Molecular Mechanism Underlying ROS-Mediated AKH Resistance to Imidacloprid in Whitefly

Simple Summary As an invasive pest, whitefly causes great losses in agricultural production, and resistance increases in whitefly to chemical insecticides are an important problem in plant protection. Our study revealed that imidacloprid stress inhibits the expression of AKH, thereby increasing ROS and the expression levels of the resistance gene CYP6CM1 and upstream regulatory factors in Bemisia tabaci. AKH silencing by RNA interference affected the resistance of whiteflies to imidacloprid. These results provide insight into the resistance mechanism in whitefly. Abstract Synthetic insecticides used to control Bemisia tabaci include organophosphorus, pyrethroids, insect growth regulators, nicotinoids, and neonicotinoids. Among these, neonicotinoids have been used continuously, which has led to the emergence of high-level resistance to this class of chemical insecticides in the whitefly, making whitefly management difficult. The adipokinetic hormone gene (AKH) and reactive oxygen species (ROS) play roles in the development of insect resistance. Therefore, the roles of AKH and ROS in imidacloprid resistance in Bemisia tabaci Mediterranean (MED; formerly biotype Q) were evaluated in this study. The expression level of AKH in resistant B. tabaci MED was significantly lower than that in sensitive B. tabaci (MED) (p < 0.05). AKH expression showed a decreasing trend. After AKH silencing by RNAi, we found that ROS levels as well as the expression levels of the resistance gene CYP6CM1 and its upstream regulatory factors CREB, ERK, and P38 increased significantly (p < 0.05); additionally, whitefly resistance to imidacloprid increased and mortality decreased (p < 0.001). These results suggest that AKH regulates the expression of resistance genes via ROS in Bemisia tabaci.


Introduction
Bemisia tabaci is one of the most important vegetable and ornamental crop pests in the world [1].It is characterized by a high reproductive capacity and wide host range, with more than 1000 vegetable crops and ornamental plants identified as hosts [2].Therefore, it is found on all continents except Antarctica [3].
Neonicotinoid insecticides have been used to control whiteflies in the past few decades owing to their low toxicity to mammals [4,5].This has led to the development of widespread resistance of whiteflies.Imidacloprid, a first-generation neonicotinoid insecticide, is still widely used.Extensive research has focused on the mechanism underlying resistance to imidacloprid [6][7][8], for example, the trans-regulation of CYP6CM1, a cytochrome P450 that confers resistance to neonicotinoid insecticides in the whitefly Bemisia tabaci, by the mitogen-activated protein kinase (MAPK)-directed activation of the transcription factor cAMP-response element binding protein (CREB) [9].In addition, recent studies have shown that miR-1517 may be involved in regulating the expression of CYP6CM1 [10].
One of these hormones, the adipokinetic hormone (Akh), is secreted by the corpora cardiaca (CC) and elicits both carbohydrate and lipid mobilization from the fat body (trehalose from glycogen and diacylglycerol from triacylglycerol, respectively), acting as a functional homolog of glucagon [11,12].This hormone signals to the G-protein coupled receptor encoded by the Akh receptor gene AkhR to elevate hemolymph lipid and/or trehalose titers and thus redirects energy to the sites and processes where it is required [13,14].
Insecticides can induce a strong oxidative stress response in insects, which is accompanied by the production of a large number of ROS.Excessive ROS will damage insect cells and even lead to insect death [15,16].The role of Akh in regulating antioxidant systems involved in insect resistance to insecticides has been reported [17].Velki et al. found that the Akh mature peptide levels in the hemolymph of the firebug Pyrrhocoris apterus increased significantly after insecticide treatment [18].AKH regulates the expression of imidacloprid resistance genes by regulating ROS levels [19].
In this study, imidacloprid resistance levels in Bemisia tabaci MED were studied.In particular, the molecular mechanism underlying imidacloprid resistance in B. tabaci MED was investigated using resistant and sensitive strains, with a focus on the roles of AKH and ROS.To evaluate the role of AKH in resistance, its response to imidacloprid as well as changes in resistance after AKH silencing by RNA interference (RNAi) were evaluated.To evaluate the role of ROS in resistance and changes in ROS in response to imidacloprid, as well as changes in imidacloprid resistance after ROS elimination, were evaluated.Furthermore, the effect of AKH on ROS was studied by silencing AKH.The results of this study provide insight into the molecular mechanism underlying the imidacloprid resistance of B. tabaci MED from the perspectives of ROS and AKH and provide a new target for the development of insecticides.

