Molecular docking analysis and biochemical characterization of metabolic detoxification enzymes in adults of Aedes aegypti L. (Diptera: Culicidae) exposed to α-cypermethrin

1 Introduction

Aedes aegypti serves as the primary vector for numerous arboviral diseases, including dengue, chikungunya, Zika, and yellow fever. Each year, millions of Aedes-borne disease cases, especially dengue, are recorded by the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC), causing a large number of deaths and considerable health issues (1, 2). India remains a major hotspot for these diseases, which is often attributed to its large population, rapid and often unplanned urbanization, and climatic conditions that favor year-round mosquito breeding. In 2025, India reported over 125,824 dengue cases, with significant transmission clusters in states such as Tamil Nadu, West Bengal, Uttar Pradesh, Kerala, and Maharashtra (3). As of 23 March 2026, global dengue cases had already surpassed 500,000, and early reports suggest that India may experience high transmission rates again this year (4). The lack of widely accessible and effective vaccines and targeted medications for these arboviral diseases, except for yellow fever, has made the situation grave. Despite continuous research on dengue vaccines, limited success has been achieved due to the complex nature of the four virus serotypes and antibody-dependent enhancement (ADE). A few vaccines, such as Dengvaxia (CYD-TDV), licensed in several countries, and Qdenga, with 80% efficacy, are still under trials in several countries and face challenges in approval (5, 6). Other candidates, such as Butantan-DV, are still in Phase 3 trials, leaving a substantial gap in public health defense (7). Consequently, the global strategy remains heavily reliant on vector control as the only viable method to break the transmission cycle.

Chemical interventions for vector control have progressed through several insecticide classes, starting with the widespread use of organochlorines (OCL) such as DDT, organophosphates (OPs) such as including malathion and temephos, and carbamates such as bendiocarb. These classes, however, are linked to increased toxicity to non-target organisms and the development of cross-resistance to multiple compounds (8). To address these issues, pyrethroids were developed and have become central to modern vector control. These are preferred for their high efficacy at low concentrations, rapid knockdown of adult mosquitoes, and better safety for humans compared with other synthetic insecticides (9).

Among various pyrethroids, α-cypermethrin, a type II pyrethroid, is considered highly effective against mosquitoes, leading to sustained depolarization of the nerve membrane, irreversible paralysis, and increased mortality (10). A few investigations have revealed the effective use of α-cypermethrin against different life stages of mosquitoes. Indoor residual spraying of α-cypermethrin at 20–30 mg/m² has demonstrated residual efficacy for 11–16 weeks against Anopheles mosquitoes (11). Use of a 0.667% α-cypermethrin-treated long lasting insecticidal net caused knockdown of 70%–90% of Anopheles culicifacies and An. fluviatilis adults after 3 min, with 100% mortality after 24 h of exposure (12). The lethal concentration (LC) value of 0.0005 ppm has been reported by Samal et al. (13) against Ae. aegypti larvae.

However, extensive use of pyrethroids has also imposed strong selective pressure on Ae. aegypti populations, facilitating the development of resistance mechanisms (14). Understanding these mechanisms is crucial for insecticide resistance surveillance and mosquito management programs. In India, surveillance studies conducted in Delhi and surrounding areas reported variable susceptibility profiles of Ae. aegypti to insecticides of public-health importance, emphasizing the necessity for continuous monitoring of resistance trends and the mechanisms responsible for insecticide detoxification (15).

Reports have suggested that pyrethroid resistance is mainly driven by metabolic detoxification and modifications of voltage-gated sodium channels (VGSCs) due to target-site mutations. It is widely known that mosquitoes possess a plethora of enzymes that confer resistance to insecticides through metabolic detoxification (16). These enzymes include glutathione S-transferases (GSTs), cytochrome P450 monooxygenases (CYP450s), α- and β-carboxylesterases (CCEs), and acetylcholinesterases (AChEs). These enzymes metabolize, sequester, or eliminate insecticides before they reach the target sites, thereby reducing insecticidal efficacy. Overexpression or structural modification of these detoxifying enzymes due to frequent insecticide exposure can enhance insecticide detoxification and reduce susceptibility through the selection of resistant strains resulting from altered gene expression, causing control failures (17, 18).

