Next Article in Journal
The Influence of Calcium Sulfate and Different Doses of Potassium on the Soil Enzyme Activity and the Yield of the Sward with a Mixture of Alfalfa and Grasses
Next Article in Special Issue
Sublethal Effects of Emamectin Benzoate on Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae)
Previous Article in Journal
Genetic Effects of Indica Lineage Introgression on Amylopectin Chain Length Distribution in Japonica Milled Rice
Previous Article in Special Issue
Effectiveness of Low Copper-Containing Chemicals against Olive Leaf Spot Disease Caused by Venturia oleaginea
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agriculture
Volume 12
Issue 4
10.3390/agriculture12040473
agriculture-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Cytpchrome P450 CYP4G68 Is Associated with Imidacloprid and Thiamethoxam Resistance in Field Whitefly, Bemisia tabaci (Hemiptera: Gennadius)

by
Jinjin Liang
1,2,
Jing Yang
2,
Jinyu Hu
2,
Buli Fu
2,3,
Peipan Gong
2,
Tianhua Du
2,
Hu Xue
2,
Xuegao Wei
2,
Shaonan Liu
1,2,
Mingjiao Huang
1,2,
Cheng Yin
2,
Yao Ji
1,2,
Chao He
2,
Wen Xie
2,4,
Ran Wang
5,
Xin Yang
2,4,* and
Youjun Zhang
1,*
1
College of Plant Protection, Hunan Agricultural University, Changsha 410125, China
2
Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China
3
The Ministry of Agriculture and Rural Affairs Key Laboratory of Integrated Pest Management of Tropical Crops, Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
4
National Research Institute of Breeding in Hainan, Chinese Academy of Agricultural Sciences, Sanya 572024, China
5
Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China
*
Authors to whom correspondence should be addressed.
Agriculture 2022, 12(4), 473; https://doi.org/10.3390/agriculture12040473
Submission received: 3 March 2022 / Revised: 21 March 2022 / Accepted: 25 March 2022 / Published: 27 March 2022
(This article belongs to the Special Issue Sustainable Use of Pesticides)
Browse Figures
Versions Notes

Abstract

:
The superfamily cytochrome P450s is involved in the evolution of insecticide resistance. However, whether CYP4G68, a differentially expressed gene identified from our transcriptomics analysis, confers resistance to the world’s heavily used insecticide class neonicotinoids is unknown. Hence, we explored the role of CYP4G68 in conferring imidacloprid and thiamethoxam resistance in Bemisia tabaci. The species B. tabaci MED developed low-to-high resistance to imidacloprid and thiamethoxam. Exposure to imidacloprid and thiamethoxam significantly increased the expression of CYP4G68. Moreover, quantitative real-time PCR analysis demonstrated that CYP4G68 was remarkably overexpressed in imidacloprid-resistant and thiamethoxam-resistant strains compared to susceptible strains. Further correlation analysis showed that CYP4G68 expression was significantly positively correlated with the associated resistance level in various strains of B. tabaci. These results suggest that the enhanced expression of CYP4G68 appears to mediate imidacloprid and thiamethoxam resistance in B. tabaci. Additionally, silencing CYP4G68 via RNA interference strongly increased the susceptibility of B. tabaci MED to imidacloprid and thiamethoxam. Collectively, this work revealed that CYP4G68 plays a vital role in imidacloprid and thiamethoxam resistance in B. tabaci MED. These findings will not only advance our understanding of the role of P450s in insecticide resistance but also provide a great potential target for the sustainable control of destructive insect pests such as whiteflies.

1. Introduction

The evolution of resistance to insecticides poses a substantial threat to agriculture, the environment, and public health [1,2,3,4,5,6,7]. In particular, the widespread resistance to neonicotinoids, one of the most heavily used insecticide class in the world, has caused global concern, which is mainly attributed to neonicotinoids abuse in recent decades [8,9,10]. Imidacloprid and thiamethoxam, the two most important members belonging to neonicotinoids, have been extensively applied to control many destructive insect pests worldwide [8,11,12,13,14,15,16]. Nevertheless, field-evolved resistance to the two neonicotinoids has emerged in diverse pests such as aphid [17,18], brown planthopper [19,20], Asian citrus psyllid [14], and whitefly [21,22,23]. To address the negative impacts of neonicotinoid resistance, there is, thus, an urgent need to understand the molecular basis that underlies imidacloprid and thiamethoxam resistance in specific insect pests.
The superfamily cytochrome P450s (P450s), among the most important phase I metabolic enzymes, participate in the metabolic detoxification of toxic xenobiotics [8,10,24,25] and the reduction in the penetration rates of insecticides [26,27,28,29]. Of the many cases reported, the CYP6 subfamilies are usually involved in detoxification resistance, which has been well documented in Bemisia tabaci [21,22,23,30,31], Nilaparvata lugens [15,19,32,33], Locusta migratoria [34,35], Drosophila melanogaster [36], and Anopheles funestus [37,38]. However, the CYP4 subfamilies are usually associated with penetration resistance in N. lugens [26], D. melanogaster [28], and Anopheles gambiae [27,29]. Notably, the CYP6 and CYP4 subfamilies play key roles in conferring neonicotinoid resistance, especially imidacloprid and thiamethoxam resistance, such as CYP6ER1 and CYP6AY1, which confer imidacloprid resistance in N. lugens [19,39]. CYP6BB2 contributes to imidacloprid resistance in Aedes aegypti [16]. CYP6CM1 is implicated in imidacloprid resistance in B. tabaci [22,23,31,40]. CYP4C64 contributes to thiamethoxam resistance in B. tabaci [21]. CYP4g15 is correlated with imidacloprid-resistance in Diaphorina citri [14].
Bemisia tabaci (Hemiptera: Gennadius) is a notorious agricultural pest that causes direct damage via phloem feeding and has a vector of more than 100 plant viruses [22,41]. Among the species, B. tabaci MED (Mediterranean, ‘Q’ type) and MEAM1 (Middle East-Asia Minor 1, ‘B’ type) are the two most widespread and destructive biotype species worldwide [42,43]. Notably, B. tabaci MED is assumed to be more resistant to pesticides than MEAM1, and this advantage is considered the key reason for the displacement of MEAM1 by MED [9,12,44,45,46].
The CYP4G enzymes are involved in reducing the penetration of toxic xenobiotics through the cuticle [26,27,28,29,47]. The overexpression of CYP4G1 contributes to DDT resistance in D. melanogaster [27]. Likewise, the mRNA expression of CYP4G68 was also significantly higher in some resistant field populations of B. tabaci [48]. Interestingly, our transcriptomic analyses revealed that the P450 gene CYP4G68 was overexpressed in resistant B. tabaci MED compared to that in susceptible B. tabaci MED [49]. In addition, we have reported that methyltransferase-like 3 (METTL3) and METTL14 may regulate CYP4G68 in neonicotinoid resistance in B. tabaci, and a more functional validation of CYP4G68 is required [21]. In view of these previous studies, it is thus plausible that CYP4G68 has a potential role in underlying neonicotinoid resistance; however, this mechanism has never been elucidated. In this work, we therefore aimed to probe the role of CYP4G68 in conferring imidacloprid and thiamethoxam resistance in B. tabaci MED. After identifying imidacloprid and thiamethoxam resistance in both laboratory and field strains, the putative CYP4G68 gene of B. tabaci MED was cloned and characterized. Using quantitative real-time PCR (qRT–PCR) and RNA interference (RNAi) technology, we further investigated the function of CYP4G68 in mediating imidacloprid and thiamethoxam resistance. The obtained results will provide a new insight into understanding the P450-mediated resistance mechanism and be invaluable in proposing the most adequate strategies for the sustainable pest control of destructive pests such as whiteflies.

2. Materials and Methods

2.1. Insect Strains

2.1.1. Laboratory Strains

The initial strain of B. tabaci MED was collected from tomato plants in Hangzhou, Zhejiang in 2011 (Table S1). This strain was divided into three lots to construct laboratory susceptible and laboratory resistant strains sharing the same genetic background by using a two-way method [11]. The resistance group was split and separately selected with imidacloprid (200 mg/L, 95% technical compound, Shanghaiyuanye BioTechnology Co., Ltd., Shanghai, China) and thiamethoxam (200 mg/L, 95% technical compound, Shanghaiyuanye BioTechnology Co., Ltd., Shanghai, China) to establish resistant strains. The remaining group has not been exposed to any pesticides for 10 years and served as the laboratory susceptible strain (S#1). These three strains were separately reared with cotton seedlings in cages and maintained in a glasshouse with a temperature of 25 °C and 14:10 photoperiod.

2.1.2. Field Populations

From 2019 to 2021, a total of 7 field populations were sampled from many vegetable-planting areas across China. Specifically, these field samples were collected from 5 provinces (Figure 1A) including Hubei, Fujian, Shandong, Beijing, and Hainan (Table S1). The sample location, sampling date, and host plant for each population were also summarized in Table S1. All strains included in our study were separately reared in the glasshouse, as described above, and used for bioassays (the methods described in Section 2.2). All field populations used in the study were B. tabaci MED identified by the method of Zheng [50]. Additional details about the strain background are summarized in Figure 1A and Table S1.

