E ﬀ ects of Sublethal Doses of Methyl Benzoate on the Life History Traits and Acetylcholinesterase (AChE) Activity of Aphis gossypii

: Safer alternatives to synthetic pesticides are essential for sustainable agriculture. Methyl benzoate (MB) is a volatile essential oil found in several plants. Recent reports of the toxicity of MB to arthropod pests suggest that MB may be a useful alternative insecticide. The present study assessed the e ﬀ ects of a sublethal concentration of MB (LC 30 , 0.22%) on the life history and reproductive characteristics of the cotton aphid, Aphis gossypii Glover, in both a treated parental generation (F 0 ) and untreated progeny (F 1 ). MB treatment signiﬁcantly decreased longevity and fecundity in both the F 0 and F 1 generations, and prolonged the developmental duration of each immature instar of the F 1 generations, compared with controls. The intrinsic rate of increase ( r ), ﬁnite rate of increase ( λ ), and net reproductive rate ( R 0 ) of the F 1 generation were signiﬁcantly reduced, compared to controls. The mode of action of MB is not known, but in aphids treated with LC 30 MB, the activity of the enzyme acetylcholinesterase (AChE) decreased by more than 65%, compared with untreated controls. AChE activity was rapidly inhibited within 1 h, and remained inhibited for 6 h after in vivo exposure to MB. Moreover, molecular docking analysis revealed that MB had a strong a ﬃ nity with the catalytic site of AChE, with a binding energy value of − 6.2 kcal / mole. Our results suggest that MB targets AChE, and that a sublethal dose of MB can have adverse transgenerational e ﬀ ects on cotton aphids.


Introduction
The cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is a common agricultural insect pest that attacks crops worldwide [1]. The cotton aphid damages plants directly by consuming maintained at 25 ± 1 • C and 60 ± 10% relative humidity (RH), with a 16:8 h light/dark photoperiod. The colony has been maintained without any exposure to insecticides since 2017.
Commercially available MB (99% purity) and emulsifiers Tween 20 and Tween 80 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The sublethal dose of MB was the LC 30 concentration (0.22%) determined in our previous study [22]. The 0.22% MB solution and the control solution were prepared in distilled water, according to our published procedure [21].

Sublethal Effects of Methyl Benzoate on the Cotton Aphid F0 Generation
The toxicity of MB against cotton aphid adults (≤24-h-old) was evaluated, according to the procedure published by Cui et al. [8]. Cucumber leaf discs (20 mm in diameter) were dipped into the control or MB solution (LC 30 ) for 5 s, then allowed to air dry for~30 min. After drying, the treated leaf discs were placed into Petri dishes (5 cm D × 1.5 cm H) containing 1% agar (Junsei Chemical Co., Ltd., Tokyo, Japan), with the leaf underside resting on the agar bed. The agar provided moisture to prevent desiccation of the leaf discs. Ventilation was provided in the Petri dishes by cutting a 2 cm diameter hole in each lid and covering the hole with nylon mesh. For each treatment, including controls, 75 apterous adult aphids were used. Mortality data were collected 24 h after the aphids were placed in the treatments. Any surviving aphids were transferred from treated leaves onto fresh untreated leaf discs using a camel hair brush. The untreated leaf discs were replaced every 3 d and mortality data were collected every 24 h until all of the remaining aphids were dead. These bioassays were conducted under laboratory conditions at 25 ± 1 • C, 60 ± 10% RH, and 16:8 h light/dark cycle.

Transgenerational Effects of Methyl Benzoate on the Cotton Aphid F1 Generation
Newly born nymphs from the MB-treated F 0 female adults were collected within 24 h and placed individually on untreated cucumber leaf discs in Petri dishes (as described in Section 2.2) to observe the transgenerational effects of MB. A total of 75 newborn F 1 nymphs were collected from each treatment (MB and control). Life-cycle characteristics, such as growth stage, fecundity, mortality, and longevity, were monitored daily. At the time of reproduction, the newly born nymphs were counted and transferred daily to fresh cucumber leaf discs until the reproducing adult died. Throughout the experiment, leaf discs were replaced every three days. All bioassays were conducted under the laboratory conditions described in the previous section.