Insect Rearing
A colony of the Bemisia tabaci Mediterranean (MED; formerly biotype Q) cryptic species was maintained on cucumber plants, which was not exposed to any insecticide during the culture period and served as a sensitive population in this experiment.Resistant whiteflies were collected from the greenhouse of the Plant Protection Institute of Hunan Academy of Agricultural Sciences (Changsha, Hunan, China, 28 • 12 ′ N, 112 • 59 ′ E).B. tabaci MED was a multi-generation population reared in the presence of imidacloprid (as the resistant population).The culture conditions were 16 h light/8 h dark, 26 ± 0.5 • C, and 75 ± 0.5% humidity.

Bioassay of Insecticide Resistance
The LC50 values and drug resistance of whiteflies after treatment with imidacloprid (pesticide registration no.: PD20131915; product standard no.: GB/T28143-2011; dosage form: emulsifiable oil; imidacloprid 5%, Bayer Crop Science (China), Hangzhou, China) were determined using the agar disk diffusion method.The test device was improved by using a small box with a lid that was ventilated with gauze mesh, leaving a small opening for insects [20].An appropriate amount of 1% agar was added to the bottom of the lid.The plate was immersed in diluted imidacloprid at various concentrations for 10 s and then placed on the agar after drying naturally.In total, 20 individuals were placed in each small box and the experiment was repeated 10 times.B. tabaci MED death was quantified after 48 h, and water was used as the control.

RNA Extraction, cDNA Synthesis, and Real-Time Quantitative Polymerase Chain Reaction
Total RNA was extracted using the TaKaRa RNAiso Plus Kit (Cat# 9109, Lot# AL42064A, TaKaRa, Beijing, China), according to the manufacturer's instructions.Then, cDNA was synthesized using the TaKaRa Reverse Transcription Kit (Cat# RR047A, Lot# AL21113A, TaKaRa, Beijing, China).To detect the expression of the resistance gene CYP6CM1, quantitative real-time PCR (qRT-PCR) was performed using TaKaRa TB GreenR Premix Ex TaqTM II (Cat# RR820A, Lot# AJF2612A, TaKaRa, Beijing, China), according to the manufacturer's instructions.

RNAi
Cloning and ligation of the adipokinin gene fragment were performed using the pMDTM18-T Vector Cloning Kit (TaKaRa, Cat#6011, Lot#AK91485A, TaKaRa, Beijing, China).After sequencing, double-stranded RNA (dsAKH) was cloned.dsGFP was synthesized using the T7 High Yield RNA Synthesis Kit (YESEN, Code No: 10623ES50, YESEN, Shanghai, China), following the kit instructions.The membrane feeding method (refer to Li et al. 2015) [21] was used to feed dsAKH and dsGFP to sensitive B. tabaci MED.The feeding concentration was 500 ng/µL, determined based on a preliminary experiment.After feeding for 48 h, the silencing efficiency and the change in the expression level of the resistance gene CYP6CM1 were analyzed by qRT-PCR with sequence-specific primers (Table 1).dsGFP was used for comparison.

Determination of ROS Contents in B. tabaci MED
The ROS content in B. tabaci MED was measured after treatment and grinding with 250 µL of phosphate buffer (pH 7.4).After full grinding, samples were centrifuged at 13,680× g for 5 min, 190 µL of the supernatant was transferred to a 96-well plate, and 10 µL of DCFH-DA (Solarbio, Cat#CA1410, Solarbio, Beijing, China) (10 µmol/L) was added and mixed well.Samples were incubated in a dark room for 1 h [22].Fluorescence was detected at an excitation wavelength of 488 nm and emission wavelength of 525 nm.The same volume of phosphate buffer and DCFH-DA-adjusted fluorescence value were used as the blank control, and dsGFP treatment was used as the control.

Scavenging Effect of N-acetylcysteine on ROS
The scavenging effect of N-acetylcysteine (NAC) on ROS in insects was evaluated following a previously reported method [19].In particular, a NAC solution of 1 mol/L was used based on preliminary analyses of concentrations up to 10 mol/L.The volume of liquid feed used was 200 µL, and pure liquid feed was used as the control.