2.2. Bioassays with Imidacloprid and Thiamethoxam

To detect the susceptibility of strains to imidacloprid and thiamethoxam, all bioassays were conducted on adults B. tabaci MED using an improved feeding method described in Figure 1B. Briefly, the technical compound of each insecticide was initially dissolved by acetone to 20,000 mg/L. Afterwards, it was diluted by a diet solution (5% yeast extract and 30% sucrose, wt/vol) to serial dilutions of the tested concentrations (1–500 mg/L). Approximately 60 μL of this solution was pipetted onto the outer surface of the Parafilm stretched over the top of a glass tube (50 mm in length and 15 mm in diameter). A second layer of Parafilm was stretched on top of the first membrane to form a feeding sachet. Then, approximately 25 adults B. tabaci MED (female:male ≈ 1:1, 3–10 days after eclosion) were introduced into the bioassay tube. After that, the remaining opening of the tube was sealed with a third layer of Parafilm. At least 6 concentrations of each insecticide were assayed with 4 replications containing 25 adults B. tabaci MED per tube. For the control, a total of 60 μL of the diet solution was used. Finally, all tubes were placed in black plastic casings (55 mm in length and 20 mm in diameter) and maintained in the greenhouse, as described above. After 48 h, the number of surviving insects and all insects was counted, then the mortality for each treatment was calculated. Insects were recognized as dead when they were unable to walk after being prodded with a fine-hair brush [51].

2.3. Molecular Cloning of CYP4G68

Total RNA from 50 whole adult (female:male ≈ 1:1, 3–10 days after eclosion) B. tabaci MED was isolated using standard TRIzol (Invitrogen, Carlsbad, CA, USA) protocols, and the RNA concentration was determined via a NanoDrop 2000c spectrophotometer (Thermo 150 Fisher Scientific Inc., Waltham, MA, USA) [11]. The extracted RNA was converted to cDNA using oligo (dT) primers and SuperScript II Reverse Transcriptase with gDNA Eraser (Takara Biotech, Tokyo, Japan). Preliminarily, the putative P450 sequence was in silico identified from the transcriptome data of B. tabaci MED [12,49] and then cloned by reverse transcription PCR (RT–PCR) using the full-length primers for CYP4G68 (Table S2).

2.4. Bioinformatic Analysis of CYP4G68

The transmembrane domains (TMDs) were predicted by TMHMM (http://www.cbs.dtu.dk/services/TMHMM/ (accessed on 10 December 2021)), and the theoretical isoelectric points (pIs) and molecular weights (Mw) were analyzed by ExPASy (https://web.expasy.org/protparam/ (accessed on 10 December 2021)). The homologous sequences of CYP4G68 in other insects, including Bombyx mori, Bombyx mandarina, Manduca sexta, Helicoverpa armigera, Frankliniella occidentails, Thrips palmi, Rhopalosiphum maidis, Nilaparvata lugens, Diaphorina citri, Leptinotarsa decemlineata, Anoplophora glabripennis, Harmonia axyridis, Solenopsis invicta, Athalia rosae, and Orussus abietinus, were downloaded from the National Center for Biotechnology Information (NCBI) database. The related amino acid sequences were subjected to phylogenetic analysis, and MEGAX was used to construct a phylogenetic tree by the neighbor-joining method with 1000 bootstrap replications.

2.5. Quantitative Real-Time PCR

The relative mRNA levels of CYP4G68 were determined by qRT–PCR. qRT–PCR was conducted according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [52]. The reaction system consisted of 20 μL composed of 10 μL of 2× SuperReal PreMix Plus (Tiangen, Beijing, China), 0.4 μL of 50× ROX Reference Dye (Tiangen, Beijing, China), 0.6 μL of each specific primer, 1 μL of diluted cDNA, and 7.4 μL of ddH2O [11]. qRT–PCR was performed using a two-step protocol as follows: a pre-denaturation at 95 °C for 1 min, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 15 s. The reaction was conducted in a QuentStudio 3 RealTime PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). The amplification efficiencies of quantitative primers were evaluated using six 2-fold serial dilutions (1:1, 1:2, 1:4, 1:8, 1:16, and 1:32) with 4 replicates of cDNA. The equation E = (10[−1/slope] − 1) was used to calculate the amplification efficiencies. Only primers that had amplification efficiencies of 95–105% and correlation coefficients of >0.95 were used [11]. The sequences of the quantitative primers and reference genes (RPL29 and EF-1α) are listed in Table S2 [53,54]. The calculations were performed according to the 2−ΔΔCt method [55]. Each group contained 3 biological replications, and each repetition was performed 4 times.

2.6. Analysis of Spatial-Temporal Specific Expression of CYP4G68

To obtain the developmental stage-specific and tissue-specific expression patterns of CYP4G68 in B. tabaci MED, we examined the expression levels of CYP4G68. Approximately 2000 eggs, 300 1st–2nd instar nymphs, 200 3rd instar nymphs, 200 4th instar nymphs, and 100 adults (female:male ≈ 1:1, 3–10 days after eclosion) of the S#1 strain were collected as replications to determine the expression levels of CYP4G68. Simultaneously, the head, thorax, and abdomen of 2000 adults (female:male ≈ 1:1, 3–10 days after eclosion) were dissected and collected as replications to quantify the mRNA levels of CYP4G68. Three biological replications were conducted for each developmental stage and body part. The methods used for total RNA extraction, cDNA synthesis, and qRT–PCR are described in Section 2.3 and Section 2.5.

2.7. Functional Validation of CYP4G68 in Laboratory Strains

2.7.1. The Expression of CYP4G68 in Response to Exposure Tests and in Resistant and Susceptible

To determine the transcriptional response of CYP4G68 to imidacloprid and thiamethoxam in B. tabaci MED, approximately 100 S#1 strain adults (female:male ≈ 1:1, 3–10 days after eclosion) were exposed to 4 mg/L (≈LC25) imidacloprid, 4 mg/L (≈LC25) thiamethoxam or control groups (diet solution alone) by the method described in Section 2.2. After 3, 6, 12, 24, and 36 h, adults were separately collected and used for expression level quantification by the methods described in Section 2.3 and Section 2.5. Each group contained 3 biological replications, and each repetition was performed 4 times.
To investigate the role of CYP4G68 in laboratory strains, we analyzed the mRNA levels of CYP4G68 in resistant and susceptible strains. The relative expression of CYP4G68 in the S#1, R#1 and R#2 strains was determined. All methods have been described above. Each group contained 3 biological replications, and each repetition was performed 4 times.

2.7.2. Functional Validation of CYP4G68 by RNAi

The dsRNA of CYP4G68 was synthesized by amplifying a positive colony using gene-specific dsRNA primers, as listed in Table S2, containing the T7 RNA polymerase promoter sequence. The products were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). The T7 RiboMAXTM Express RNAi System (Promega, Madison, WI, USA) was used to obtain the dsRNA in vitro by strictly following the manufacturer’s instructions. dsRNA was quantified using a NanoDrop 2000c spectrophotometer, and its integrity was confirmed using 1.5% agarose gel electrophoresis. The dsRNA products were stored at −80 °C before use [11].
The dsRNAs were fed to adults (female:male ≈ 1:1, 3–10 days after eclosion) using the methodology previously described in Section 2.2. In brief, dsRNAs were diluted to 0.5 μg/μL with the diet solution. Some adults were collected after 48 h of feeding to expression analysis by qRT–PCR described in Section 2.5. The mRNA level of CYP4G68 was significantly decreased after 48 h of feeding, and then living adults were transferred into new tubes for the bioassay via the feeding method (as described in Section 2.2). Two concentrations (50 and 100 mg/L) of imidacloprid and thiamethoxam were used for testing the dsRNA-treated R#1 and R#2 strains, and the mortality of adults was recorded at 12 and 24 h. Enhanced green fluorescent protein (EGFP) was used as a control. Each group contained 4 biological replications.

2.8. Functional Validation of CYP4G68 in Field Populations

To further confirm the effect of CYP4G68 on imidacloprid and thiamethoxam resistance in field-resistant populations of B. tabaci MED, the relative mRNA levels of CYP4G68 in seven field strains (S#2, R#3, R#4, R#5, R#6, R#7, and R#8 strains; more details about these strains are summarized in Section 2.1.2 and Table 1) were determined, the correlation between relative mRNA levels of CYP4G68 and resistance (imidacloprid and thiamethoxam) levels was analyzed, and adult (female:male ≈ 1:1, 3–10 days after eclosion) mortality was examined upon exposure to imidacloprid and thiamethoxam after RNAi in the R#8 strain. The methods of total RNA extraction, cDNA synthesis, qRT–PCR, dsRNA synthesis, and RNAi were conducted as previously described. Each group contained three biological replications, and each repetition was performed 4 times.

2.9. Statistical Analysis

LC50 values were calculated via the bioassay data and their 95% confidence limits according to Probit regressions via POLO Plus 2.0 software (LeOra Software, Berkeley, CA, USA) [50]. The insecticide resistance level was quantified as described by Torres-Vila [56] as susceptible (resistance ratio; RR = 1), low resistance (RR = 2–10), moderate resistance (RR = 11–30), and high resistance (RR = 31–100). The statistical analyses were performed with SPSS 26.0.0.0 (SPSS, Chicago, IL, USA). The effects of different developmental stages and body parts on the relative expression level of CYP4G68 were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test at p < 0.05. The differences in expression levels of CYP4G68 and mortality between treatments and control were compared using Student’s t-test. The correlation between the relative mRNA levels of CYP4G68 and resistance levels was analyzed by linear model II regression analysis [57].