Despite the importance of α-cypermethrin in mosquito control programs, information regarding its interaction with major detoxification enzymes and the consequent development of potential resistance remains limited. Hence, it is important to assess the baseline susceptibility of mosquitoes to an insecticide and understand the enzymes involved and the underlying mechanisms that enable them to survive insecticide exposure. Previous studies have suggested the role of CCEs in causing OP and carbamate resistance, GSTs in DDT resistance, and esterases and monooxygenases in resistance against organophosphates, pyrethroids, and, to some extent, carbamates (17–19). However, it has been shown that mosquitoes may have multiple resistance mechanisms contributed by different enzymes, probably due to cross-resistance with other groups of insecticides. Cross-resistance between DDT and various pyrethroids has been widely reported, leading to a high frequency of DDT and pyrethroid resistance in Ae. aegypti (20). This has resulted in the involvement of multifarious detoxifying enzymes in imparting resistance in mosquitoes. In Costa Rica, Ae. aegypti adults with deltamethrin resistance showed high esterase levels, while CYP450 or GSTs did not show significant alterations (21). In contrast, in Brazil, temephos resistance was found to be associated with all three of these enzymes (22).

The resistance levels in mosquitoes have been monitored by several bioassays recommended by the WHO and CDC. These include the WHO tube bioassay, based on mosquito exposure to insecticide-impregnated papers; the CDC bottle bioassay, which measures the knockdown time of mosquitoes exposed to a particular insecticide dose coated on a glass bottle; and the WHO bottle bioassay, which is similar to the CDC bottle bioassay but recommends exposure of mosquitoes to insecticides for 1 h and records mortality after 24 h (23–25). Limited studies have reported the susceptibility status of mosquitoes measured through the WHO bottle bioassay, while no studies are available that assess susceptibility to α-cypermethrin. In Indonesia, development of α-cypermethrin resistance has been reported in Ae. aegypti assessed through the CDC bottle bioassay, revealing adult mortality rates of 65.0%, 68.3%, 74.2%, and 82.5% in the Plaosan, Ngujung, Candirejo, and Taji districts, respectively (26). Similarly, cypermethrin resistance in Ae. aegypti (< 80% mortality) has been documented in other Indonesian districts using the CDC bottle bioassay.

Hence, the present study attempted to assess the susceptibility of an Indian strain of Ae. aegypti to α-cypermethrin through the standardized WHO Bottle Bioassay (23). Additionally, the possible role of detoxification mechanisms in the development of α-cypermethrin resistance was investigated by performing molecular docking studies between α-cypermethrin and five enzymes: glutathione-S-transferase (GST), cytochrome P450 monooxygenases, α- and β-esterases, and acetylcholinesterase. It is suggested that this computational approach can help provide a systematic understanding of resistance at the molecular level and complement experimental observations. The validation of the findings was carried out through biochemical characterization to understand the metabolic defense mechanisms in mosquitoes. We believe that understanding these pathways will help in mosquito management by identifying target sites and facilitating the development of modified insecticides with enhanced action.

2 Materials and methods

2.1 Rearing and maintenance of Aedes aegypti under controlled conditions

The susceptible strain of Ae. aegypti was procured from ICGEB (International Center for Genetic Engineering and Biotechnology), New Delhi, India, in 2009 and was supplemented in 2017. The colonies were reared in the Insect Pest and Vector Control Laboratory, Acharya Narendra Dev College, University of Delhi, India (27). The culture has been maintained under controlled temperature (27 ± 2 °C), relative humidity (80 ± 10%), and a photoperiod of (14L:10D) without any insecticide selection pressure. A sucrose solution (10%) was used as a nutrient source for the adults kept in screened muslin cloth cages (18” × 16” × 16”). For egg maturation and development, the female adults were also given a blood meal from an albino mouse kept in the cage every alternate day. Eggs were collected on moist Whatman paper strips lining bowls filled with dechlorinated water. Larvae were reared in dechlorinated water contained in enamel/plastic trays and provided with nutrition through ground dog biscuits and yeast (3:1 w/w). Care was taken to keep the water clean. The pupae were collected on a regular basis for adult emergence.

2.2 Chemicals used

The pyrethroid insecticide, α-cypermethrin (purity ≥98.0%), used in the present study, was purchased from Sigma-Aldrich, India (Product No. 45806, CAS Number: 67375-30-8). Other chemicals used for performing detoxification enzyme bioassays were also purchased from Sigma-Aldrich, India.

A stock solution of α-cypermethrin was prepared using analytical-grade acetone. The serial dilutions were made to obtain 12 different concentrations: 0.00001% (0.1 µg/bottle), 0.00002% (0.2 µg/bottle), 0.00004% (0.4 µg/bottle), 0.00006% (0.6 µg/bottle), 0.00008% (0.8 µg/bottle), 0.0001% (1 µg/bottle), 0.0002% (2 µg/bottle), 0.0004% (4 µg/bottle), 0.0006% (6 µg/bottle), 0.0008% (8 µg/bottle), 0.001% (10 µg/bottle), and 0.0012% (12 µg/bottle), which includes the diagnostic dose of 0.001% (10 µg/bottle). All solutions were freshly prepared prior to the experiment to ensure their stability and effectiveness.

2.3 World Health Organization (WHO) bottle bioassay

The susceptibility/resistance status of the Ae. aegypti population was assessed through a WHO bottle bioassay (WHO, 2022). Female adults that were 2–3 days old and starved for 24 h were used for the bioassay.