Statistical Analysis
IBM SPSS Statistics 20.0 software (IBM Corp., Armonk, NY, USA) was used for the numerical analyses of all experimental data [23], and independent samples t-tests were used for comparisons between groups [24].The quantitative fluorescence data were analyzed using the 2 −∆∆Ct method.The LC50 of imidacloprid was calculated using the SPSS Probit function [25], and then the regression model was fitted and R 2 was calculated.Plots were generated using GraphPad Prism 8.0.2 [26].The experimental results are expressed as the mean ± standard error.

Bioassay of Imidacloprid Sensitivity of B. tabaci
We first estimated imidacloprid LC50 values in B. tabaci MED.The LC50 of the sensitive B. tabaci MED was 51.820 mg/L, and the LC50 of the resistant B. tabaci MED was 535.932 mg/L, with a resistance ratio of 10.34.The resistant B. tabaci MED was moderately resistant (Table 2).

The Role of AKH in the Response of B. tabaci MED to Imidacloprid
We compared adipokinin gene expression levels in resistant and sensitive whiteflies.We also evaluated changes in adipokinin in sensitive whiteflies after treatment with imidacloprid.The expression level of AKH in resistant B. tabaci MED was significantly lower than that in sensitive B. tabaci MED (p < 0.05) (Figure 1A).After treatment with 50 mg/L imidacloprid, the relative expression level of AKH decreased but did not differ significantly from that of the control.Under treatment with 100 mg/L, relative AKH expression increased but did not differ significantly from that of the control.After treatment with 150 mg/L imidacloprid, the relative expression of AKH decreased but was not significantly different from that in the control (Figure 1B).
IBM SPSS Statistics 20.0 software (IBM Corp., Armonk, NY, USA) was used for the numerical analyses of all experimental data [23], and independent samples t-tests were used for comparisons between groups [24].The quantitative fluorescence data were analyzed using the 2 −ΔΔCt method.The LC50 of imidacloprid was calculated using the SPSS Probit function [25], and then the regression model was fitted and R 2 was calculated.Plots were generated using GraphPad Prism 8.0.2 [26].The experimental results are expressed as the mean ± standard error.

Bioassay of imidacloprid sensitivity of B. tabaci
We first estimated imidacloprid LC50 values in B. tabaci MED.The LC50 of the sensitive B. tabaci MED was 51.820 mg/L, and the LC50 of the resistant B. tabaci MED was 535.932 mg/L, with a resistance ratio of 10.34.The resistant B. tabaci MED was moderately resistant (Table 2).

The Role of AKH in the Response of B. tabaci MED to Imidacloprid
We compared adipokinin gene expression levels in resistant and sensitive whiteflies.We also evaluated changes in adipokinin in sensitive whiteflies after treatment with imidacloprid.The expression level of AKH in resistant B. tabaci MED was significantly lower than that in sensitive B. tabaci MED (p < 0.05) (Figure 1A).After treatment with 50 mg/L imidacloprid, the relative expression level of AKH decreased but did not differ significantly from that of the control.Under treatment with 100 mg/L, relative AKH expression increased but did not differ significantly from that of the control.After treatment with 150 mg/L imidacloprid, the relative expression of AKH decreased but was not significantly different from that in the control (Figure 1B).

Effect of AKH on the Expression of CYP6CM1 and Its Upstream Regulatory Factors
After preliminary experimental data processing and a literature review, we predicted that the lipid-motility hormone is related to imidacloprid resistance in B. tabaci MED.