3. Results

3.1. Resistance to Imidacloprid and Thiamethoxam in Field B. tabaci MED Populations

Relative to the S#1 strain, two strains, S#2 (RR = 0.49-fold) and R#6 (RR = 0.72-fold), were susceptible to imidacloprid, while low-to-high imidacloprid resistance was observed in strains R#3, R#4, R#5, R#7, and R#8, with RRs varying from 5.03-fold to 37.85-fold (Table 1). The highest LC50 value was 335.34 mg/L for imidacloprid (for the R#8 strain, isolated in 2019).
For thiamethoxam, two strains remained susceptible (S#2, RR = 1.27-fold; R#5, RR = 1.71-fold) relative to the S#1 strain, three strains (R#3, RR = 2.10-fold; R#4, RR = 2.61-fold; R#6, RR = 7.58-fold) had low resistance, and two strains (R#7, RR = 32.82-fold; R#8, RR = 57.57-fold) showed high resistance (Table 1). The highest LC50 value was 403.59 mg/L for thiamethoxam (for the R#8 strain, isolated in 2019).

3.2. Gene Structure and Phylogenetic Analysis

The structural features of CYP4G68 are shown in Figure 2A–C. The full-length gene that encodes CYP4G68 (http://www.whiteflygenomics.org/cgi-bin/bta/index.cgi (accessed on 10 December 2021): Bta14906) in B. tabaci contains a 1764 bp ORF encoding 587 amino acid residues (Figure 2C). The theoretical isoelectric point (pI) was 7.92, and the molecular weight (Mw) was 66.53 kDa. A hydrophobic transmembrane domain of CYP4G68, at the N-terminus, contains approximately 23 hydrophobic amino acid residues. Genomic structure analysis indicated that CYP4G68 contains 13 exons and 12 introns (Figure 2A). Conserved domains present in this gene are common to cytochrome P450s (Figure 2B,C), including the WxxxR motif (positions 101–105 and 151–155), the GxE/DTT/S motif (positions 382–386), the ExLR motif (positions 439–442), the conserved amino acid sequence PxxFxP motif (positions 495–500), and the heme-binding motif (PFxxGxxxCxG, positions 513–523).
The phylogenetic relationships of CYP4G68 from B. tabaci and related P450s from other insects are shown in Figure 2D. The P450 sequences of Lepidoptera, Thysanoptera, Hemiptera, Coleoptera, and Hymenoptera species showed a distinct clustering of the five species groups, which could imply that they might play some specific role in these species.

3.3. Spatiotemporal Expression Profiles

In the stage-specific expression profile analysis, CYP4G68 in the S#1 strain had higher expression levels from the first to second instar nymphs, third instar nymphs, and adult instar nymphs than those in the other stages (F4,25 = 403.18, p < 0.001, Figure 3A). The tissue-specific expression of CYP4G68 in the head was significantly higher than that in the thorax and abdomen (F2,15 = 20.99, p < 0.001, Figure 3B).

3.4. Functional Analysis of CYP4G68 in Laboratory Strains

3.4.1. Response of CYP4G68 Expression to Imidacloprid and Thiamethoxam Exposure

The expression levels of CYP4G68 in the S#1 strain were markedly upregulated after exposure to imidacloprid (Figure 4A) and thiamethoxam (Figure 4B). Compared with that of the control group, the relative expression levels of CYP4G68 were significantly increased by 1.94- (p = 0.029), 3.88- (p < 0.001), 1.70- (p = 0.001), and 1.42-fold (p = 0.002) after imidacloprid treatment at 6, 12, 24, and 36 h, respectively. Likewise, after exposure to thiamethoxam for 6, 12, 24, and 36 h, the mRNA levels of this gene were remarkably upregulated by 2.94- (p = 0.006), 3.64- (p = 0.001), 1.18- (p = 0.033), and 2.43-fold (p = 0.006), respectively. The transcript levels of CYP4G68 peaked at 12 h after imidacloprid and thiamethoxam treatment.

3.4.2. Expression Analysis of CYP4G68 in Resistant and Susceptible Strains

The mRNA levels of CYP4G68 were significantly upregulated in the R#1 and R#2 strains compared with those in the S#1 strain (Figure 4C). The relative expression levels of CYP4G68 in the R#1 and R#2 strains were more than 2.75- (p = 0.013) and 2.13-fold (p = 0.009) higher, respectively, than those in the S#1 strain.

3.4.3. Functional Analysis of CYP4G68 by RNAi

Interference with the CYP4G68 gene induced an observable increase in mortality when the adults of the imidacloprid resistance strain (R#1, Figure 4E,F) and thiamethoxam resistance strain (R#2, Figure 4G,H) were exposed to imidacloprid and thiamethoxam, respectively. After 48 h of feeding on a diet containing 0.5 μg/μL dsRNA specific for CYP4G68, the mRNA levels of this gene significantly decreased by 47.4% compared with those in the control (fed dsEGFP) (p = 0.002, Figure 4D). After RNAi knockdown of CYP4G68, mortality relative to that of the control was observably elevated upon treatment with 50 mg/L imidacloprid (12 h: 17.08%, p < 0.001; 24 h: 20.26%, p = 0.008; Figure 4E) and 100 mg/L imidacloprid (12 h: 18.86%, p = 0.003; 24 h: 30.81%, p = 0.001; Figure 4F). Similarly, mortality after dsCYP4G68 feeding was also notably enhanced relative to that with dsEGFP feeding upon exposure to 50 mg/L thiamethoxam (12 h: 25.21%, p = 0.001; 24 h: 11.39%, p = 0.003; Figure 4G) and 100 mg/L thiamethoxam (12 h: 29.35%, p = 0.001; 24 h: 8.17%, p= 0.019; Figure 4H).

3.5. Field Validation of CYP4G68 Function

3.5.1. Relative Expression of CYP4G68 in Resistant and Susceptible Strains

CYP4G68 was strongly overexpressed in the imidacloprid-resistant strains compared with in the susceptible strains (Figure 5A). In thiamethoxam-resistant and thiamethoxam-susceptible strains, the trend of CYP4G68 expression was consistent with that for imidacloprid (Figure 5B). The relative expression levels of CYP4G68 in the R#3, R#4, R#5, R#6, R#7, and R#8 strains were 1.75- (p = 0.017), 1.84- (p = 0.037), 2.55- (p = 0.005), 2.37- (p < 0.001), 3.78- (p = 0.006), and 3.95-fold (p < 0.001) higher than those in the S#2 strain. The correlation between the relative mRNA expression levels of CYP4G68 (Figure 5A,B) and resistance (imidacloprid and thiamethoxam) levels (RR values, Table 1) was positive and highly significant (p = 9.00 × 10−3, p = 7.00 × 10−3) (Figure 5C,D).

3.5.2. Further Functional Validation of CYP4G68

Knockdown of CYP4G68 prominently increased the mortality of adults of the R#8 strain relative to that of control adults upon exposure to 50 mg/L imidacloprid (12 h: 32.61%, p < 0.001; 24 h: 30.08%, p < 0.001; Figure 5E) and 100 mg/L imidacloprid (12 h: 30.88%, p < 0.001; 24 h: 34.76%, p < 0.001; Figure 5F). Furthermore, the adults fed dsCYP4G68 had a significantly higher mortality than relevant control adults upon treatment with 50 mg/L thiamethoxam (12 h: 17.23%, p = 0.004; 24 h: 11.78%, p = 0.011; Figure 5G) and 100 mg/L thiamethoxam (12 h: 20.71%, p = 0.002; 24 h: 12.72%, p = 0.004; Figure 5H).