Preparation of Cotton Aphid Proteins
Cotton aphid adults were treated with the LC 30 dose of MB (0.22%). Aphids (n = 60) were exposed to MB for 0, 1, 3, and 6 h. After the exposures, the surviving aphids were collected and homogenized using 300 µL of ice-cold phosphate buffer (PBS) (0.1 M, pH 7.4). Next, the homogenate material was centrifuged at 12,000 rpm for 15 min at 4 • C, and the supernatant was collected for the protein source. The protein concentration of the enzyme extract was evaluated using the Bradford method [28], with bovine serum albumin as the standard.

Determination of Acetylcholinesterase Activity
To measure AChE activity, acetylthiocholine iodide (ATChI, Sigma-Aldrich, St. Louis, MO, USA) was used as a substrate, following the methods described by Ellman et al. [29]. Next, 20 µL of enzyme solution and 160 µL of PBS (0.1 M, pH 7.4) were added to each well of a 96-well microplate (SPL, Pocheon, Korea). The mixtures were then incubated in a shaker for 15 min at 27 • C. After this procedure, 10 µL of 75 mM ATChI and 10 µL of 0.1 M dithionitrobenzoic acid (DTNB, Sigma-Aldrich, St. Louis, MO, USA) were added to each well. The absorbance was measured at 412 nm using a microplate reader (Victor3, PerkinElmer, Waltham, MA, USA). The change in absorbance was recorded every 30 s for 20 min. All treatments were performed in triplicate. AChE activity was indicated as nmol of ATChI hydrolyzed/min/mg protein using an extinction coefficient of 1.36 × 10 4 M −1 cm −1 .

Molecular Docking Analysis of the Protein-Ligand
The crystal structure of AChE (PDB ID 1QON) was obtained from the Protein Data Bank (PDB) (http://www.rcsb.org/pdb/home/home.do) [30]. To investigate whether MB has the ability to inhibit AChE, a molecular docking test was performed using AutoDock Vina. The protein structures were prepared using UCSF Chimera 1.13.1 (http://www.cgl.ucsf.edu/chimera) to remove all non-receptor atoms, including water, ions, and miscellaneous compounds. The ligand (MB) was prepared using ChemBio3D Ultra, version 12.0 (PerkinElmer, Waltham, MA, USA), then an MMFF94 energy minimization was performed. AutoDock Vina (The Scripps Research Institute, La Jolla, CA, USA) was used for the molecular docking simulations. The ligand was removed, and a site sphere was specified to define the active site of AChE. The center of the grid box was 33, 67, and 10 Å on the x, y, and z axes, respectively, whereas the dimension (Å) was 16.02, 15.98, and 16.25 on the x, y, and z axes, respectively. The best confirmation with the lowest binding energy was selected. After the docking search was completed, it was visualized using a PyMOL Molecular Graphics System (version 1.7.4, Schrödinger, Inc., New York, NY, USA). The binding results were visualized as 3D and 2D diagrams using Discovery Studio Visualization, version 4.5 (Accelrys, Inc., San Diego, CA 92121, CA, USA) and LigPlot viewer (v.2.2, EMBL-EBI, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SD, UK, http://www.ebi.ac.uk/thornton-srv/software/LIGPLOT), respectively.

Sublethal Effects of Methyl Benzoate on the F 0 Generation
Treatment of adult cotton aphids with 0.22% MB using the leaf disc method significantly affected longevity and fecundity ( Figure 1). Compared with the untreated control group, the adult longevity of F 0 individuals treated with MB was significantly reduced from 20.37 d to 8.56 d (p < 0.0001; Figure 1). Fecundity of the F 0 generation also significantly decreased from 27.88 offspring/female in the control to 9.63 offspring/female in the MB treatment (p < 0.0001; Figure 1).