Effect of AKH on the Expression of CYP6CM1 and Its Upstream Regulatory Factors
After preliminary experimental data processing and a literature review, we predicted that the lipid-motility hormone is related to imidacloprid resistance in B. tabaci MED.Therefore, we used RNAi technology to disrupt AKH expression and evaluated the effects on CYP6CM1 and the three upstream regulatory factors.After feeding double-stranded RNA at a concentration of 500 ng/µL for 48 h by the membrane feeding technique, the relative expression of AKH decreased significantly (p < 0.0001) (Figure 2A) and the expression of the resistance gene CYP6CM1 was significantly higher than that in the control (p < 0.01) (Figure 2B).The expression levels of CREB (p < 0.05) (Figure 2C), ERK (p < 0.05) (Figure 2D), and P38 (Figure 2E) were significantly higher (p < 0.05) in the RNAi group than in the Therefore, we used RNAi technology to disrupt AKH expression and evaluated the effects on CYP6CM1 and the three upstream regulatory factors.After feeding double-stranded RNA at a concentration of 500 ng/µL for 48 h by the membrane feeding technique, the relative expression of AKH decreased significantly (p < 0.0001) (Figure 2A) and the expression of the resistance gene CYP6CM1 was significantly higher than that in the control (p < 0.01) (Figure 2B).The expression levels of CREB (p < 0.05) (Figure 2C), ERK (p < 0.05) (Figure 2D), and P38 (Figure 2E) were significantly higher (p < 0.05) in the RNAi group than in the control group, indicating that AKH could have an impact on resistance genes and their signaling pathways (Abbreviations).

Effect of AKH Silencing on Imidacloprid Sensitivity
Previous analyses indicated that AKH can affect the expression of an imidacloprid resistance gene, which is crucial for the metabolism of imidacloprid.Therefore, we evaluated the imidacloprid sensitivity of B. tabaci fed dsAKH.After 2 days of feeding with dsAKH, the B. tabaci MED was transferred to a bioassay unit and treated with imidacloprid at the LC50 of sensitive B. tabaci MED for 48 h.B. tabaci MED mortality was significantly lower in the treatment group than in the control group (p < 0.001) (Figure 3).

Effect of AKH Silencing on Imidacloprid Sensitivity
Previous analyses indicated that AKH can affect the expression of an imidacloprid resistance gene, which is crucial for the metabolism of imidacloprid.Therefore, we evaluated the imidacloprid sensitivity of B. tabaci fed dsAKH.After 2 days of feeding with dsAKH, the B. tabaci MED was transferred to a bioassay unit and treated with imidacloprid at the LC50 of sensitive B. tabaci MED for 48 h.B. tabaci MED mortality was significantly lower in the treatment group than in the control group (p < 0.001) (Figure 3).

ROS Response to Imidacloprid
Insects subjected to insecticide stress will produce a large number of ROS, thus reducing the harmful effects of pesticides [27].Therefore, sensitive B. tabaci MED was treated with imidacloprid to evaluate changes in ROS levels.After treatment with 100 mg/L imidacloprid, the ROS content in B. tabaci in the treatment group was significantly higher

ROS Response to Imidacloprid
Insects subjected to insecticide stress will produce a large number of ROS, thus reducing the harmful effects of pesticides [27].Therefore, sensitive B. tabaci MED was treated with imidacloprid to evaluate changes in ROS levels.After treatment with 100 mg/L imidacloprid, the ROS content in B. tabaci in the treatment group was significantly higher than that in the control group (p < 0.05) (Figure 4A).In addition, to further study the role of ROS in resistance, the ROS scavenger NAC was used to treat resistant B. tabaci MED [19].The results showed that under treatment with 1, 5, and 10 mmol/L NAC, the ROS contents in resistant whiteflies decreased; however, a significant difference was only observed between 10 mmol/L and the control (p < 0.05) (Figure 4B).For subsequent studies of resistance mechanisms, a concentration of 10 mmol/L was optimal.
mortality rate of B. tabaci.Groups were compared using an independent samples t-test nificance level of 0.05.Data are presented as the mean ± standard error (*** p < 0.001).

ROS Response to Imidacloprid
Insects subjected to insecticide stress will produce a large number of RO ducing the harmful effects of pesticides [27].Therefore, sensitive B. tabaci MED w with imidacloprid to evaluate changes in ROS levels.After treatment with 100 idacloprid, the ROS content in B. tabaci in the treatment group was significan than that in the control group (p < 0.05) (Figure 4A).In addition, to further stu of ROS in resistance, the ROS scavenger NAC was used to treat resistant B. tabac The results showed that under treatment with 1, 5, and 10 mmol/L NAC, the RO in resistant whiteflies decreased; however, a significant difference was only ob tween 10 mmol/L and the control (p < 0.05) (Figure 4B).For subsequent stu sistance mechanisms, a concentration of 10 mmol/L was optimal.