4. Discussion

Herbivorous insects have evolved multiple resistance mechanisms to protect themselves against xenobiotics [11,58,59,60,61], particularly those used as insecticides [21,22,62,63]. In case studies, P450s-mediated resistance has been one of the most commonly reported mechanisms underlying resistance to various insecticide classes, especially neonicotinoids [21,64]. Of the P450s reported, the CYP6 subfamilies are widely proved to be associated with insecticide resistance in the whitefly [23,31,40,65]. Here, we uncovered that CYP4G68 plays an essential role in conferring imidacloprid and thiamethoxam resistance in the whitefly B. tabaci MED. Significantly, this work together with previous studies highlights the importance of the superfamily of cytochrome-P450s, mostly the CYP4 and CYP6 subfamilies, conferring neonicotinoid resistance in numerous insect pests from different orders [13,14,19,21,22,23,39,40].
In recent decades, imidacloprid and thiamethoxam, the most commonly used neonicotinoid insecticides, have been extensively applied to combat B. tabaci worldwide [9,48,50]. An extreme level of resistance to thiamethoxam in B. tabaci has been reported in Israel (RR > 1000) [66], China (RR > 1000) [46,67], and Pakistan (RR > 500) [68]. The resistance level of imidacloprid is similar to that of thiamethoxam [67,69,70]. Notably, many studies have demonstrated low-to-moderate resistance to imidacloprid and thiamethoxam in B. tabaci across China [48,50,71]. Likewise, we found that most field B. tabaci developed low-to-high levels of resistance to the two neonicotinoids (Table 1). Such resistance is mainly attributed to the overuse of neonicotinoids in collection sites. Although a high level of neonicotinoid resistance has rarely been detected in field B. tabaci in China, attention also needs to be paid to field-evolved resistance given the ever-increasing resistance to neonicotinoids worldwide. Thus, it is necessary to carry out long-term monitoring of neonicotinoid resistance in the field population. To address this resistance issue, our results provide valuable information for implementing sustainable pest control via the deliberate rotation and mixed use of insecticides with differing modes of action.
The expression patterns of P450s in special developmental stages and body parts are usually linked to their protein function. For Tribolium castaneum, CYP6BQ7 has been found to be highly expressed in the adult and larval stages, suggesting that CYP6BQ7 may be involved in the resistance of exogenous toxic substances [72]. Similarly, the spatiotemporal expression profiles of CYP6HC1 and CYP6HL1 in L. migratoria suggest that CYP6HC1 and CYP6HL1 play key roles in insecticide resistance [34]. In the current work, CYP4G68 showed higher expression in first to second instar nymphs, third instar nymphs, and adults than in other developmental stages (Figure 3A). In addition, CYP4G68 was most highly expressed in the head (Figure 3B). Some studies have implied that P450s, which are highly expressed in the brains of insects, are closely correlated with the resistance of exogenous toxic substances, including CYP6FD1 in L. migratoria [35,73], CYP6BQ9 in T. castaneum [74], CYP6BQ7 in T. castaneum [72], and CYP4G15 in D. melanogaster [75]. Therefore, the spatial-temporal expression pattern of CYP4G68 indicated that it may be associated with insecticide resistance in B. tabaci MED.
In previous studies, the essential oil of Artemisia vulgaris (EOAV) significantly induced overexpression of CYP6BQ7 in T. castaneum [72], CYP6FF1 was observably upregulated by deltamethrin in L. migratoria [35], and the expression level of CYP6FV12 notably increased after imidacloprid treatment in Bradysia odoriphaga [76]. Similarly, the expression of CYP4G68 in the S#1 strain was markedly elevated after exposure to imidacloprid (Figure 4A) and thiamethoxam (Figure 4B) in the present study. However, there is an increase in expression following a sharp decline after thiamethoxam exposure that is unlike imidacloprid treatment. The possible reasons are that 5-hydroxy-imidacloprid, 4-hydroxy-imidacloprid, and 4,5-dihydroxy-imidacloprid are the main metabolites of imidacloprid in insects, which generally have lower affinity for nicotinic acetylcholine receptors (nAChRs) and lower toxicity than imidacloprid [65,77,78,79,80]. By contrast, the main metabolite of thiamethoxam in insects is clothianidin, a higher affinity for insect nAChRs and higher activity than thiamethoxam [81,82], which may induce the increased expression of CYP4G68 again. Thus, the induction experiment revealed that CYP4G68 may be involved in imidacloprid and thiamethoxam resistance in B. tabaci MED.
Here, CYP4G68 was prominently overexpressed in the imidacloprid-resistant strains (Figure 4C and Figure 5A) in both laboratory and field populations, with the level of expression positively correlating with the resistance of these strains to imidacloprid (Figure 5C). There was a similar trend for thiamethoxam (Figure 4C and Figure 5B,D). Therefore, the overexpression of CYP4G68 was closely linked to field-evolved resistance to imidacloprid and thiamethoxam. Moreover, the mortality after dsCYP4G68 feeding dramatically increased when the adults were treated with imidacloprid (Figure 4E,F and Figure 5E,F) and thiamethoxam (Figure 4G,H and Figure 5G,H) relative to that of the control. Likewise, the overexpression of CYP4G relates to insecticide resistance [28,47,83,84,85,86], and the overexpression of CYP4C64 has been shown to be conducive to imidacloprid resistance in B. tabaci field populations [57]. CYP4C64 is overexpressed in field populations of B. tabaci, with the level of expression positively correlating with the thiamethoxam resistance level [21]. RNAi of CYP4G1 in D. melanogaster resistance strain increases the susceptibility of DDT [28]. RNAi experiments suggest that CYP4G19 and CYP4G14 are involved in pyrethroids resistance [87,88,89]. Hence, these results further illustrated that CYP4G68 is strongly correlated with imidacloprid and thiamethoxam resistance in B. tabaci MED.
We reported that METTL3 and METTL14 knockdown resulted in the downregulation of CYP4G68, which contains a predicted m6A consensus sequence in 5′UTR. METTL3 and METTL14 may regulate CYP4G68 in neonicotinoid resistance in B. tabaci [21]. Accordingly, the further study of this gene is needed to confirm the extent to which these methyltransferases act as posttranscriptional regulators associated with the xenobiotic response in insects.
Previously, the leaf dipping method has always been conducted for the B. tabaci bioassays [21,22,44,48,50], and the previous feeding method has also used for the B. tabaci bioassays and RNAi [11,53,90]. However, in our work, we improved the feeding method for all insecticide bioassays and RNAi experiments (Figure 1B). Compared with the leaf dipping method [44,50], the feeding method reported here is considered to be more labor-saving and time-saving. To our best knowledge, our bioassay method showed good reproducibility, which is more suitable for resistance bioassays. Additionally, less solution was needed than that for the old feeding method for insecticide bioassays and RNAi experiments; hence, the improved feeding method can conserve experimental materials. From this view, the improved feeding method could be recommended for use in insecticide bioassays and RNAi experiments, especially for sap-sucking pests such as whitefly.

5. Conclusions

In summary, the role of CYP4G68 in imidacloprid and thiamethoxam resistance was partially characterized in our study. This work strongly implied that CYP4G68 plays an important role in imidacloprid and thiamethoxam resistance in B. tabaci MED. Thus, CYP4G68 can be used to monitor and manage neonicotinoid resistance in field populations. Our results provide new insights into the potential roles of P450s in neonicotinoid resistance, and the results preeminently enrich studies of P450s. In addition, this work is beneficial for implementing insecticide resistance management (IRM) and integrated pest management (IPM) strategies for maintaining sustainable pest control.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/agriculture12040473/s1, Table S1: Origins of the B. tabaci strains used in this study, Table S2: Primers used in this study.