2.3.1 Preparation of bottles for bioassay

Standard 200 mL Wheaton glass bottles with screw caps, as recommended by the WHO, were used for conducting the bottle bioassay with slight modifications. A total of 49 clean, air-dried, capped bottles were used: four bottles (replicates) for each of the twelve concentrations and one for the control.

The control bottle was coated with 1 mL of acetone while the test bottles were coated with 1 mL of the appropriate α-cypermethrin concentration to achieve the target dose. The solutions were poured into the respective bottles. The bottles were gently swirled and rotated to uniformly coat the inner surfaces (bottom, sides, and cap). The prepared bottles were covered with foil to protect them from light.

2.3.2 Bioassay procedure

In each bottle, 20 unfed 2–3-day old female mosquitoes were introduced with the help of a mechanical aspirator. Bottles were immediately covered to prevent the escape of mosquitoes. After exposure to α-cypermethrin coated on the bottle surface for 1 h, mosquitoes were transferred to beakers covered with muslin cloth. A sucrose solution was provided as a nutrient source.

Mortality was recorded after 24 h. The mosquitoes unable to fly or sit on the surface and those showing uncoordinated movement were considered dead.

2.3.3 Data recording and analysis

Percent mean adult mortality was calculated using the formula given in Equation 1:

(1) Adult mortality(%)=Number of dead mosquitoesTotal mosquitoes exposed×100

The ≥98% adult mortality indicated that the population was susceptible; 90%–98% mortality suggested possible resistance requiring confirmation, while <90% mortality confirmed that the population used for the bioassay was resistant to α-cypermethrin (23). Control mortality was monitored in each bioassay. When control mortality ranged between 5% and 20%, mortality data were corrected using Abbott’s formula (29). Bioassays with control mortality exceeding 20% were discarded and repeated.

The mortality data were analyzed using Probit regression in SPSS 19.0 software. The LC (lethal concentration) values at different levels (30, 50, 70, and 90) were computed with confidence limits and additional statistical parameters, such as slope and goodness-of-fit.

2.4 Effect of α-cypermethrin on the detoxification enzymes of Aedes aegypti

The effect of α-cypermethrin on the five detoxification enzymes of Ae. aegypti was assessed through in silico molecular docking followed by biochemical estimation. The selected detoxifying enzymes (target proteins) included glutathione S-transferase (GST), cytochrome P450 monooxygenase (CYP450), carboxylesterases (α-esterase and β-esterase), and acetylcholinesterase (AChE).

2.4.1 In silico molecular docking of detoxification enzymes

2.4.1.1 Protein preparation

Three-dimensional crystal structures of the target detoxification enzymes were retrieved from the Protein Data Bank in PDB format. The downloaded protein structures were carefully examined for structural completeness and suitability for docking analysis. Proteins were purified using UCSF Chimera 1.19 (https://www.cgl.ucsf.edu/chimerax/) by removing all non-essential co-crystallized ligands, water molecules, and ions to avoid interference during docking. Missing hydrogen atoms were added to stabilize the protein structure and ensure proper protonation of amino acid residues. Partial charges were assigned to the proteins, and the prepared structures were energy-minimized using the default minimization parameters available in Chimera in order to obtain stable conformations suitable for docking studies. The processed protein structures were subsequently saved in PDB format for further analysis.

2.4.1.2 Ligand preparation

The chemical structure of α-cypermethrin was obtained from the PubChem database in SDF format. The ligand structure was imported into PyRx (https://pyrx.sourceforge.io/) and subjected to geometry optimization to obtain a stable conformer. Hydrogen atoms and Gasteiger charges were added, and the ligand was converted into PDBQT format for docking analysis.

2.4.1.3 Molecular docking analysis

Molecular docking was performed using the PyRx Virtual Screening Tool. The prepared protein and ligand files were imported into the PyRx workspace. Prior to docking, the receptor molecules were converted into macromolecule format, while the ligand molecule was converted into ligand format using the AutoDock tools integrated within PyRx.

A grid box was generated around the protein to define the docking search space. Docking simulations were then carried out using the default AutoDock Vina parameters implemented in PyRx. The binding affinity of α-cypermethrin toward each detoxifying enzyme was evaluated based on the docking score expressed in kcal/mol. The best docked conformations were selected according to the lowest binding energy and favorable interaction patterns.