Effects of ROS Scavengers on CYP6CM1 and Its Upstream Regulators
ROS levels changed in response to stimulation with imidacloprid, and R gers significantly reduced ROS levels in resistant B. tabaci MED.Therefore, evaluated whether ROS could affect the expression of resistance genes.After with 10 mmol/L NAC, the expression level of CYP6CM1 in resistant B. tabaci

Effects of ROS Scavengers on CYP6CM1 and Its Upstream Regulators
ROS levels changed in response to stimulation with imidacloprid, and ROS scavengers significantly reduced ROS levels in resistant B. tabaci MED.Therefore, we further evaluated whether ROS could affect the expression of resistance genes.After treatment with 10 mmol/L NAC, the expression level of CYP6CM1 in resistant B. tabaci MED was significantly lower than that in the control group (p < 0.05) (Figure 5A).The expression levels of ERK, P38, and CREB were significantly lower than those in the control (all p < 0.05) (Figure 5B-D).These results indicate that ROS levels influence the expression of resistance genes and related regulatory factors.

Effect of an ROS Scavenger on Imidacloprid Sensitivity
The previous analyses showed that ROS can affect the expression of the resistance gene CYP6CM1, which is crucial for the metabolism of imidacloprid.Therefore, a bioassay of imidacloprid sensitivity was performed after whiteflies were fed NAC.After 2 days of NAC feeding, whiteflies were treated with imidacloprid at the LC50 dose for 48 h.B. tabaci MED mortality was significantly lower in the treatment group than in the control group (p < 0.001) (Figure 6).
significantly lower than that in the control group (p < 0.05) (Figure 5A).The expression levels of ERK, P38, and CREB were significantly lower than those in the control (all p < 0.05) (Figure 5B-D).These results indicate that ROS levels influence the expression of resistance genes and related regulatory factors.

Effect of an ROS Scavenger on Imidacloprid Sensitivity
The previous analyses showed that ROS can affect the expression of the resistance gene CYP6CM1, which is crucial for the metabolism of imidacloprid.Therefore, a bioassay of imidacloprid sensitivity was performed after whiteflies were fed NAC.After 2 days of NAC feeding, whiteflies were treated with imidacloprid at the LC50 dose for 48 h.B. tabaci MED mortality was significantly lower in the treatment group than in the control group (p < 0.001) (Figure 6).The horizontal axis represents different treatments, and the vertical axis represents the mortality rate of B. tabaci MED.Groups were compared using the independent samples t-test, with a significance level of 0.05.Data are presented as the mean ± standard error (*** p < 0.001).

Effect of AKH on ROS Levels in B. tabaci MED
It has been reported that AKH exerts an antioxidant effect in insects [17].We found that AKH and ROS can affect the expression of resistance genes.Therefore, we speculated that AKH may regulate the expression of resistance genes by influencing ROS.To evaluate this prediction, levels of ROS in whiteflies were measured after AKH was silenced by RNAi.The results showed that the ROS content in the treatment group was significantly higher than that in the control group (p < 0.05) (Figure 7), indicating that AKH could affect ROS in B. tabaci MED.

Effect of AKH on ROS Levels in B. tabaci MED
It has been reported that AKH exerts an antioxidant effect in insects [17].We found that AKH and ROS can affect the expression of resistance genes.Therefore, we speculated that AKH may regulate the expression of resistance genes by influencing ROS.To evaluate this prediction, levels of ROS in whiteflies were measured after AKH was silenced by RNAi.The results showed that the ROS content in the treatment group was significantly higher than that in the control group (p < 0.05) (Figure 7), indicating that AKH could affect ROS in B. tabaci MED.rate of B. tabaci MED.Groups were compared using the independent samples t-test, with a significance level of 0.05.Data are presented as the mean ± standard error (*** p < 0.001).

Effect of AKH on ROS Levels in B. tabaci MED
It has been reported that AKH exerts an antioxidant effect in insects [17].We found that AKH and ROS can affect the expression of resistance genes.Therefore, we speculated that AKH may regulate the expression of resistance genes by influencing ROS.To evaluate this prediction, levels of ROS in whiteflies were measured after AKH was silenced by RNAi.The results showed that the ROS content in the treatment group was significantly higher than that in the control group (p < 0.05) (Figure 7), indicating that AKH could affect ROS in B. tabaci MED.