Author Contributions

Conceptualization, Y.Z., X.Y. and J.L.; methodology, X.Y. and J.L.; software, J.L.; validation, J.L., J.Y., J.H., X.W., H.X., C.Y., Y.J. and C.H.; formal analysis, J.L. and P.G.; writing—original draft preparation, J.L. and T.D.; writing—review and editing, X.Y., B.F., R.W. and S.L.; visualization, J.L. and M.H.; supervision, X.Y.; funding acquisition, Y.Z., W.X., X.Y. and R.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (Grant No. 32122073, 31972266), Beijing Natural Science Foundation (Grant No. 6212031), China Agriculture Research System (Grant No. CARS-24-C-02), The 2020 Research Program of Sanya Yazhou Bay Science and Technology City (Grant No. SKJC -2020-02-012), The Beijing Key Laboratory for Pest Control and Sustainable Cultivation of Vegetables, and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (Grant No. CAAS-ASTIP-IVFCAAS).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Gould, F.; Brown, Z.S.; Kuzma, J. Wicked Evolution: Can We Address the Sociobiological Dilemma of Pesticide Resistance? Science 2018, 360, 728–732. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. WHO. Global Plan for Insecticide Resistance Management in Malaria Vectors (GPIRM); World Health Organization: Geneva, Switzerland, 2012; p. 130. [Google Scholar]
  3. Ranson, H.; Lissenden, N. Insecticide Resistance in African Anopheles Mosquitoes: A Worsening Situation That Needs Urgent Action to Maintain Malaria Control. Trends Parasitol. 2016, 32, 187–196. [Google Scholar] [CrossRef] [PubMed]
  4. WHO. Pesticides and Their Application: For the Control of Vectors and Pests of Public Health Importance; World Health Organization: Geneva, Switzerland, 2006. [Google Scholar]
  5. Hemingway, J.; Ranson, H.; Magill, A.; Kolaczinski, J.; Fornadel, C.; Gimnig, J.; Coetzee, M.; Simard, F.; Roch, D.K.; Hinzoumbe, C.K.; et al. Averting a Malaria Disaster: Will Insecticide Resistance Derail Malaria Control? Lancet 2016, 387, 1785–1788. [Google Scholar] [CrossRef]
  6. Scott, J.G. Evolution of Resistance to Pyrethroid Insecticides in Musca Domestica. Pest Manag. Sci. 2017, 73, 716–722. [Google Scholar] [CrossRef]
  7. FAO. Guidelines on Prevention and Management of Pesticide Resistance; FAO: Rome, Italy, 2012; p. 55. [Google Scholar]
  8. Bass, C.; Denholm, I.; Williamson, M.S.; Nauen, R. The Global Status of Insect Resistance to Neonicotinoid Insecticides. Pestic. Biochem. Physiol. 2015, 121, 78–87. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  9. Horowitz, A.R.; Ghanim, M.; Roditakis, E.; Nauen, R.; Ishaaya, I. Insecticide Resistance and Its Management in Bemisia tabaci Species. J. Pest Sci. 2020, 93, 893–910. [Google Scholar] [CrossRef]
  10. Matsuda, K.; Ihara, M.; Sattelle, D.B. Neonicotinoid Insecticides: Molecular Targets, Resistance, and Toxicity. Annu. Rev. Pharmacol. 2019, 60, 241–255. [Google Scholar] [CrossRef]
  11. Du, T.; Fu, B.; Wei, X.; Yin, C.; Yang, J.; Huang, M.; Liang, J.; Gong, P.; Liu, S.; Xue, H.; et al. Knockdown of UGT352A5 Decreases the Thiamethoxam Resistance in Bemisia tabaci (Hemiptera: Gennadius). Int. J. Biol. Macromol. 2021, 186, 100–108. [Google Scholar] [CrossRef]
  12. Yang, N.; Xie, W.; Yang, X.; Wang, S.; Wu, Q.; Li, R.; Pan, H.; Liu, B.; Shi, X.; Fang, Y.; et al. Transcriptomic and Proteomic Responses of Sweetpotato Whitefly, Bemisia tabaci, to Thiamethoxam. PLoS ONE 2013, 8, e61820. [Google Scholar] [CrossRef] [Green Version]
  13. Sun, X.; Gong, Y.; Ali, S.; Hou, M. Mechanisms of Resistance to Thiamethoxam and Dinotefuran Compared to Imidacloprid in the Brown Planthopper: Roles of Cytochrome P450 Monooxygenase and a P450 Gene CYP6ER1. Pestic. Biochem. Physiol. 2018, 150, 17–26. [Google Scholar] [CrossRef]
  14. Tian, F.; Li, C.; Wang, Z.; Liu, J.; Zeng, X. Identification of Detoxification Genes in Imidacloprid-Resistant Asian Citrus Psyllid (Hemiptera: Lividae) and Their Expression Patterns under Stress of Eight Insecticides. Pest Manag. Sci. 2019, 75, 1400–1410. [Google Scholar] [CrossRef] [PubMed]
  15. Ding, Z.; Wen, Y.; Yang, B.; Zhang, Y.; Liu, S.; Liu, Z.; Han, Z. Biochemical Mechanisms of Imidacloprid Resistance in Nilaparvata lugens: Over-Expression of Cytochrome P450 CYP6AY1. Insect Biochem. Mol. Biol. 2013, 43, 1021–1027. [Google Scholar] [CrossRef] [PubMed]
  16. Zoh, M.G.; Gaude, T.; Prud’homme, S.M.; Riaz, M.A.; David, J.P.; Reynaud, S. Molecular Bases of P450-Mediated Resistance to the Neonicotinoid Insecticide Imidacloprid in the Mosquito Ae. aegypti. Aquat. Toxicol. 2021, 236, 105860. [Google Scholar] [CrossRef]
  17. Ullah, F.; Gul, H.; Tariq, K.; Desneux, N.; Gao, X.; Song, D. Functional Analysis of Cytochrome P450 Genes Linked with Acetamiprid Resistance in Melon Aphid, Aphis gossypii. Pestic. Biochem. Physiol. 2020, 170, 104687. [Google Scholar] [CrossRef] [PubMed]
  18. Chen, A.; Zhang, H.; Shan, T.; Shi, X.; Gao, X. The Overexpression of Three Cytochrome P450 Genes CYP6CY14, CYP6CY22 and CYP6UN1 Contributed to Metabolic Resistance to Dinotefuran in Melon/Cotton Aphid, Aphis gossypii Glover. Pestic. Biochem. Physiol. 2020, 167, 104601. [Google Scholar] [CrossRef] [PubMed]
  19. Tang, B.; Cheng, Y.; Li, Y.; Li, W.; Ma, Y.; Zhou, Q.; Lu, K. Adipokinetic Hormone Regulates Cytochrome P450-Mediated Imidacloprid Resistance in the Brown Planthopper, Nilaparvata lugens. Chemosphere 2020, 259, 127490. [Google Scholar] [CrossRef]
  20. Pang, R.; Xing, K.; Yuan, L.; Liang, Z.; Chen, M.; Yue, X.; Dong, Y.; Ling, Y.; He, X.; Li, X.; et al. Peroxiredoxin Alleviates the Fitness Costs of Imidacloprid Resistance in an Insect Pest of Rice. PLoS Biol. 2021, 19, e3001190. [Google Scholar] [CrossRef]
  21. Yang, X.; Wei, X.; Yang, J.; Du, T.; Yin, C.; Fu, B.; Huang, M.; Liang, J.; Gong, P.; Liu, S.; et al. Epitranscriptomic Regulation of Insecticide Resistance. Sci. Adv. 2021, 7, eabe5903. [Google Scholar] [CrossRef]
  22. Yang, X.; Deng, S.; Wei, X.; Yang, J.; Zhao, Q.; Yin, C.; Du, T.; Guo, Z.; Xia, J.; Yang, Z.; et al. MAPK-directed Activation of the Whitefly Transcription Factor CREB Leads to P450-mediated Imidacloprid Resistance. Proc. Natl. Acad. Sci. USA 2020, 117, 10246–10253. [Google Scholar] [CrossRef]
  23. Karunker, I.; Benting, J.; Lueke, B.; Ponge, T.; Nauen, R.; Roditakis, E.; Vontas, J.; Gorman, K.; Denholm, I.; Morin, S. Over-Expression of Cytochrome P450 CYP6CM1 Is Associated with High Resistance to Imidacloprid in the B and Q Biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae). Insect Biochem. Mol. Biol. 2008, 38, 634–644. [Google Scholar] [CrossRef]
  24. Nauen, R.; Bass, C.; Feyereisen, R.; Vontas, J. The Role of Cytochrome P450s in Insect Toxicology and Resistance. Annu. Rev. Entomol. 2022, 67, 105–124. [Google Scholar] [CrossRef]
  25. Scott, J.G. Cytochromes P450 and Insecticide Resistance. Insect Biochem. Mol. Biol. 1999, 29, 757–777. [Google Scholar] [CrossRef]
  26. Wang, S.; Li, B.; Zhang, D. NlCYP4G76 and NlCYP4G115 Modulate Susceptibility to Desiccation and Insecticide Penetration through Affecting Cuticular Hydrocarbon Biosynthesis in Nilaparvata lugens (Hemiptera: Delphacidae). Front. Physiol. 2019, 10, 913. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Balabanidou, V.; Kampouraki, A.; Maclean, M.; Blomquist, G.J.; Tittiger, C.; Juárez, M.P.; Mijailovsky, S.J.; Chalepakis, G.; Anthousi, A.; Lynd, A.; et al. Cytochrome P450 Associated with Insecticide Resistance Catalyzes Cuticular Hydrocarbon Production in Anopheles gambiae. Proc. Natl. Acad. Sci. USA 2016, 113, 9268–9273. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Kim, J.H.; Moreau, J.A.; Zina, J.M.; Mazgaeen, L.; Yoon, K.S.; Pittendrigh, B.R.; Clark, J.M. Identification and Interaction of Multiple Genes Resulting in DDT Resistance in the 91-R Strain of Drosophila melanogaster by RNAi Approaches. Pestic. Biochem. Physiol. 2018, 151, 90–99. [Google Scholar] [CrossRef] [PubMed]
