Expression profile changes of cytochrome P450 genes between thiamethoxam susceptible and resistant strains of Aphis gossypii Glover

Accepted Manuscript Expression profile changes of cytochrome P450 genes between thiamethoxam susceptible and resistant strains of Aphis gossypii Glover

Yongqiang Wu, Hongfei Xu, Yiou Pan, Xiwu Gao, Jinghui Xi, Juhong Zhang, Qingli Shang PII: DOI: Reference:

S0048-3575(18)30051-8 doi:10.1016/j.pestbp.2018.05.007 YPEST 4224

To appear in:

Pesticide Biochemistry and Physiology

Received date: Revised date: Accepted date:

9 February 2018 5 May 2018 16 May 2018

Please cite this article as: Yongqiang Wu, Hongfei Xu, Yiou Pan, Xiwu Gao, Jinghui Xi, Juhong Zhang, Qingli Shang , Expression profile changes of cytochrome P450 genes between thiamethoxam susceptible and resistant strains of Aphis gossypii Glover. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ypest(2017), doi:10.1016/j.pestbp.2018.05.007

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ACCEPTED MANUSCRIPT Expression profile changes of cytochrome P450 genes between thiamethoxam susceptible and resistant strains of Aphis gossypii Glover Yongqiang Wu1# , Hongfei Xu1# , Yiou Pan1 , Xiwu Gao2 , Jinghui Xi1 , Juhong Zhang1 ,

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Qingli Shang1*

College of Plant Science, Jilin University, Changchun 130062, PR China

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Department of Entomology, China Agricultural University, Beijing 100193, PR

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1

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#

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China

These authors are co-fist author

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* Corresponding author

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E-mail: [email protected] (QS);

ACCEPTED MANUSCRIPT Abstract Cytochrome P450 monooxygenases represent a key detoxification mechanism in neonicotinoids resistance in Aphis gossypii Glover. Synergism analysis has indicates that P450s are involved in thiamethoxam resistance. In this study, expression changes in the transcripts of P450 genes were determined in thiamethoxam-susceptible and

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thiamethoxam-resistant strains. Nine P450 genes in CYP3 clade were significantly overexpressed in the resistant strain (especially CYP6CY14, which was increased

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17.67-fold) compared with the susceptible strain. Transcripts of ecdysone

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synthesis-related P450 genes, including CYP302A1, CYP306A1, CYP307A1 and CYP315A1, were up-regulated in the resistant strain, which may accelerate molting

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hormone production. The ecdysone response genes (ecdysone receptor (EcR), ultra-spiracle (USP) and Broad-complex protein (Br-C) were overexpressed in the

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resistant strain. RNA interference (RNAi) targeting CYP6CY14 significantly increased the sensitivity of the resistant aphid to thiamethoxam. The results of the present study indicate the possible involvement of these P450 genes in thiamethoxam resistance.

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Our findings may facilitate further work to validate the roles of these P450s in thiamethoxam resistance based on heterologous expression, and show that screening

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the expression changes in P450 genes can reveal the impact of thiamethoxam on ecdysone synthesis-related P450 genes. These results are useful for understanding the

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mechanism of thiamethoxam resistance and will contribute to the management of insecticide-resistant cotton aphids in China. Keywords: Aphis gossypii, insecticide resistance, P450

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1. Introduction Thiamethoxam, a second- generation neonicotinoid insecticide that irreversibly binds to the nicotinic acetylcholine receptors (nAChRs) of cells in the nervous system, interferes with the transmission of nerve impulses in insects [1].

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Thiamethoxam is effective for controlling Aphis gossypii Glover (Hemiptera: Aphididae), which is one of the most economically important insect pests in

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agriculture [2]. The extensive and repetitive use of neonicotinoids has led to the development of resistance in the Nilaparvata lugens [3], Myzus persicae [4],

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Bemisia tabaci [5-7] and A. gossypii [8,9].

Target-site insensitivity and increased metabolic detoxification are significant

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mechanisms underlying resistance to neonicotinoid insecticides in insects. Mutation (Y151S) in α1 and α3 of nAChRs confers imidacloprid resistance in N. lugens [3]. A

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mutation (R81T) in the β1 subunit of nAChR contributes to target-site insensitivity to imidacloprid in M. persicae and A. gossypii [9,10]. In addition to target-site

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insensitivity, the metabolic mechanisms related to resistance involve cytochrome P450 monooxygenases (P450s or CYPs), representing an important biochemical

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mechanism for xenobiotic tolerance. P450s metabolize a wide variety of substrates, including both endogenous (steroids, fatty acids) and exogenous substances (plant secondary metabolites and pesticides) by catalyzing oxidation reactions [11].

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Enhanced detoxification of P450 enzymes contributes to high levels of insecticide tolerance, and overexpression of P450 gene transcripts is a general mechanism for elevating enzyme levels in insects [4, 12-14]. The overexpression of CYP6CM1 and CYP6CY3 is responsible for high levels of resistance to neonicotinoids in B. tabaci and M. persicae, respectively [15-17]. CYP6AY1, CYP6ER1, CYP6CS1, CYP4CE1 and

CYP6CW1

have

been

found

to

be

significantly

up-regulated

in

imidacloprid-resistant N. lugens [18,19], the CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1 have been functionally proven to be important in imidacloprid resistance [20]. The up-regulated CYP6CY22 and CYP6CY13 in a field-collected multiple neonicotinoids resistant A. gossypii strain (Kushima resistant strain, KR strain) could

ACCEPTED MANUSCRIPT metabolize all of the neonicotinoids including thiamethoxam [21]. Our previous research indicated that piperonyl butoxide (PBO) significantly increases the toxicity of thiamethoxam in resistant cotton aphids (ThR strain), indicating that up-regulation of P450s may be an important mechanism for thiamethoxam resistance [22]. Whereas, the ThR strain didn’t show cross resistance to the other neonicotinoids including

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imidacloprid, sulfoxaflor and clothianidin [22], which illustrating the mechanisms of resistance to neonicotinoids are likely to be different between the ThR and the KR

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strain. In the present study, expression of P450 genes [including the Halloween genes that function in ecdysone biosynthesis: CYP302A1 (Disembodied, Dib), CYP306A1

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(Phantom, Phm), CYP307A1 (Spook, Spo), CYP314A1 (Shade, Shd) and CYP315A1 (Shadow, Sad) [23]] was determined in both susceptible and resistant strains via

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reverse transcription quantitative real-time PCR (RT-qPCR) based on our transcriptome database (https://www.ncbi.nlm.nih.gov/sra/?term=SRX683625 ). This

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strategy potentially allows the identification of most putative P450 genes related to the response to thiamethoxam stress. The involvement of overexpressed P450s was

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examined through RNA interference (RNAi). Our data provide preliminary insight into the changes in the gene expression of the P450s and their involvement in

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thiamethoxam resistance. The results may facilitate further stud ies on the functions of P450s in insecticide resistance.

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2. Materials and methods 2.1. Insects

Two cotton aphid (A. gossypii) strains were used in this study. One strain was resistant to thiamethoxam (ThR), and the other was susceptible (SS) [22, 24]. The SS strain was collected in 2008 from Jilin Province, where limited insecticides have been applied. This strain has been maintained without any insecticide treatment since its collection. The ThR strain was established from the SS population via consecutive selection with increasing concentrations of thiamethoxam (LC30 ) via the leaf-dipping method. Both the resistant and susceptible strains were reared on cotton plants (Gossypium hirsutum (L.)) in the laboratory at 20 -23 °C, with a photoperiod of 16 h:8 h (light:dark).

ACCEPTED MANUSCRIPT 2.2. Chemicals Thiamethoxam (25% WDG) was purchased from Syngenta (Switzerland). All other chemicals and solvents were reagent grade. The PrimeScriptT M First-Strand cDNA Synthesis kit, SYBR® Premix Ex Taq™ II (Tli RNaseH Plus), oligo(dT)18 , Ex Taq DNA polymerase, RNase- free DNase I, RNase Inhibitor, DNA Marker DL2000 and agarose were purchased from Takara (Dalian, China). The pGEM- T vector and the

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T7 RiboMAX™ Express RNAi System were purchased from Promega (USA).

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2.3. Conserved domains of detoxification and ecdysone synthesis in P450 proteins

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Multiple alignments of representative overexpressed protein sequences among the A. gossypii P450s were performed using ClustalW, and structural domains such as P450

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signature motif were detected based on comparison with other sequences, the primary structures of which have been characterized.

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2.4. Total RNA isolation and cDNA synthesis

Total RNA was extracted from adult apterous ThR and SS aphids using TRIzol (Invitrogen, USA) according to the manufacturer’s instructions and then treated with

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RNase- free DNase I (Takara, Japan). The RNA samples were quantified by measuring the absorbance at 260 nm, and the quality was checked via agarose gel

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electrophoresis. First-strand cDNA was synthesized from the total RNA using the PrimeScriptT M First-Strand cDNA Synthesis kit (Takara, Japan) with oligo(dT) 18 as a primer.

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2.5. Quantitative RT-PCR and data analysis Quantitative real-time PCR was performed on the ABI 7500 platform (Applied Biosystems) using SYBR ® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, Japan). Gene-specific primers for real-time PCR (Table S1) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The thermal cycling protocol included an initial denaturation at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 34 s. The fluorescence signal was measured at the end of each extension step at 60 °C. After amplification, one dissociation step cycle of 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s was performed to confirm that only the specific products were amplified. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

ACCEPTED MANUSCRIPT and elongation factor 1-alpha (EF1a) housekeeping genes were used as internal reference genes for A. gossypii. Relative gene expression was calculated via the 2−ΔΔCT method [25]. The experiment included three independent biological replicates for each strain. Significant differences were analyzed using GraphPad InStat3 statistical software (GraphPad Software, 2000). 2.6. Rearing on artificial diet and dsRNA feeding

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Based on P450 sequences (Supplementary data 1) and predicted possible interference

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sites obtained from online prediction software (http://www.dkfz.de/signaling/e-rnai3/), we designed specific primers using DNAMAN 6.0 software. The gene fragments were

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amplified from cDNA and then cloned into pGEM-T (Promega, USA). The purified plasmids served as templates for RNA synthesis using the T7 RiboMAX™ Express

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RNAi System (Promega, USA). ECFP dsRNA was employed as the control and was synthesized under the same conditions as the primers (Table S1). The artificial diet

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and the rearing method used in this study were reported previously [26,27]. The diet was prepared in DEPC-treated water to ensure the absence of RNase activity. For the

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dsRNA feeding experiments, dsRNA was added to the artificial diet at a 150 ng/μL concentration. An artificial diet containing dsRNA-ECFP was employed as a control.

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Apterous thiamethoxam-resistant adult A. gossypii were transferred onto the artificial diet for rearing. To analyze the efficiency of dsRNA knockdown of P450 expression, the aphids were fed an artificial diet containing dsRNA (100 ng/μL) for 48 h, and

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samples were then collected for RT-qPCR. To assess the sensitivity of the cotton aphids to thiamethoxam after P450 RNAi, eighty resistant adult aphids were transferred to the artificial diet containing thiamethoxam (1.0mg/L) mixed with dsRNA-CYP6CY14/dsRNA-ECFP (100 ng/μL); dsRNA-ECFP was used as the control. Cotton aphid mortality was recorded after 48 h. Each treatment included three replicates, with 80 aphids in each replication.

3. Results 3.1. Dynamic changes in P450 gene expression in the SS and ThR strains.

ACCEPTED MANUSCRIPT As reported by Wei et al. (2017) [22], PBO increased thiamethoxam toxicity by 3.00-fold in the ThR strain, which displayed 13.79-fold greater resistance to thiamethoxam than the SS strain (Table 1, Wei et al., 2017) [22]. This finding indicated that the cytochrome P450s are major detoxification enzymes involved in thiamethoxam resistance. The quantitative real-time PCR results indicated that the

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mRNA transcription levels of CYP6CY14, CYP6DC1, CYP6CZ1, CYP6DD1 CYP6CY9, CYP6CY13-2, CYP6CY5, CYP6CY18 and CYP6CY12 (in CYP3 clade)

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were up-regulated 17.67-, 4.27-, 2.05-, 1.69-, 1.54-, 1.47-, 1.46-, 1.41- and 1.18- fold respectively, in the resistant strain, compared with the susceptible strain (Figure 1).

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The transcripts of ecdysone synthesis-related P450 genes (Figure 2), including CYP302A1, CYP306A1, CYP307A1 and CYP315A1 were increased to 1.67-, 1.78-,

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1.71- and 1.28- fold in the resistant strain, respectively (Figure 1). In contrast, expression of CYP18A1, which metabolizes 20 hydroxyecdysone (20-E) to

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20,26-dihydroxyecdysone, decreased to 0.87- fold in the resistant strain compared with that in the susceptible strain. Expression of CYP4G51 (in CYP4 clade) and

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CYP305E1 (in the Mito clade) was up re-gulated 2.26- and 2.82- fold, respectively in thiamethoxam-resistant aphids (Figure 1). Our results indicated that the ecdysone

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response genes, ecdysone receptor (EcR), ultra-spiracle (USP) and Broad-complex protein (Br-C) were overexpressed in the resistant strain (Figure 3).