Discussion
As a major pest worldwide, B. tabaci seriously endangers food security [28,29].To control B. tabaci MED, a large number of chemical pesticides (organophosphorus, pyrethroids, insect growth regulators, nicotinoids, and neonicotinoids.)are used, causing environmental pollution and posing a threat to human health.Pesticide residues are becoming more and more serious; at the same time, whitefly resistance is increasing.
In this study, we found that the AKH gene of B. tabaci MED contributes to the regulation of resistance to imidacloprid.In particular, AKH expression levels were lower in resistant than in sensitive whiteflies After treatment with 50, 100, and 150 mg/L imidacloprid in sensitive B. tabaci MED, AKH expression exhibited variable levels.This trend was

Discussion
As a major pest worldwide, B. tabaci seriously endangers food security [28,29].To control B. tabaci MED, a large number of chemical pesticides (organophosphorus, pyrethroids, insect growth regulators, nicotinoids, and neonicotinoids.)are used, causing environmental pollution and posing a threat to human health.Pesticide residues are becoming more and more serious; at the same time, whitefly resistance is increasing.
In this study, we found that the AKH gene of B. tabaci MED contributes to the regulation of resistance to imidacloprid.In particular, AKH expression levels were lower in resistant than in sensitive whiteflies After treatment with 50, 100, and 150 mg/L imidacloprid in sensitive B. tabaci MED, AKH expression exhibited variable levels.This trend was consistent with the results of Tang et al., showing that the AKH expression level in the brown planthopper (Nilaparvata lugens) is low in resistant strains and decreases after treatment with imidacloprid.However, in this study, the AKH expression level increased after treatment with 100 mg/L imidacloprid.It is possible that excess ROS activated the antioxidant system of whiteflies, which has been reported previously [19].
Imidacloprid resistance in B. tabaci is mainly caused by the P450 family gene CYP6CM1 [30].Consistent with our expectations, we found that CYP6CM1 expression in B. tabaci was significantly increased.Furthermore, CYP6CM1 is regulated by the MAPK pathway, and the expression levels of the three regulatory factors ERK, P38, and CREB also increased significantly [31].This suggests that AKH may act as a negative regulator of resistance genes.However, the increased levels of resistance genes do not necessarily mean that B. tabaci MED tolerance to imidacloprid increased.Therefore, we tested the sensitivity of B. tabaci MED to insecticides after silencing AKH, revealing that the sensitivity of B. tabaci MED to insecticides decreased.This suggests that AKH can indeed influence the expression of CYP6CM1 and thus the resistance of B. tabaci MED to imidacloprid.
We found that AKH exerts a negative regulatory effect on CYP6CM1 in B. tabaci MED.However, the pathways through which AKH exerts these effects are still unclear.We speculate that AKH exerts its effects through its antioxidant stress function [32,33].Therefore, we treated the resistant B. tabaci MED with the antioxidant NAC and found that the expression levels of the resistance gene CYP6CM1 and the upstream regulatory factors ERK, P38, and CREB decreased.Moreover, the sensitivity of B. tabaci MED to imidacloprid Insects 2024, 15, 436 9 of 11 increased (Scheme 1).These results are consistent with those of Tang et al. (2020), who showed that the resistance of the brown planthopper to imidacloprid is mediated by ROS.In another study on the brown planthopper, the expression levels of Akh and AkhR increased significantly under stimulation with chlorpyrifos.Interference with the expression of Akh or AkhR significantly reduced the activity of carboxylesterase and the resistance of the brown planthopper to chlorpyrifos [34].This suggests that the role of AKH in insecticide resistance depends on the type of insecticide and the degree of insecticide stimulation [17,35].
the expression of CYP6CM1 and thus the resistance of B. tabaci MED to imidacloprid.
We found that AKH exerts a negative regulatory effect on CYP6CM1 in B. tabaci MED.However, the pathways through which AKH exerts these effects are still unclear.We speculate that AKH exerts its effects through its antioxidant stress function [32,33].Therefore, we treated the resistant B. tabaci MED with the antioxidant NAC and found that the expression levels of the resistance gene CYP6CM1 and the upstream regulatory factors ERK, P38, and CREB decreased.Moreover, the sensitivity of B. tabaci MED to imidacloprid increased (Scheme 1).These results are consistent with those of Tang et al. (2020), who showed that the resistance of the brown planthopper to imidacloprid is mediated by ROS.In another study on the brown planthopper, the expression levels of Akh and AkhR increased significantly under stimulation with chlorpyrifos.Interference with the expression of Akh or AkhR significantly reduced the activity of carboxylesterase and the resistance of the brown planthopper to chlorpyrifos [34].This suggests that the role of AKH in insecticide resistance depends on the type of insecticide and the degree of insecticide stimulation [17,35].In conclusion, this study demonstrated that exposure to imidacloprid stimulation decreased AKH expression, inhibited antioxidant activity mediated by this gene, and In conclusion, this study demonstrated that exposure to imidacloprid stimulation decreased AKH expression, inhibited antioxidant activity mediated by this gene, and increased ROS in B. tabaci MED, thereby activating the MAPK signaling pathway and increasing the expression of the resistance gene CYP6CM1 [31].