  29. Balabanidou, V.; Grigoraki, L.; Vontas, J. Insect Cuticle: A Critical Determinant of Insecticide Resistance. Curr. Opin. Insect Sci. 2018, 27, 68–74. [Google Scholar] [CrossRef] [PubMed]
  30. Jones, C.M.; Daniels, M.; Andrews, M.; Slater, R.; Lind, R.J.; Gorman, K.; Williamson, M.S.; Denholm, I. Age-Specific Expression of a P450 Monooxygenase (CYP6CM1) Correlates with Neonicotinoid Resistance in Bemisia tabaci. Pestic. Biochem. Physiol. 2011, 101, 53–58. [Google Scholar] [CrossRef]
  31. Nauen, R.; Vontas, J.; Kaussmann, M.; Wölfel, K. Pymetrozine Is Hydroxylated by CYP6CM1, a Cytochrome P450 Conferring Neonicotinoid Resistance in Bemisia tabaci. Pest Manag. Sci. 2013, 69, 457–461. [Google Scholar] [CrossRef]
  32. Mao, K.; Zhang, X.; Ali, E.; Liao, X.; Jin, R.; Ren, Z.; Wan, H.; Li, J. Characterization of Nitenpyram Resistance in Nilaparvata lugens (Stål). Pestic. Biochem. Physiol. 2019, 157, 26–32. [Google Scholar] [CrossRef]
  33. Jin, R.; Mao, K.; Liao, X.; Xu, P.; Li, Z.; Ali, E.; Wan, H.; Li, J. Overexpression of CYP6ER1 Associated with Clothianidin Resistance in Nilaparvata lugens (Stål). Pestic. Biochem. Physiol. 2019, 154, 39–45. [Google Scholar] [CrossRef]
  34. Zhang, X.; Dong, J.; Wu, H.; Zhang, H.; Zhang, J.; Ma, E. Knockdown of Cytochrome P450 CYP6 Family Genes Increases Susceptibility to Carbamates and Pyrethroids in the Migratory Locust, Locusta migratoria. Chemosphere 2019, 223, 48–57. [Google Scholar] [CrossRef]
  35. Guo, Y.; Wu, H.; Zhang, X.; Ma, E.; Guo, Y.; Zhu, K.Y.; Zhang, J. RNA Interference of Cytochrome P450 CYP6F Subfamily Genes Affects Susceptibility to Different Insecticides in Locusta migratoria. Pest Manag. Sci. 2016, 72, 2154–2165. [Google Scholar] [CrossRef] [PubMed]
  36. le Goff, G.; Hilliou, F. Resistance evolution in Drosophila: The case of CYP6G1. Pest Manag. Sci. 2017, 73, 493–499. [Google Scholar] [CrossRef] [PubMed]
  37. Tchouakui, M.; Riveron Miranda, J.; Mugenzi, L.M.J.; Djonabaye, D.; Wondji, M.J.; Tchoupo, M.; Tchapga, W.; Njiokou, F.; Wondji, C.S. Cytochrome P450 Metabolic Resistance (CYP6P9a) to Pyrethroids Imposes a Fitness Cost in the Major African Malaria Vector Anopheles funestus. Heredity 2020, 124, 621–632. [Google Scholar] [CrossRef] [PubMed]
  38. Weedall, G.D.; Mugenzi, L.M.J.; Menze, B.D.; Tchouakui, M.; Ibrahim, S.S.; Amvongo-Adjia, N.; Irving, H.; Wondji, M.J.; Tchoupo, M.; Djouaka, R.; et al. A Cytochrome P450 Allele Confers Pyrethroid Resistance on a Major African Malaria Vector, Reducing Insecticide-Treated Bednet Efficacy. Sci. Transl. Med. 2019, 11, eaat7386. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  39. Bao, H.; Gao, H.; Zhang, Y.; Fan, D.; Fang, J.; Liu, Z. The Roles of CYP6AY1 and CYP6ER1 in Imidacloprid Resistance in the Brown Planthopper: Expression Levels and Detoxification Efficiency. Pestic. Biochem. Physiol. 2016, 129, 70–74. [Google Scholar] [CrossRef] [PubMed]
  40. Roditakis, E.; Morou, E.; Tsagkarakou, A.; Riga, M.; Nauen, R.; Paine, M.; Morin, S.; Vontas, J. Assessment of the Bemisia tabaci CYP6CM1vQ Transcript and Protein Levels in Laboratory and Field-Derived Imidacloprid-Resistant Insects and Cross-Metabolism Potential of the Recombinant Enzyme. Insect Sci. 2011, 18, 23–29. [Google Scholar] [CrossRef]
  41. Jones, D.R. Plant Viruses Transmitted by Whiteflies. Eur. J. Plant Pathol. 2003, 109, 195–219. [Google Scholar] [CrossRef]
  42. Horowitz, A.R.; Denholm, I.; Gorman, K.; Cenis, J.L.; Kontsedalov, S.; Ishaaya, I. Biotype Q of Bemisia tabaci Identified in Israel. Phytoparasitica 2003, 31, 94–98. [Google Scholar] [CrossRef]
  43. de Barro, P.J.; Liu, S.S.; Boykin, L.M.; Dinsdale, A.B. Bemisia tabaci: A Statement of Species Status. Annu. Rev. Entomol. 2011, 56, 1–19. [Google Scholar] [CrossRef]
  44. Feng, Y.; Wu, Q.; Wang, S.; Chang, X.; Xie, W.; Xu, B.; Zhang, Y. Cross-resistance study and biochemical mechanisms of thiamethoxam resistance in B-biotype Bemisia tabaci (Hemiptera: Aleyrodidae). Pest Manag. Sci. 2010, 66, 313–318. [Google Scholar] [CrossRef] [PubMed]
  45. Zhang, L.; Zhang, Y.; Zhang, W.; Wu, Q.; Xu, B.; Chu, D. Analysis of Genetic Diversity among Different Geographical Populations and Determination of Biotypes of Bemisia tabaci in China. J. Appl. Entomol. 2005, 129, 121–128. [Google Scholar] [CrossRef]
  46. Luo, C.; Jones, C.M.; Devine, G.; Zhang, F.; Denholm, I.; Gorman, K. Insecticide Resistance in Bemisia tabaci Biotype Q (Hemiptera: Aleyrodidae) from China. Crop Prot. 2010, 29, 429–434. [Google Scholar] [CrossRef]
  47. Feyereisen, R. Origin and Evolution of the CYP4G Subfamily in Insects, Cytochrome P450 Enzymes Involved in Cuticular Hydrocarbon Synthesis. Mol. Phylogenet. Evol. 2020, 143, 106695. [Google Scholar] [CrossRef]
  48. Wang, Q.; Wang, M.; Jia, Z.; Ahmat, T.; Xie, L.; Jiang, W. Resistance to Neonicotinoid Insecticides and Expression Changes of Eighteen Cytochrome P450 Genes in Field Populations of Bemisia tabaci from Xinjiang, China. Entomol. Res. 2020, 50, 205–211. [Google Scholar] [CrossRef]
  49. Xie, W.; Yang, X.; Chen, C.; Yang, Z.; Guo, L.; Wang, D.; Huang, J.; Zhang, H.; Wen, Y.; Zhao, J.; et al. The Invasive MED/Q Bemisia tabaci Genome: A Tale of Gene Loss and Gene Gain. BMC Genomics 2018, 19, 68. [Google Scholar] [CrossRef] [Green Version]
  50. Zheng, H.; Xie, W.; Fu, B.; Xiao, S.; Tan, X.; Ji, Y.; Cheng, J.; Wang, R.; Liu, B.; Yang, X.; et al. Annual Analysis of Field-Evolved Insecticide Resistance in Bemisia tabaci across China. Pest Manag. Sci. 2021, 77, 2990–3001. [Google Scholar] [CrossRef]
  51. Fu, B.; Tao, M.; Xue, H.; Jin, H.; Liu, K.; Qiu, H.; Yang, S.; Yang, X.; Gui, L.; Zhang, Y.; et al. Spinetoram Resistance Drives Interspecific Competition between Megalurothrips usitatus and Frankliniella intonsa. Pest Manag. Sci. 2022. [Google Scholar] [CrossRef]
  52. Bustin, S.A.; Benes, V.; Garson, J.A.; Hellemans, J.; Huggett, J.; Kubista, M.; Mueller, R.; Nolan, T.; Pfaffl, M.W.; Shipley, G.L.; et al. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clin. Chem. 2009, 55, 611–622. [Google Scholar] [CrossRef] [Green Version]
  53. Yang, X.; Xie, W.; Li, R.M.; Zhou, X.M.; Wang, S.L.; Wu, Q.J.; Yang, N.N.; Xia, J.X.; Yang, Z.Z.; Guo, L.T.; et al. RNA Interference-Mediated Knockdown of the Hydroxyacid-Oxoacid Transhydrogenase Gene Decreases Thiamethoxam Resistance in Adults of the Whitefly Bemisia tabaci. Sci. Rep. 2017, 7, 41201. [Google Scholar] [CrossRef]
  54. Li, R.; Xie, W.; Wang, S.; Wu, Q.; Yang, N.; Yang, X.; Pan, H.; Zhou, X.; Bai, L.; Xu, B.; et al. Reference Gene Selection for QRT-PCR Analysis in the Sweetpotato Whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae). PLoS ONE 2013, 8, e53006. [Google Scholar] [CrossRef] [PubMed]
  55. Livak, K.J.; Schmittgen, T.D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  56. Torres-Vila, L.M.; Rodríguez-Molina, M.C.; Lacasa-Plasencia, A.; Bielza-Lino, P.; Rodríguez-Del-Rincón, Á. Pyrethroid Resistance of Helicoverpa armigera in Spain: Current Status and Agroecological Perspective. Agric. Ecosyst. Environ. 2002, 93, 55–60. [Google Scholar] [CrossRef]
  57. Yang, X.; Xie, W.; Wang, S.; Wu, Q.; Pan, H.; Li, R.; Yang, N.; Liu, B.; Xu, B.; Zhou, X.; et al. Two Cytochrome P450 Genes Are Involved in Imidacloprid Resistance in Field Populations of the Whitefly, Bemisia tabaci, in China. Pestic. Biochem. Physiol. 2013, 107, 343–350. [Google Scholar] [CrossRef]
  58. Zhu, F.; Moural, T.W.; Nelson, D.R.; Palli, S.R. A Specialist Herbivore Pest Adaptation to Xenobiotics through Up-Regulation of Multiple Cytochrome P450s. Sci. Rep. 2016, 6, 20421. [Google Scholar] [CrossRef] [Green Version]
  59. Kennedy, C.; Tierney, K. Encyclopedia of Sustainability Science and Technology; Springer: New York, NY, USA, 2012. [Google Scholar]
  60. Lu, K.; Song, Y.; Zeng, R. The Role of Cytochrome P450-Mediated Detoxification in Insect Adaptation to Xenobiotics. Curr. Opin. Insect Sci. 2021, 43, 103–107. [Google Scholar] [CrossRef]