Sublethal Effects of Methyl Benzoate on the F 1 Generation
The effects on the F 1 generation of treating the F 0 generation of aphids with LC 30 MB are presented in Table 1. Compared with the untreated control group, the mean longevity and fecundity of the F 1 generation significantly decreased, due to the treatment of the parental generation with LC 30 of MB (p < 0.0001). Furthermore, after exposing the F 0 individuals to MB, the growth period of each immature stage of the F 1 generation significantly increased (p < 0.0001). This shows that F 0 MB treatment significantly increased the pre-adult duration (p < 0.0001), APRP (p < 0.0001), and TPRP (p < 0.0001) of the F 1 progeny, compared to the control. A significant difference in the number of reproductive days of the F 1 generation was also observed between the MB and control treatment groups (p < 0.0001; Table 1). Table 1. Transgenerational effects of methyl benzoate on developmental times, longevity, adult pre-reproductive period (APRP), total pre-reproductive period (TPRP), and mean fecundity of F 1 progeny Aphis gossypii when parental individuals (F 0 ) were exposed to the sublethal concentration (LC 30  a Standard errors (SE) were estimated using the bootstrap technique with 100,000 re-samplings; b differences between two treatments were compared using a paired bootstrap test implemented in TWOSEX-MSChart. The means in the same rows followed by different lower-case letters indicate significant differences between treatments (p < 0.05).

Transgenerational Effects of Methyl Benzoate on Population Parameters
The transgenerational effects of MB (LC 30 ) on population parameters of the F1 generation were assessed using a life table-based bootstrap technique. Compared to the control group, the intrinsic rate of increase (r), finite rate of increase (λ), the net reproductive rate (R 0 ), and gross reproductive rate (GRR) significantly decreased (p < 0.0001), while the mean generation time (T) (13.71 ± 0.24) significantly increased in the MB treatment group, compared with the control group (12.42 ± 0.23) (p < 0.0001; Table 2). a r: intrinsic rate of increase; λ: finite rate of increase; R 0 : net reproductive rate; T: mean generation time (offspring/individual); GRR: gross reproduction rate; b standard errors (SE) were estimated using the bootstrap technique with 100,000 re-samplings; c differences between two treatments were compared using a paired bootstrap test implemented in TWOSEX-MSChart. The means in the same rows followed by different lower-case letters indicate significant differences between treatments (p < 0.05).

Transgenerational Effects of Methyl Benzoate on Age-Stage Specific Rate of Survival and Fecundity
The age-stage specific rate of survival (s xj ) is the probability that a newly-born individual will survive to age x and stage j (Figure 2). Due to variable growth rates between individuals, considerable overlaps were observed between the life stages in the control and MB treatment groups (Figure 2). The pre-adult survival rate (s a ) was not significantly different between treatments ( Table 1).
The age-survival rate (l x ) provides a simplistic description of the rate of survival without considering the stage distinction ( Figure 3). In this study, the l x curve of MB-treated groups significantly decreased from 16-days-old, while the l x in the control group dropped from 22-days-old ( Figure 3). The l x value for LC 30 treatment with MB declined earlier, compared to the control. Based on the curve m x (age-specific fecundity) the control group's maximum age-specific fecundity rate (2.2 offspring) occurred at the age of 7 d. Additionally, the MB-treated group had peak fecundity at 14-days-old (1.51 offspring) (Figure 3). Based on both l x and m x , the maximum net maternity l x m x value of 1.93 offspring occurred in the control group at 7-days-old, while the l x m x 1.31 in the MB-treated groups occurred at 14-days-old ( Figure 3).  Age-specific survival rate (I x ), age-specific fecundity of total population (m x ), and age-specific maternity (I x m x ) for F 1 Aphis gossypii descended from F 0 exposed to LC 30 methyl benzoate (MB).
The age-specific life expectancy (e xj ) is the length of time that an individual of age x and stage j is expected to survive after age x (Figure 4). The life expectancy (e xj ) of cotton aphids treated with MB was lower than the control group (Figure 4). . Age-stage-specific life expectancy (e xj ) for F 1 Aphis gossypii descended from F 0 exposed to LC 30 methyl benzoate.
In addition, the age-stage reproductive value (v xj ) is an indicator of an individual's contribution to the future population at various ages and stages ( Figure 5). In the MB-treated group, the maximum v xj value occurred significantly later (6.86/d at 8-days-old), compared to that in the control group (9.53/d at 5-days-old) ( Figure 5).

Figure 5.
Age-stage reproductive value (v xj ) of F 1 Aphis gossypii descended from F 0 exposed to LC 30 methyl benzoate.

Effect of Methyl Benzoate on AChE Activity
A direct correlation was observed between AChE activity and MB exposure period, thereby emphasizing the time-dependence of MB activity (F = 7.99; df = 3, 23; p < 0.0001) ( Figure 6). In the time-course inhibition studies, AChE activity of A. gossypii was quickly inhibited by MB treatment, and remained inhibited following in vivo exposure ( Figure 6). The high level of AChE inhibition by 1 h bioactivation occurred rapidly after exposure to MB. AChE activity decreased by more than 65% in MB-exposed aphids, compared to the control (p < 0.0001; Figure 6).

Protein-Ligand Molecular Docking Analysis
The molecular docking protocol must be validated before performing molecular docking studies. Therefore, to validate the protocols used in this analysis, the ligand crystallographic information was subjected to the development of docking until the spatial conformation was found by comparison with the original crystallographic structure of the AChE inhibitors (PDB ID 1QON). The validation was performed by retrieving the structure of an AChE inhibitor (I40) by calculating the root-mean-square deviation (RMSD) of 0.98 Å. The binding mode predicted using docking indicates that when the RMSD is less than 2.0 Å regarding the crystallographic pose of a respective ligand, the validation is considered satisfactory [40,41]. The satisfactory results are illustrated in Figure 7a.
The binding affinity between MB and the AChE protein was expressed as a binding energy value of −6.2 kcal/mol, while the actual ligand I40 (9-(3-iodobenzylamino)-1,2,3,4-tetrahydroacridine) binding with AChE had a value of −12.5 kcal/mol. As shown in Figure 7b-d, the individually-observed interaction after MB docking was identical to I40 in the active site of AChE, found around the α-helix between amino acid residues Tyr-370, Tyr-374, and Tyr-71, as well as the β-sheet with amino acid residue Trp-83. Quantitative data on residues, distances, and types between MB and the insect AChE are presented in Table S1 and Figure S1.

Discussion
Natural products can often provide a more environmentally-friendly approach to managing insect pests and plant diseases [42,43]. MB, which is a naturally occurring compound found in many plants, exhibits potent insecticidal activity against a variety of insect pests [18,[21][22][23][24]44]. Therefore, MB could provide an alternative to synthetic insecticides for agricultural control of cotton aphids in the near future. However, the sublethal and transgenerational effects of MB on cotton aphids were previously unknown.
This study showed the effects of a sublethal dose of MB on the biological traits and AChE activity of the cotton aphid. We observed a significant decrease in adult survival and fecundity of A. gossypii directly exposed to the previously determined LC 30 of MB. Similar effects were documented in an earlier study, in which the fecundity and longevity of A. gossypii significantly declined with exposure to sublethal doses of buprofezin [45]. Additionally, adult survival and fertility were significantly reduced when cotton aphids were treated with LC 10 and LC 40 cycloxaprid [46]. Aphid longevity and fertility in the aforementioned studies substantially decreased, but similar decreases were not observed in B. tabaci exposed to sublethal doses of imidacloprid [47]. Likewise, no decrease in longevity or fecundity was observed in Apolygus lucorum Meyer-Dür (Hemiptera: Miridae) exposed to a sublethal concentration of cycloxapride [16]. In addition, the reduced longevity and fecundity patterns observed in cotton aphids suggest a lack of hormesis, which is a common sublethal effect of insecticide exposure. Hormesis is a concentration-response biphasic process, which is normally characterized by stimulation at low concentrations and inhibition at high concentrations [48,49]. Insecticide-induced hormesis has been recorded in several insect species, for example, higher fecundity in Myzus persicae Sulzer (Hemiptera: Aphididae) subjected to low imidacloprid concentration [50] and Oligonychus ilicis McGregor (Acari: Tetranychidae) outbreaks caused by low doses of pyrethroid [51]. However, hormesis effects on the longevity and fecundity of the parental generation (F 0 ) of A. gossypii exposed to a sublethal concentration of MB were not observed in this study.
Transgenerational effects of MB on cotton aphid progeny (F 1 ) were identified when the parental generation (F 0 ) was exposed to LC 30 MB. The length of each nymphal stage and the pre-adult duration of the F 1 progeny of MB-exposed F 0 were significantly extended. The delayed development rate we observed is comparable to earlier reports, such as the delayed development of Plutella xylostella L. (Lepidoptera: Plutellidae) exposed to LC 25 chlorantraniliprole [13]. Similarly, the duration of each developmental stage in Rhopalosiphum padi L. (Hemiptera: Aphididae) increased when exposed to sublethal concentrations of beta-cypermethrin or indoxacarb [52]. We found that the demographic parameters in the F 1 generation were substantially reduced, relative to the control, when the F 0 generation was subjected to MB exposure. Similar transgenerational effects on the offspring of white-backed planthopper, Sogatella furcifera Horváth (Hemiptera: Delphacidae) [53], and brown planthopper, Laodelphax striatellus Fallén (Hemiptera: Delphacidae) [54], were recorded after parental exposure to sublethal concentrations of buprofezin and sulfoxaflor. Although we have reported the GRR in this study, it is necessary to point out that because GRR excludes the survival rate, a higher GRR does not necessarily represent higher fitness; thus, it should be interpreted with caution.
Plotting the s xj , l x , m x , l x m x , and e xj curves revealed the adverse effects of a sublethal dose of MB on cotton aphid population growth parameters. In the control treatment, the reproduction began on age 5 day, the peak of reproduction was on age 7 day, and the reproductive period extended from age 5 to 40 day. Consequently, the intrinsic rate of increase and finite rate of increase of the control treatment are significantly higher than that of MB treatment. Due to various physical and chemical processes, the pre-adult and mean generation time (T) increased after exposure to MB. In a few other studies on A. gossypii, similar effects were reported at the demographic level [12,55].
MB has been demonstrated as an excellent organic pesticide; however, the insecticidal mechanism of action of MB was not previously elucidated. Few studies have been conducted on the role of MB in human and animal subjects. A related compound methyl hydroxybenzoate has been shown to act on nervous conduction in the spinal root fibers of cats [56]. In rabbits, a sublethal dose of MB (500 mg/kg) increased blood cell counts but reduced cholinesterase activity; frequent application of high doses resulted in damage to the central nervous system [57]. AChE in the central nervous system of insects is an important target molecule for organophosphate and carbamate insecticides [58]. Our study showed that A. gossypii AChE activity decreased following treatment with MB. This finding indicates that MB works directly or indirectly to suppress AChE activity, and therefore, AChE inhibition may be the mechanism for cotton aphid mortality. Furthermore, we simulated the molecular interaction between MB and AChE using a molecular docking program. Our results showed that MB docked at the catalytic site of the AChE molecule. Additionally, MB exhibited hydrophobic interactions with at least five AChE amino acids.

Conclusions
The results of this study demonstrate that the application of a sublethal concentration of MB adversely affects cotton aphid growth by extending the pre-adult period and suppressing the population growth of the progeny. In addition, this study is the first to explore the constructed atomistic AChE model for A. gossypii and its potential binding of MB through molecular docking analyses. Further research on the mode of action of MB on insects is recommended. Importantly, biopesticides such as MB could become key components of crop protection programs, reducing the use of synthetic and harmful chemical products. Adopting a more sustainable approach to pest management will benefit human health, the environment, and biodiversity.