Figure 1 .
Figure 1.AKH expression in sensitive and resistant strains and in response to imidacloprid.Note: (A) Comparison of AKH expression levels between resistant and sensitive Bemisia tabaci, R indicates resistant B. tabaci, and S indicates sensitive B. tabaci.(B) Effect of imidacloprid treatment on the expression level of AKH in sensitive B. tabaci; the horizontal axis indicates the concentration of imidacloprid, CK represents the control (water).Groups were compared using independent samples t-tests, with a significance level of 0.05.Data are presented as the mean ± standard error (* p < 0.05, ns indicates insignificant difference).

Figure 1 .
Figure 1.AKH expression in sensitive and resistant strains and in response to imidacloprid.Note: (A) Comparison of AKH expression levels between resistant and sensitive Bemisia tabaci, R indicates resistant B. tabaci, and S indicates sensitive B. tabaci.(B) Effect of imidacloprid treatment on the expression level of AKH in sensitive B. tabaci; the horizontal axis indicates the concentration of imidacloprid, CK represents the control (water).Groups were compared using independent samples t-tests, with a significance level of 0.05.Data are presented as the mean ± standard error (* p < 0.05, ns indicates insignificant difference).
Insects 2024, 15, 436 5 of 11 control group, indicating that AKH could have an impact on resistance genes and their signaling pathways (Abbreviations).

Figure 2 .
Figure 2. Effect of RNAi silencing targeting AKH on the expression of CYP6CM1 and upstream regulatory factors in sensitive Bemisia tabaci MED.Note: (A) Expression level of AKH after feeding with dsAKH; (B) Expression level of CYP6CM1 after feeding with dsAKH; (C) Expression level of CREB after feeding with dsAKH; (D) Expression level of ERK after feeding with dsAKH; (E) Expression level of P38 after feeding with dsAKH.Groups were compared using independent samples t-tests, with a significance level of 0.05.Data are presented as the mean ± standard error (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Figure 2 .
Figure 2. Effect of RNAi silencing targeting AKH on the expression of CYP6CM1 and upstream regulatory factors in sensitive Bemisia tabaci MED.Note: (A) Expression level of AKH after feeding with dsAKH; (B) Expression level of CYP6CM1 after feeding with dsAKH; (C) Expression level of CREB after feeding with dsAKH; (D) Expression level of ERK after feeding with dsAKH;(E) Expression level of P38 after feeding with dsAKH.Groups were compared using independent samples t-tests, with a significance level of 0.05.Data are presented as the mean ± standard error (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Insects 2024 , 11 Figure 3 .
Figure 3.Effect of RNAi silencing targeting AKH on the sensitivity of Bemisia tabaci MED to imidacloprid.Note: The horizontal axis represents the treatments, and the vertical axis represents the mortality rate of B. tabaci.Groups were compared using an independent samples t-test, with a significance level of 0.05.Data are presented as the mean ± standard error (*** p < 0.001).

Figure 3 .
Figure 3.Effect of RNAi silencing targeting AKH on the sensitivity of Bemisia tabaci MED to imidacloprid.Note: The horizontal axis represents the treatments, and the vertical axis represents the mortality rate of B. tabaci.Groups were compared using an independent samples t-test, with a significance level of 0.05.Data are presented as the mean ± standard error (*** p < 0.001).