  61. Li, X.; Schuler, M.A.; Berenbaum, M.R. Molecular Mechanisms of Metabolic Resistance to Synthetic and Natural Xenobiotics. Annu. Rev. Entomol. 2007, 52, 231–253. [Google Scholar] [CrossRef]
  62. Liu, N. Insecticide Resistance in Mosquitoes: Impact, Mechanisms, and Research Directions. Annu. Rev. Entomol. 2015, 60, 537–559. [Google Scholar] [CrossRef]
  63. Zimmer, C.T.; Garrood, W.T.; Singh, K.S.; Randall, E.; Lueke, B.; Gutbrod, O.; Matthiesen, S.; Kohler, M.; Nauen, R.; Davies, T.G.E.; et al. Neofunctionalization of Duplicated P450 Genes Drives the Evolution of Insecticide Resistance in the Brown Planthopper. Curr. Biol. 2018, 28, 268–274. [Google Scholar] [CrossRef] [Green Version]
  64. Manikandan, P.; Nagini, S. Cytochrome P450 Structure, Function and Clinical Significance: A Review. Curr. Drug Targets 2017, 19, 38–54. [Google Scholar] [CrossRef]
  65. Nauen, R.; Wölfel, K.; Lueke, B.; Myridakis, A.; Tsakireli, D.; Roditakis, E.; Tsagkarakou, A.; Stephanou, E.; Vontas, J. Development of a Lateral Flow Test to Detect Metabolic Resistance in Bemisia tabaci Mediated by CYP6CM1, a Cytochrome P450 with Broad Spectrum Catalytic Efficiency. Pestic. Biochem. Physiol. 2015, 121, 3–11. [Google Scholar] [CrossRef] [PubMed]
  66. Kontsedalov, S.; Abu-Moch, F.; Lebedev, G.; Czosnek, H.; Horowitz, A.R.; Ghanim, M. Bemisia tabaci Biotype Dynamics and Resistance to Insecticides in Israel During the Years 2008–2010. J. Integr. Agr. 2012, 11, 312–320. [Google Scholar] [CrossRef]
  67. Wang, Z.; Yan, H.; Yang, Y.; Wu, Y. Biotype and Insecticide Resistance Status of the Whitefly Bemisia tabaci from China. Pest Manag. Sci. 2010, 66, 1360–1366. [Google Scholar] [CrossRef] [PubMed]
  68. Ahmad, M.; Khan, R.A. Field-Evolved Resistance of Bemisia tabaci (Hemiptera: Aleyrodidae) to Carbodiimide and Neonicotinoids in Pakistan. J. Econ. Entomol. 2017, 110, 1235–1242. [Google Scholar] [CrossRef] [PubMed]
  69. Wang, R.; Fang, Y.; Mu, C.; Qu, C.; Li, F.; Wang, Z.; Luo, C. Baseline Susceptibility and Cross-Resistance of Cycloxaprid, a Novel Cis-Nitromethylene Neonicotinoid Insecticide, in Bemisia tabaci MED from China. Crop Prot. 2018, 110, 283–287. [Google Scholar] [CrossRef]
  70. Vassiliou, V.; Emmanouilidou, M.; Perrakis, A.; Morou, E.; Vontas, J.; Tsagkarakou, A.; Roditakis, E. Insecticide Resistance in Bemisia tabaci from Cyprus. Insect Sci. 2011, 18, 30–39. [Google Scholar] [CrossRef]
  71. Chen, J.; Wang, Z.; Cao, L.; Gong, Y.; Hoffmann, A.A.; Wei, S. Toxicity of Seven Insecticides to Different Developmental Stages of the Whitefly Bemisia tabaci MED (Hemiptera: Aleyrodidae) in Multiple Field Populations of China. Ecotoxicology 2018, 27, 742–751. [Google Scholar] [CrossRef]
  72. Zhang, Y.; Gao, S.; Xue, S.; An, S.; Zhang, K. Disruption of the Cytochrome P450 CYP6BQ7 Gene Reduces Tolerance to Plant Toxicants in the Red Flour Beetle, Tribolium castaneum. Int. J. Biol. Macromol. 2021, 172, 263–269. [Google Scholar] [CrossRef]
  73. Zhang, X.; Kang, X.; Wu, H.; Silver, K.; Zhang, J.; Ma, E.; Zhu, K.Y. Transcriptome-Wide Survey, Gene Expression Profiling and Exogenous Chemical-Induced Transcriptional Responses of Cytochrome P450 Superfamily Genes in Migratory Locust (Locusta migratoria). Insect Biochem. Mol. Biol. 2018, 100, 66–77. [Google Scholar] [CrossRef]
  74. Zhu, F.; Parthasarathy, R.; Bai, H.; Woithe, K.; Kaussmann, M.; Nauen, R.; Harrison, D.A.; Palli, S.R. A Brain-Specific Cytochrome P450 Responsible for the Majority of Deltamethrin Resistance in the QTC279 Strain of Tribolium castaneum. Proc. Natl. Acad. Sci. USA 2010, 107, 8557–8562. [Google Scholar] [CrossRef] [Green Version]
  75. Chung, H.; Sztal, T.; Pasricha, S.; Sridhar, M.; Batterham, P.; Daborn, P.J. Characterization of Drosophila melanogaster Cytochrome P450 Genes. Proc. Natl. Acad. Sci. USA 2009, 106, 5731–5736. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  76. Chen, C.; Shan, T.; Liu, Y.; Wang, C.; Shi, X.; Gao, X. Identification and Functional Analysis of a Cytochrome P450 Gene Involved in Imidacloprid Resistance in Bradysia odoriphaga Yang et Zhang. Pestic. Biochem. Physiol. 2019, 153, 129–135. [Google Scholar] [CrossRef] [PubMed]
  77. Fan, Y.; Shi, X.; Gao, X. Research Progresses on the Metabolism of Neonicotinoids Imidacloprid and Thiamethoxam. Chin. J. Pesticide Sci. 2012, 14, 587–596. [Google Scholar]
  78. Suchail, S.; de Sousa, G.; Rahmani, R.; Belzunces, L.P. In Vivo Distribution and Metabolisation of 14C-Imidacloprid in Different Compartments of Apis Mellifera L. Pest Manag. Sci. 2004, 60, 1056–1062. [Google Scholar] [CrossRef]
  79. Joußen, N.; Heckel, D.G.; Haas, M.; Schuphan, I.; Schmidt, B. Metabolism of Imidacloprid and DDT by P450 CYP6G1 Expressed in Cell Cultures of Nicotiana Tabacum Suggests Detoxification of These Insecticides in Cyp6g1-Overexpressing Strains of Drosophila Melanogaster, Leading to Resistance. Pest Manag. Sci. 2008, 64, 65–73. [Google Scholar] [CrossRef]
  80. Byrne, F.J.; Castle, S.; Prabhaker, N.; Toscano, N.C. Biochemical Study of Resistance to Imidacloprid in B Biotype Bemisia tabaci from Guatemala. Pest Manag. Sci. 2003, 59, 347–352. [Google Scholar] [CrossRef] [PubMed]
  81. Benzidane, Y.; Touinsi, S.; Motte, E.; Jadas-Hécart, A.; Communal, P.Y.; Leduc, L.; Thany, S.H. Effect of Thiamethoxam on Cockroach Locomotor Activity Is Associated with Its Metabolite Clothianidin. Pest Manag. Sci. 2010, 66, 1351–1359. [Google Scholar] [CrossRef] [Green Version]
  82. Nauen, R.; Ebbinghaus-Kintscher, U.; Salgado, V.L.; Kaussmann, M. Thiamethoxam Is a Neonicotinoid Precursor Converted to Clothianidin in Insects and Plants. Pestic. Biochem. Physiol. 2003, 76, 55–69. [Google Scholar] [CrossRef]
  83. Jones, C.M.; Haji, K.A.; Khatib, B.O.; Bagi, J.; Mcha, J.; Devine, G.J.; Daley, M.; Kabula, B.; Ali, A.S.; Majambere, S.; et al. The Dynamics of Pyrethroid Resistance in Anopheles arabiensis from Zanzibar and an Assessment of the Underlying Genetic Basis. Parasit. Vectors 2013, 6, 343. [Google Scholar] [CrossRef] [Green Version]
  84. Pittendrigh, B.; Aronstein, K.; Zinkovsky, E.; Andreev, O.; Campbell, B.; Daly, J.; Trowell, S.; Ffrench, R.H. Cytochrome P450 Genes from Helicoverpa armigera: Expression in a Pyrethroid-Susceptible and-Resistant Strain. Insect Biochem. Molec. Biol. 1997, 27, 507–512. [Google Scholar] [CrossRef]
  85. Müller, P.; Chouaïbou, M.; Pignatelli, P.; Etang, J.; Walker, E.D.; Donnelly, M.J.; Simard, F.; Ranson, H. Pyrethroid Tolerance Is Associated with Elevated Expression of Antioxidants and Agricultural Practice in Anopheles arabiensis Sampled from an Area of Cotton Fields in Northern Cameroon. Mol. Ecol. 2008, 17, 1145–1155. [Google Scholar] [CrossRef] [PubMed]
  86. Pridgeon, J.W.; Zhang, L.; Liu, N. Overexpression of CYP4G19 Associated with a Pyrethroid-Resistant Strain of the German Cockroach, Blattella germanica (L.). Gene 2003, 314, 157–163. [Google Scholar] [CrossRef]
  87. Chen, N.; Pei, X.J.; Li, S.; Fan, Y.L.; Liu, T.X. Involvement of Integument-Rich CYP4G19 in Hydrocarbon Biosynthesis and Cuticular Penetration Resistance in Blattella germanica (L.). Pest Manag. Sci. 2020, 76, 215–226. [Google Scholar] [CrossRef] [PubMed]
  88. Kalsi, M.; Palli, S.R. Cap n Collar Transcription Factor Regulates Multiple Genes Coding for Proteins Involved in Insecticide Detoxification in the Red Flour Beetle, Tribolium castaneum. Insect Biochem. Mol. Biol. 2017, 90, 43–52. [Google Scholar] [CrossRef]
  89. Zhang, R.L.; Guo, G.Z.; Geng, Y.J.; Huang, D.N.; Xue, C.F. Level of CYP4G19 Expression Is Associated with Pyrethroid Resistance in Blattella germanica. J. Parasitol. Res. 2010, 2010, 517534. [Google Scholar]
  90. He, C.; Liang, J.; Liu, S.; Wang, S.; Wu, Q.; Xie, W.; Zhang, Y. Changes in the Expression of Four ABC Transporter Genes in Response to Imidacloprid in Bemisia tabaci Q (Hemiptera: Aleyrodidae). Pestic. Biochem. Physiol. 2019, 153, 136–143. [Google Scholar] [CrossRef]