2.4.1.4 Visualization and interaction analysis

The docked complexes obtained from PyRx were visualized and analyzed using UCSF Chimera 1.19 and other visualization tools where necessary. The interactions between α-cypermethrin and amino acid residues within the active site of each enzyme were examined, including hydrogen bonding, hydrophobic interactions, van der Waals interactions, and π–π interactions. These interactions were used to evaluate the possible involvement of detoxification enzymes in α-cypermethrin resistance mechanisms in Ae. aegypti. The docking scores and interaction profiles of all target enzymes were comparatively analyzed to determine the relative binding affinity of α-cypermethrin toward different detoxification enzymes. Enzymes exhibiting stronger binding interactions with the insecticide were considered to have a potentially significant role in insecticide metabolism and resistance development.

2.4.2 Biochemical characterization of detoxification enzymes

The docking results were validated through estimation of detoxification enzyme levels in Ae. aegypti according to the WHO protocol with minor modifications (28, 30). Adult mosquitoes were exposed to the LC₅₀ and LC₉₀ concentrations of α-cypermethrin, and surviving adults were separated for the assay.

Individual adults were homogenized in 500 µL of autoclaved water kept on ice. Two volumes of 25 µL (to conduct replicates) of the homogenized mixture were kept separately for the AChE estimation, while the remaining homogenate was spun in a refrigerated microfuge at 17000 rpm for 30 s. The supernatant obtained was used for the estimation of proteins, α-esterase, β-esterase, GST, AChE, and CYP450.

2.4.2.1 Estimation of proteins

For protein estimation, 10 µL supernatant from each sample was pipetted into two wells of a microtiter plate, and 300µL of BIORAD protein reagent was added. A blank was run simultaneously using water in place of the homogenate. The absorbance was read at 570 nm, and the standard curve was plotted. The protein concentration of control adults and experimental adults exposed to LC₅₀ and LC₉₀ of α-cypermethrin was calculated in mg/mL and in nmol.

2.4.2.2 Estimation of non-specific esterase

For the estimation of non-specific esterase, 10µL (in replicates) of the supernatant from each sample was pipetted into a microtiter plate. Then, 200 µL of α-naphthyl acetate was added to each well, and the plate was incubated for 15 min to estimate the levels of α-esterase. Thereafter, 50 µL of freshly prepared fast blue stain was added to each well, and the absorbance was read at 570 nm (31). The activity of α-esterase was expressed as mol naphthol/min/mg protein.

The level of β-esterase in each Ae. aegypti adult was estimated following a similar procedure using β-naphthyl acetate in place of α-naphthyl acetate.

2.4.2.3 Estimation of glutathione-S-transferase

The GST estimation involved adding 50 µL of 2mM reduced glutathione (GSH) and 50 µL of 1mM CDNB (1-chloro-2,4-dinitrobenzene) to 20 µL of supernatant from each sample (in replicates). The absorbance was read at 340 nm for 5 consecutive min to determine enzyme kinetics (32). The GST activity was expressed as nmol/min/mg protein.

2.4.2.4 Estimation of acetylcholinesterase

The AChE estimation was carried out using 25 µL (in replicates) of mosquito crude homogenate, to which 145 µL of 1% Triton X-100 was added to solubilize AChE, followed by 10 µL of DTNB (dithio-bis 2-nitrobenzoic acid). The ASCHI solution was divided into two 10 mL aliquots. One aliquot was supplemented with 20µL of 0.1M propoxur. Thereafter, one replicate was mixed with 25 µL of ASCHI (acetylthiocholine iodide), while the other replicate was mixed with 25 µL of ASCHI + propoxur (drawn from 10 mL ASCHI + 20 µL of 0.1M propoxur). The plate was incubated for 1 h, and the absorbance was read at 405 nm (33). The endpoint of the reaction was calculated using the formula given in Equation 2:

(2) Endpoint=Absorbance of the mixture containing propoxurAbsorbance of the mixture without propoxur

Percent inhibition of acetylcholinesterase was calculated using the following formula (Equation 3):

(3) % AChE inhibition=100−(100% X Endpoint)

2.4.2.5 Estimation of cytochrome P450 monooxygenase

For the estimation of cytochrome P450 monooxygenase, 20 μL supernatant from each adult homogenate (two replicates) was pipetted into microtiter plate wells and mixed with 80 μL of 0.625 M potassium phosphate buffer. Subsequently, 200 μL of a solution [drawn from a mixture of 15 mL of 0.25 M sodium acetate buffer and 5 mL of a methanolic solution of TMBZ (3,3’,5,5’-tetramethylbenzidine)] was added, followed by 25 μL of 3% hydrogen peroxide. The plate was incubated for 10–15 min, and the absorbance was read at 620 nm. The activity of the CYP450 monooxygenase enzyme was expressed as OD/min/mg of protein.

2.6 Statistical analysis of data

The mortality and enzyme data were analyzed statistically using SPSS 19.0 software using ANOVA (one-way analysis of variance) followed by Tukey’s all-pairs multiple comparison test to compare means and determine the statistical significance of data at p < 0.05.

The complete methodological workflow is presented in Figure 1.

3 Results

3.1 WHO bottle bioassay

The susceptibility status of adult female Ae. aegypti against α-cypermethrin was evaluated using the WHO bottle bioassay across 12 different concentrations, including the diagnostic dose of 0.001% (25). Linear dose-dependent mortality ranging from 8.33% to 100.00% (p < 0.05) was recorded with increasing insecticide concentration (Figures 2, 3).

The lowest tested concentration of α-cypermethrin (0.1 µg/bottle or 0.00001%) resulted in 8.33 ± 0.70% adult mortality, whereas 12 µg α-cypermethrin/bottle (0.0012%) caused 100% adult mortality (p < 0.05). No mortality was observed in the control. The diagnostic dose concentration (10 µg/bottle or 0.001%) produced 97.91 ± 0.65% mortality. Because the mortality fell within the WHO category of suspected or possible resistance (90%–98% mortality), it suggests that the Ae. aegypti population maintained in the laboratory is slightly resistant or at the borderline of the susceptibility threshold.

Probit analysis of the mortality data using SPSS software revealed an LC₅₀ value of α-cypermethrin of 0.00007%, whereas the LC₉₀ value was 0.00059% (Table 1). The slope value (1.419 ± 0.114) indicated moderate heterogeneity in the response of mosquitoes to α-cypermethrin exposure. The chi-square value (χ2 = 16.903; df = 7) suggested an acceptable goodness-of-fit for the Probit regression model. The non-overlapping confidence limits indicated significant differences between LC values.

Table 1. Lethal concentrations of α-cypermethrin against adult Aedes aegypti after 24 h of WHO bottle bioassay.

LC₃₀, Lethal concentration at which 30% adults are killed; LC₅₀, Lethal concentration at which 50% adults are killed; LC₇₀, Lethal concentration at which 70% adults are killed; LC₉₀, Lethal concentration at which 90% adults are killed; *Figures in parentheses depict 95% fiducial limits; SE, Standard error; χ2, Chi-square; df, degree of freedom. Test samples were transformed into log covariant (Log10) for analysis. ^c means "centered".

3.2 In silico molecular docking

In silico molecular docking was performed between α-cypermethrin and five major detoxification enzymes of Ae. aegypti: glutathione-S-transferase (GST), cytochrome P450 monooxygenase (CYP450), acetylcholinesterase (AChE), α-esterase, and β-esterase. The structure of α-cypermethrin (CID 93357) was extracted from PubChem in SDF format (Figure 4). The structures of three detoxifying enzymes (GST, AChE, and CYP450) were obtained from the Protein Data Bank (PDB) via UniProt accession. Since the three-dimensional crystal structures of α-esterase and β-esterase of Ae. aegypti were not available in the PDB, homology models were generated using the FASTA amino acid sequences through the Swiss-Model server. The generated models showed 100% sequence identity and high structural similarity with Ae. aegypti esterases and were subsequently used for molecular docking studies with α-cypermethrin.

The docking results demonstrated differential binding affinities of α-cypermethrin toward the selected detoxification enzymes (Table 2). Among all enzymes studied, β-esterase exhibited the strongest interaction with α-cypermethrin, with a binding affinity score of −9.4 kcal/mol, followed by monooxygenase (−8.8 kcal/mol), GST (−7.5 kcal/mol), α-esterase (−7.1 kcal/mol), and AChE (−6.6 kcal/mol).

Table 2. Molecular docking of α-cypermethrin with different detoxifying enzymes in Aedes aegypti.

Interaction between receptor-ligand demonstrated potential interaction of α-cypermethrin within the active sites of the selected detoxification enzymes. Two-dimensional interaction analysis revealed the involvement of several amino acid residues in hydrophobic interactions, hydrogen bonding, and van der Waals interactions, contributing to ligand stabilization within the binding pocket (Figure 5).

α-cypermethrin was found to interact with alanine (ALA), tyrosine (TYR), phenylalanine (PHE), leucine (LEU), and histidine (HIS) of α-esterase; with phenylalanine (PHE), leucine (LEU), histidine (HIS), and isoleucine (ILE) of β-esterase; with alanine (ALA), phenylalanine (PHE), leucine (LEU), and threonine (THR) of GST; with alanine (ALA), tyrosine (TYR), aspartate (ASP), and lysine (LYS) of AChE; and with leucine (LEU), phenylalanine (PHE), isoleucine (ILE), histidine (HIS), and cystine (CYS) of CYP450 monooxygenase (Figures 6A–E; Table 3).

Table 3. Interactions between amino acid residues of detoxifying enzymes and α-cypermethrin.

3.3 Detoxification enzyme assays

Biochemical estimation of detoxification enzymes in Ae. aegypti adults exposed to α-cypermethrin at LC₅₀ and LC₉₀ concentrations is presented in Table 4 and Figure 7.

Table 4. Total proteins and activity of various detoxifying enzymes in adult Aedes aegypti following exposure to LC₅₀ and LC₉₀ of α-cypermethrin.

Statistical analyses were performed using GraphPad Prism, which includes one−way ANOVA followed by Tukey’s all pairs multiple comparison tests. Values followed by different letters in each column of an enzyme are significantly different (p < 0.05).

3.3.1 α-esterase activity

Exposure to α-cypermethrin showed a dose-dependent increase in α-esterase activity in Ae. aegypti (p < 0.05). The control set of mosquitoes exhibited α-esterase activity of 7.52 nmol/min/mg protein, which increased by 7.2-fold (54.14 nmol/min/mg protein; p < 0.05) following exposure to the LC₅₀ value and 7.4-fold (55.52 nmol/min/mg protein; p < 0.05) following exposure to the LC₉₀ value of α-cypermethrin (Figure 7A).

3.3.2 β-esterase activity

The change in β-esterase activity in adult Ae. aegypti following exposure to α-cypermethrin was the most significant (p < 0.05) in comparison to the rest of the enzymes (Table 4; Figure 7B). Exposure to the LC₅₀ value increased β-esterase activity to 238.59 nmol/min/mg protein (a 21.41-fold increase) compared with 11.14 nmol/min/mg protein in control mosquitoes (p < 0.05). However, enzyme activity showed a significant decline following exposure to LC₉₀ α-cypermethrin, decreasing by 17.46-fold relative to the control (p < 0.05).

3.3.3 Glutathione S-transferase activity

Glutathione S-transferase activity in adults Ae. aegypti, on the other hand, decreased following exposure to α-cypermethrin (Figure 7C). GST activity was recorded as 851.96 nmol/min/mL in control mosquitoes, which decreased by 1.11-fold to 767.82 nmol/min/mL after LC₅₀ exposure (p > 0.05), and declined markedly by 5.45-fold after LC₉₀ exposure to α-cypermethrin (p < 0.05).

3.3.4 Acetylcholinesterase inhibition

A dose-dependent decrease in the percentage inhibition in acetylcholinesterase activity was recorded following exposure of adult Ae. aegypti to α-cypermethrin, indicating higher AChE activity (p > 0.05). Compared with the 23.29% inhibition observed in the control set, mosquitoes exposed to LC₅₀ and LC₉₀ concentrations of α-cypermethrin showed 2.26-fold and 3.55-fold decreases, respectively, in % AChE inhibition (p > 0.05), indicating a rise in activity (Table 4; Figure 7D).

3.3.5 Cytochrome P450 monooxygenase activity

Cytochrome P450 monooxygenase activity increased in adult Ae. aegypti exposed to LC₅₀ and LC₉₀ values of α-cypermethrin (2.04-fold). The increase was significant (p < 0.05) in comparison to the activity in the control group (Table 4). The rise in α-cypermethrin concentration caused a change in the CYP450 activity (Figure 7E).

4 Discussion

The control of Ae. aegypti is a major challenge in public health, as this species acts as a primary vector for several arboviral diseases, such as dengue, chikungunya, and Zika. Climate change and the mosquito’s ability to adapt to changing conditions have contributed to the increased prevalence of Ae. aegypti, thereby increasing disease burden and morbidity. Adulticide studies against Ae. aegypti are limited; the majority of studies have reported the efficacy of insecticides against Ae. aegypti larvae. Hence, the present study aimed to assess the effect of α-cypermethrin on the survival, susceptibility status, and detoxification enzymes of adult Ae. aegypti. The present study was conducted using a laboratory-susceptible strain of Ae. aegypti to assess baseline physiological and biochemical responses to α-cypermethrin.

The effects of α-cypermethrin on adult survival were evaluated through the WHO bottle bioassay, which showed dose-dependent mortality in adult Ae. aegypti, revealing LC₃₀, LC₅₀, LC₇₀, and LC₉₀ values of 0.00003%, 0.00007%, 0.00017%, and 0.00059%, respectively. The bioassay has been recommended by the WHO to determine the status of insecticide resistance in mosquitoes based on their survival following insecticide exposure (23). Few studies have evaluated the susceptibility status of mosquitoes to α-cypermethrin using the WHO bottle bioassay, although a few studies have employed CDC bottle bioassays to estimate the resistance status in Ae. aegypti (26). A bioassay involving a 30-min exposure to 0.5% α-cypermethrin-impregnated paper was conducted using field-collected Ae. albopictus in West Malaysia (34). The authors reported 50% knockdown times (KT₅₀) ranging from 34.19 to 56.53 min and 90%–99% mortality, revealing variable susceptibility status across different regions. The adulticide effects of α-cypermethrin on yellow fever mosquitoes were evaluated using Wheaton bottles, resulting in an LC₅₀ value of ≤ 0.001, which supports the findings of our study (35).

Several other insecticides have been assessed for their toxic effects on adult Ae. aegypti, revealing 5.6% mortality induced by 1.5% malathion, 32.6% by 0.03% deltamethrin, 18.5% by 0.4% permethrin, and 64.3% by 0.2% bendiocarb (36). Exposure to imidacloprid resulted in an LC₅₀ value of 0.558 ng/mg mosquito for Ae. aegypti, compared with 2.217 ng/mg mosquito for An. stephensi, and 0.009 ng/mg mosquito for Culex quinquefasciatus (37).

The variations in data reported across studies can be attributed to the specific variation, geographical region, and susceptibility status of the mosquitoes. The present study showed that the adult Ae. aegypti population exhibited 97.91% mortality at the diagnostic dose, indicating that the population used in the study was possibly slightly resistant or near the susceptibility threshold (23). It is suggested that the slight reduction from complete mortality may be due to minor fluctuations in environmental conditions and natural biological variations, such as differences in mosquito age, physiological status, or nutritional condition, in addition to genetic drift occurring during long-term colony maintenance, rather than established resistance.

In Ecuador, Ae. aegypti populations showed confirmed resistance to α-cypermethrin through WHO susceptibility tests, displaying 80% mortality (38). In Mexico, 99.5% α-cypermethrin resistance was reported in six field populations (39). Similar reports demonstrating variable levels of resistance to different groups of insecticides are available from Peru (40) and Colombia (41).

It is well known that all organisms possess a defense system to counter various forms of stress. Insects are equipped with an active defense system of detoxification enzymes. It has been found that inherent phenotypic variation within susceptible populations can result in a small proportion of individuals possessing naturally enhanced protective traits, such as slightly thicker cuticles or elevated basal detoxification activity, allowing survival without indicating a heritable resistance mechanism (42). Hence, the present study also evaluated the impact of α-cypermethrin on the detoxifying enzymes of Ae. aegypti through molecular docking and biochemical assays to understand the defense mechanism in Ae. aegypti. To the best of our knowledge, this is the first study examining the molecular docking of α-cypermethrin with the detoxifying enzymes of Ae. aegypti. The study showed the affinity of α-cypermethrin in the order of β-esterases (–9.4 kcal/mol) > CYP450 monooxygenase (–8.8 kcal/mol) > GST (–7.5 kcal/mol) > α-esterases (−7.1 kcal/mol) > AChE (–6.6 kcal/mol). This shows the variable role of each enzyme in combating insecticide stress, although β-esterases appears to play the most significant and prominent role.

The present results are consistent with those of Saha et al. (43), who documented the highest binding affinity of cyphenothrin with esterases (–9.511 kcal/mol) in Cx. quinquefasciatus, followed by enzymes belonging to the cytochrome monooxygenase family (−8.367 to −7.526 kcal/mol) and α-esterases (−5.593 kcal/mol). Taktak et al. (44) docked four pyrethroids, namely, deltamethrin, alpha-cypermethrin, lambda-cyhalothrin, and permethrin, with three enzymes in Cx. pipiens: carboxylesterase (CaE), adenosine triphosphatase (ATPase), and glutathione-S-transferase (GST). They found binding affinity in the order of CaE > GST > ATPase, with docking scores ranging from −4.33 to −10.01 kcal/mol.

Pyrethroid insecticides contain ester linkages that can be metabolically degraded by esterase enzymes, thereby reducing their toxicity before the compounds reach the neuronal target site. Increased interaction between pyrethroid insecticides and esterase enzymes has previously been associated with enhanced hydrolysis and metabolic degradation of insecticides, leading to reduced insecticidal efficacy (45). This observation was further validated by biochemical estimation of esterases in Ae. aegypti exposed to α-cypermethrin at LC₅₀ and LC₉₀ levels, representing moderate and high toxicological stress conditions. The results showed a significant rise of 17.46 to 21.41-fold in β-esterases, suggesting the potential of mosquitoes to rapidly hydrolyze the ester bonds present in α-cypermethrin through effective interaction with the enzyme’s active site. Zuharah et al. (46) also reported a 9–10-fold increase in β-esterase in adult female of Ae. aegypti exposed to α-cypermethrin.

The study also showed a decrease in β-esterase activity at α-cypermethrin LC₉₀ exposure compared with LC₅₀ exposure, although the enzyme level still remained higher than in the control group. This reduction may be due to excessive toxic stress caused by the higher dose of insecticide, which probably have partially saturated or physiologically exhausted the detoxification enzymes due to continuous metabolic demand. Previous reports have also suggested that intense pyrethroid exposure can induce oxidative stress, protein denaturation, and enzyme inhibition, thereby reducing the efficiency of enzymatic detoxification in surviving mosquitoes (47, 48). The strong correlation between docking affinity and elevated biochemical levels suggests that β-esterase may play a dominant role in α-cypermethrin metabolism and resistance development in Ae. aegypti.

The study also revealed substantial interaction between GST and α-cypermethrin, suggesting possible conjugation-mediated detoxification. The decrease in GST activity with increasing insecticide concentration suggests that higher α-cypermethrin exposure may impair glutathione-mediated detoxification pathways. The moderate decline in GST activity at LC₅₀ α-cypermethrin and the sharp decrease observed at LC₉₀ indicate plausible excessive toxic stress, utilization of glutathione reserves during detoxification, enzyme inhibition, oxidative damage, or depletion of intracellular glutathione levels under higher insecticide exposure (44, 49).

Despite the moderate decline in biochemical activity at LC₅₀ and the sharp decline at LC₉₀, molecular docking analysis demonstrated considerable binding affinity between GST and α-cypermethrin (−7.5 kcal/mol), possibly reflecting inhibitory interactions between GST and the insecticide molecule. It is suggested that although GST participates in the insecticide detoxification process in Ae. aegypti, prolonged or high-level insecticide exposure may overwhelm the GST defense system, resulting in reduced enzymatic activity. The role of GSTs in imparting DDT resistance has been well described in mosquitoes, while a more variable role has been reported for insecticides belonging to other groups (50). Limited reports are available on detoxification enzyme assays in adult mosquitoes; however, similar results have been reported in insect larvae. Suppressed GST activity has been shown in Ae. aegypti larvae exposed to diflubenzuron (51) and Spodoptera littoralis larvae exposed to chlorflurazon and lufenuron (52). In contrast, lufenuron treatment induced a significant rise in GST activity in Ae. aegypti (53), suggesting variable contributory roles of different enzymes in insect defense mechanisms.

Likewise, decreased % AChE inhibition in α-cypermethrin-exposed adult Ae. aegypti suggests its apparent role in detoxification. This observation was further supported by molecular docking analysis, where AChE exhibited the lowest binding affinity with α-cypermethrin (−6.6 kcal/mol) among all tested detoxification enzymes. The comparatively weaker interaction suggests limited binding stability between α-cypermethrin and the active site of AChE. Similar results have been reported in Ae. aegypti larvae treated with diflubenzuron (51). Since pyrethroid insecticides primarily target voltage-gated sodium channels rather than acetylcholinesterase, the lower docking affinity suggests that although α-cypermethrin exposure may influence AChE activity, its direct role in metabolic detoxification remains unclear.

Cytochrome P450 monooxygenases are considered one of the major detoxification enzyme systems involved in the metabolism of pyrethroid insecticides by catalyzing oxidation reactions that convert lipophilic insecticide molecules into more polar metabolites, thereby facilitating their excretion from mosquitoes (54). Unlike GST levels, α-cypermethrin exposure significantly increased CYP450 activity in the Ae. aegypti adults. In mosquitoes, involvement of CYP enzymes in the detoxification of various pyrethroids has been reported in Ae. albopictus (55). The present findings are consistent with these reports and are further supported by the strong docking interaction observed in the current study. It indicates stable binding of the α-cypermethrin molecule within the active site of the enzyme, supporting its active involvement in oxidative metabolism, which was validated by the increased levels found in biochemical estimation.

The present study investigated the interaction of α-cypermethrin with selected detoxification enzymes through biochemical analyses complemented with molecular docking analyses to provide preliminary mechanistic insights. Future studies incorporating synergist bioassays using inhibitors such as piperonyl butoxide (PBO), diethyl maleate (DEM), or S,S,S-tributyl phosphorotrithioate (DEF), together with field-derived mosquito populations with diverse resistance backgrounds, could further validate the detoxification pathways implicated in α-cypermethrin metabolism and resistance development.

5 Conclusion

The present study demonstrates the significant effects of α-cypermethrin on the survival and detoxification system of Ae. aegypti adults. The WHO bottle bioassay revealed dose-dependent mortality in Ae. aegypti induced by α-cypermethrin. The in vitro and in silico studies demonstrated the variable effects of α-cypermethrin on various detoxification enzymes, indicating involvement of all the enzymes in the defense mechanism to some extent. The most significant rise in the β-esterase induced by α-cypermethrin suggests its prime role in the process of metabolic detoxification, while there is also a significant contribution of CYP450 in stimulating the defense system in Ae. aegypti adults. Further studies using field strains are required to evaluate these effects under selection pressure of other insecticides and to better understand their implications on the environment.

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Footnotes

Edited by: Marta Ferreira Maia, University of Oxford, United Kingdom

Reviewed by: Juliana Maccagnan, Regional Community University of Chapecó, Brazil

Abhirup Saha, University of North Bengal, India