Figure 4 .
Figure 4. ROS changes in response to imidacloprid.Note: (A) ROS response to imida represents the control (water), and 100 mg/L imidacloprid was applied in the treatmen Changes in ROS contents in resistant B. tabaci MED after N-acetylcysteine treatment; C trol group (pure liquid feed), and 1, 5, and 10 mmol/L are the concentrations of N-acety the liquid feed.Groups were compared using independent samples t-tests, with a signi of 0.05.Data are presented as the mean ± standard error (* p < 0.05, ns indicates insigni ence).

Figure 4 .
Figure 4. ROS changes in response to imidacloprid.Note: (A) ROS response to imidacloprid, CK represents the control (water), and 100 mg/L imidacloprid was applied in the treatment group; (B) Changes in ROS contents in resistant B. tabaci MED after N-acetylcysteine treatment; CK is the control group (pure liquid feed), and 1, 5, and 10 mmol/L are the concentrations of Nacetylcysteine in the liquid feed.Groups were compared using independent samples t-tests, with a significance level of 0.05.Data are presented as the mean ± standard error (* p < 0.05, ns indicates insignificant difference).

Figure 5 .
Figure 5.Effect of ROS scavengers on the expression of CYP6CM1 and upstream regulatory factors in resistant Bemisia tabaci MED.Note: (A) Expression level of CYP6CM1 after treatment with 10 mmol/L N-acetylcysteine; (B) Expression level of CREB after treatment with 10 mmol/L N-acetylcysteine; (C) Expression level of ERK after treatment with 10 mmol/L N-acetylcysteine; (D) Expression level of P38 after treatment with 10 mmol/L N-acetylcysteine.Groups were compared using independent samples t-tests, with a significance level of 0.05.Data are presented as the mean ± standard error (* p < 0.05).

Figure 5 .
Figure 5.Effect of ROS scavengers on the expression of CYP6CM1 and upstream regulatory factors in resistant Bemisia tabaci MED.Note: (A) Expression level of CYP6CM1 after treatment with 10 mmol/L N-acetylcysteine; (B) Expression level of CREB after treatment with 10 mmol/L N-acetylcysteine; (C) Expression level of ERK after treatment with 10 mmol/L N-acetylcysteine; (D) Expression level of P38 after treatment with 10 mmol/L N-acetylcysteine.Groups were compared using independent samples t-tests, with a significance level of 0.05.Data are presented as the mean ± standard error (* p < 0.05).Insects 2024, 15, x FOR PEER REVIEW 8 of 11

Figure 6 .
Figure 6.Effect of ROS scavengers on the sensitivity of Bemisia tabaci MED to imidacloprid.Note: The horizontal axis represents different treatments, and the vertical axis represents the mortality rate of B. tabaci MED.Groups were compared using the independent samples t-test, with a significance level of 0.05.Data are presented as the mean ± standard error (*** p < 0.001).

Figure 6 .
Figure 6.Effect of ROS scavengers on the sensitivity of Bemisia tabaci MED to imidacloprid.Note: The horizontal axis represents different treatments, and the vertical axis represents the mortality rate of B. tabaci MED.Groups were compared using the independent samples t-test, with a significance level of 0.05.Data are presented as the mean ± standard error (*** p < 0.001).

Figure 7 .
Figure 7. Effect of RNAi silencing AKH on the ROS content in Bemisia tabaci MED.Note: The horizontal axis represents different treatments, and the vertical axis represents the ROS content in B.tabaci.Groups were compared using the independent samples t-test, with a significance level of 0.05.Data are presented as the mean ± standard error (* p < 0.05).

Figure 7 .
Figure 7. Effect of RNAi silencing AKH on the ROS content in Bemisia tabaci MED.Note: The horizontal axis represents different treatments, and the vertical axis represents the ROS content in B. tabaci.Groups were compared using the independent samples t-test, with a significance level of 0.05.Data are presented as the mean ± standard error (* p < 0.05).

Table 1 .
Primers of related genes for qRT-PCR and RT-PCR.

Table 2 .
Virulence equation and resistance multiplicity for Bemisia tabaci.

Table 2 .
Virulence equation and resistance multiplicity for Bemisia tabaci.