Agriculture 12 00473 g001 550
Figure 1. Map of China showing the provinces (shaded gray) where field populations of B. tabaci were collected (A) and a diagram of the feeding method used in the insecticide bioassay for B. tabaci (B). S: susceptible strain; R: resistant strain. The red shading indicates the specific collection locations.
Figure 1. Map of China showing the provinces (shaded gray) where field populations of B. tabaci were collected (A) and a diagram of the feeding method used in the insecticide bioassay for B. tabaci (B). S: susceptible strain; R: resistant strain. The red shading indicates the specific collection locations.
Agriculture 12 00473 g001
Agriculture 12 00473 g002 550
Figure 2. Bioinformatic analysis of CYP4G68. (A) Genomic structure analysis of CYP4G68. (B) Five conserved domains present in CYP4G68 that are common to cytochrome P450s. (C) The full-length gene that encodes CYP4G68 in B. tabaci contains a 1764-bp ORF encoding 587 amino acid residues and the specific locations of the five conserved domains. (D) Phylogenetic tree of CYP4G68 from B. tabaci and related P450s from other insects. Bootstrap values (1000 replicates) are indicated next to the branches, and GenBank accession numbers are shown in parentheses. The red dot indicates CYP4G68 in B. tabaci.
Figure 2. Bioinformatic analysis of CYP4G68. (A) Genomic structure analysis of CYP4G68. (B) Five conserved domains present in CYP4G68 that are common to cytochrome P450s. (C) The full-length gene that encodes CYP4G68 in B. tabaci contains a 1764-bp ORF encoding 587 amino acid residues and the specific locations of the five conserved domains. (D) Phylogenetic tree of CYP4G68 from B. tabaci and related P450s from other insects. Bootstrap values (1000 replicates) are indicated next to the branches, and GenBank accession numbers are shown in parentheses. The red dot indicates CYP4G68 in B. tabaci.
Agriculture 12 00473 g002
Agriculture 12 00473 g003 550
Figure 3. Spatiotemporal expression profiles of CYP4G68 in B. tabaci. (A) Developmental stage expression patterns of CYP4G68 in B. tabaci. E: egg; N1–2: 1st and 2nd instar nymph; N3: 3rd instar nymph; N4: 4th instar nymph; A: adult. (B) Tissue-specific expression patterns of CYP4G68 in B. tabaci adults. H: head; T: thorax; A: abdomen. Values are means ± SEM (n = 3), with different lowercase letters above the bars representing significant differences (p < 0.05).
Figure 3. Spatiotemporal expression profiles of CYP4G68 in B. tabaci. (A) Developmental stage expression patterns of CYP4G68 in B. tabaci. E: egg; N1–2: 1st and 2nd instar nymph; N3: 3rd instar nymph; N4: 4th instar nymph; A: adult. (B) Tissue-specific expression patterns of CYP4G68 in B. tabaci adults. H: head; T: thorax; A: abdomen. Values are means ± SEM (n = 3), with different lowercase letters above the bars representing significant differences (p < 0.05).
Agriculture 12 00473 g003
Agriculture 12 00473 g004 550
Figure 4. Functional analysis of CYP4G68 in laboratory strains. (A,B) Relative expression of CYP4G68 in B. tabaci adults after exposure to imidacloprid (A) and thiamethoxam (B). (C) Relative expression of CYP4G68 in resistant and susceptible adults. (D) Relative expression of CYP4G68 in B. tabaci adults fed dsCYP4G68 for 48 h. (E–H) Mortality of the imidacloprid-resistant strain (R#1, E,F) and thiamethoxam-resistant strain (R#2, G,H). Values are the means ± SEM (n = 3), with asterisks representing significant differences between the treatments and the control (* p < 0.05, ** p < 0.01, *** p < 0.001).
Figure 4. Functional analysis of CYP4G68 in laboratory strains. (A,B) Relative expression of CYP4G68 in B. tabaci adults after exposure to imidacloprid (A) and thiamethoxam (B). (C) Relative expression of CYP4G68 in resistant and susceptible adults. (D) Relative expression of CYP4G68 in B. tabaci adults fed dsCYP4G68 for 48 h. (E–H) Mortality of the imidacloprid-resistant strain (R#1, E,F) and thiamethoxam-resistant strain (R#2, G,H). Values are the means ± SEM (n = 3), with asterisks representing significant differences between the treatments and the control (* p < 0.05, ** p < 0.01, *** p < 0.001).
Agriculture 12 00473 g004
Agriculture 12 00473 g005 550
Figure 5. Functional validation of CYP4G68 in field populations. (A,B) The expression profiles of CYP4G68 in resistant (imidacloprid or thiamethoxam) strains and susceptible strains. (C,D) Linear regression analysis between resistance (imidacloprid or thiamethoxam) levels and relative CYP4G68 expression. (E–H) Susceptibility of the resistant strain (R#8) of B. tabaci MED to imidacloprid (E,F) and thiamethoxam (G,H) after RNAi knockdown of CYP4G68. Values are the means ± SEM (n = 3), with asterisks representing significant differences between the treatments and the control (* p < 0.05, ** p < 0.01, *** p < 0.001).
Figure 5. Functional validation of CYP4G68 in field populations. (A,B) The expression profiles of CYP4G68 in resistant (imidacloprid or thiamethoxam) strains and susceptible strains. (C,D) Linear regression analysis between resistance (imidacloprid or thiamethoxam) levels and relative CYP4G68 expression. (E–H) Susceptibility of the resistant strain (R#8) of B. tabaci MED to imidacloprid (E,F) and thiamethoxam (G,H) after RNAi knockdown of CYP4G68. Values are the means ± SEM (n = 3), with asterisks representing significant differences between the treatments and the control (* p < 0.05, ** p < 0.01, *** p < 0.001).
Agriculture 12 00473 g005
Table
Table 1. Susceptibility of B. tabaci adults to imidacloprid and thiamethoxam.
Table 1. Susceptibility of B. tabaci adults to imidacloprid and thiamethoxam.
InsecticideStrainN aSlope (±SE)LC50 (mg/L)95% FL bDf cχ2RR dResistance Level
ImidaclopridS#17211.72 (±0.13)8.867.67–10.1832.78-Susceptible
S#27871.86(±0.12)4.322.86–6.25727.760.49Susceptible
R#15421.79(±0.17)171.35108.05–320.6938.9919.34Moderate
R#34582.47 (±0.21)44.5833.34–58.0846.765.03Low
R#46531.57 (±0.14)67.8939.04–98.53510.227.66Low
R#53882.80 (±0.22)72.3463.28–82.6531.698.16Low
R#64301.93 (±0.16)6.374.17–9.1348.730.72Susceptible
R#76711.39 (±0.10)264.47175.12–424.48514.5429.85Moderate
R#86891.37 (±0.15)335.34137.32–520.35512.0837.85High
ThiamethoxamS#15483.05 (±0.24)7.015.72–8.4345.08-Susceptible
S#24712.14 (±0.15)8.895.30–16.90311.741.27Susceptible
R#24993.43 (±0.29)114.1285.57–143.8937.6116.28Moderate
R#34861.62 (±0.12)14.7512.24–17.8131.832.1Low
R#44822.44 (±0.14)18.39.54–27.7036.992.61Low
R#56021.36 (±0.11)11.987.18–17.4547.781.71Susceptible
R#64913.05 (±0.23)53.1137.37–76.0943.357.58Low
R#78951.44 (±0.15)230.09141.94–689.6636.9132.82High
R#85291.72 (±0.22)403.59250.54–546.4642.6157.57High
a N = Number of B. tabaci used in each bioassay; b FL = fiducial limit; c df = degrees of freedom; d RR (resistance ratio) = LC50 of the sample strain/LC50 of strain S#1.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Liang, J.; Yang, J.; Hu, J.; Fu, B.; Gong, P.; Du, T.; Xue, H.; Wei, X.; Liu, S.; Huang, M.; et al. Cytpchrome P450 CYP4G68 Is Associated with Imidacloprid and Thiamethoxam Resistance in Field Whitefly, Bemisia tabaci (Hemiptera: Gennadius). Agriculture 2022, 12, 473. https://doi.org/10.3390/agriculture12040473

AMA Style

Liang J, Yang J, Hu J, Fu B, Gong P, Du T, Xue H, Wei X, Liu S, Huang M, et al. Cytpchrome P450 CYP4G68 Is Associated with Imidacloprid and Thiamethoxam Resistance in Field Whitefly, Bemisia tabaci (Hemiptera: Gennadius). Agriculture. 2022; 12(4):473. https://doi.org/10.3390/agriculture12040473

Chicago/Turabian Style

Liang, Jinjin, Jing Yang, Jinyu Hu, Buli Fu, Peipan Gong, Tianhua Du, Hu Xue, Xuegao Wei, Shaonan Liu, Mingjiao Huang, and et al. 2022. "Cytpchrome P450 CYP4G68 Is Associated with Imidacloprid and Thiamethoxam Resistance in Field Whitefly, Bemisia tabaci (Hemiptera: Gennadius)" Agriculture 12, no. 4: 473. https://doi.org/10.3390/agriculture12040473

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop