Biochem Physiol 188:105230. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pestbp.2022.105230\u003c/span\u003e\u003cspan address=\"10.1016/j.pestbp.2022.105230\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Apis mellifera, molecular dynamics simulation, AMED analysis, glutathione S-transferase, odorant-binding protein, ecotoxicology","lastPublishedDoi":"10.21203/rs.3.rs-8945595/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8945595/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe extensive use of insecticides in modern agriculture raises concerns regarding their sublethal molecular effects on non-target pollinators such as the honeybee (Apis mellifera). This study employed an integrated in silico approach combining AMED profiling, molecular docking, and molecular dynamics (MD) simulations to investigate the interactions of chlorantraniliprole, imidacloprid, and thiamethoxam with two key honeybee proteins: glutathione S-transferase (GST) and odorant-binding protein (OBP). Docking analyses revealed selective binding patterns, with chlorantraniliprole showing the strongest affinity toward GST, suggesting a prominent interaction with detoxification pathways. Thiamethoxam exhibited the highest affinity for OBP, indicating potential interference with olfactory signaling, whereas imidacloprid demonstrated moderate binding to both proteins. MD simulation of the GST\u0026ndash;chlorantraniliprole complex confirmed structural stability based on RMSD, RMSF, Rg, SASA, and hydrogen bond analyses. These findings provide mechanistic insights into how insecticides may simultaneously affect detoxification and sensory systems in \u003cem\u003eA. mellifera\u003c/em\u003e. The integration of AMED prediction with docking and MD simulations offers a complementary computational framework for ecotoxicological risk assessment of agrochemicals.\u003c/p\u003e","manuscriptTitle":"Mechanistic insights into the toxicological effects of insecticides with distinct modes of action on Apis mellifera: An integrated in silico study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-25 10:33:12","doi":"10.21203/rs.3.rs-8945595/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a17efd6e-52e0-4648-91e2-ef4f25f526e8","owner":[],"postedDate":"March 25th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-04-30T18:56:25+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-30T19:09:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-25 10:33:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8945595","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8945595","identity":"rs-8945595","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}