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3.2. Conserved domains of overexpressed P450 proteins Alignment of the amino acid sequences of CYP6CY14, CYP6DC1, CYP6CZ1, CYP6CY16 and CYP6CY9 (Figure 4), Halloween genes (CYP306A1, CYP307A1, CYP314A1) and CYP18A1 (Figure 5) indicated that these P450s contain conserved motifs. The conserved structural motifs of the P450 enzymes are shown in the alignment, including helix-C (WxxxR; a heme- interacting region), helix- I (AGxxT; a putative oxygen-binding pocket), helix-K (ExxR; a putative hydrogen binding sequence), the aromatic region (known also as ‘PERF’; PxxFxPExF) and the heme-binding region (PFxxGxRxCxG) [28]. 3.3. Knockdown of P450 genes increases thiamethoxam toxicity

ACCEPTED MANUSCRIPT P450s in the CYP3 and CYP4 clades usually account for xenobiotic tolerance [28]. Expression of CYP4G51 (in CYP4 clade) was increased 2.26- fold in the resistant aphids; however, its function is associated with hydrocarbon production in aphids [29]. Hence, five P450 genes from CYP3 clade (CYP6CY14, CYP6DC1, CYP6CZ1, CYP6DD1 and CYP6CY9) showing greater than 1.50- fold up-regulation were selected

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to determine their relationship with thiamethoxam resistance, and CYP6CY14 was successfully knocked down by oral- feeding of corresponding dsRNA. The successful

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RNAi reduced the expression levels of CYP6CY14 to 0.42-fold in dsRNA-CYP6CY14 treated (100 ng/μL) aphids after 48 h, compared with control expression levels

dsRNA-CYP6CY14- fed aphids, respectively,

in the presence of 1.0

mg/L

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thiamethoxam (Figure 6).

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(Figure 6). Mortality significantly increased, from 26.43% in the control to 59.86% in

4. Discussion

Thiamethoxam has been used as an alternative neonicotinoid insecticide for

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controlling cotton aphids, and no cases of serious resistance to thiamethoxam have been reported in cotton aphids to date. Wei et al. (2017) reported that PBO increased

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thiamethoxam toxicity by 3.00-fold in the ThR strain, which displayed 13.79- fold greater resistance to thiamethoxam than the SS strain (Table 1) [22]. This finding

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indicated that cytochrome P450s are major detoxification enzymes involved in thiamethoxam resistance. P450s constitute important metabolic systems involved in the detoxification of xenobiotics (pesticides, plant toxins), in addition to regulating the levels of endogenous compounds (hormones, fatty acids) [11]. The enhanced activities of cytochrome P450 monooxygenases conferring neonicotinoid insecticide resistance have been thoroughly documented in insects such as B. tabaci [15,16], M. persicae [4,17] and N. lugens [18,19]. To obtain a comprehensive view of the expression changes in P450 metabolic factors in the development of resistance, thirty five P450 genes were examined via RT-qPCR. Our results indicated that the mRNA transcription levels of the P450 genes in CYP3 clade, including CYP6CY14,

ACCEPTED MANUSCRIPT CYP6DC1, CYP6CZ1, CYP6DD1 CYP6CY9, CYP6CY13-2, CYP6CY5, CYP6CY18 and CYP6CY12, were up-regulated in the resistant strain, compared with those in the susceptible strain (Figure 1). This finding is consistent with reports that the P450s of the CYP3 clade are involved in the oxidative detoxification of plant secondary metabolites and synthetic insecticides [11,12,30]. Over-expression of CYP6CY13-2 in

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ThR strain of this study was consistent with the report of Hirata et al. (2017) [21], in which CYP6CY13 was up-regulated in neonicotinoids resistant strains (KR strain) and

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could metabolize thiamethoxam. On the other sides, significant overexpression of CYP6CY14 (17.67- fold) (Figure 1) and other P450 genes of CYP3 clade were

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identified in ThR strain instead of the KR strain [21], which indicating the mechanisms of resistance to thiamethoxam are likely to be different between the ThR

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and the KR strain.

In addition to the P450s in CYP3 clade, Halloween genes (CYP302A1, CYP306A1,

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CYP307A1 and CYP315A1) in the ecdysone biosynthesis pathway were also overexpressed in the resistant strain (Figure 1, Figure 2). In contrast, the transcript of

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CYP18A1, which encodes an enzyme that metabolizes 20 hydroxyecdysone to 20, 26-dihydroxyecdysone, showed a reduced level in the resistant strain. The mRNA

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level of CYP4G51, which is involved in cuticular hydrocarbon production in aphids [29] was significantly up-regulated in the resistant strain (Figure 1). This up-regulation may be caused by overexpression of ecdysone due to accelerated

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functions in its biosynthesis pathway. The active form of the molting hormone in insects consists of 20-hydroxyecdysone (20E) and ecdysone, the immediate precursor of 20E [31]. 20E binds to a heterodimer of the ecdysone receptor/ultra-spiracle protein (EcR-USP) in the nuclear receptor complex. With the assistance of the chaperone complex, EcR-USP binds to 20E response elements (EcRE) present in the promoter regions of 20E primary response genes, including Br-C and E74 [31,32]. Our results indicated that the EcR, USP and Br-C genes were overexpressed in the resistant strain (Figure 3). These findings illustrate that thiamethoxam may modulate molting hormone production in cotton aphids.

ACCEPTED MANUSCRIPT The suppression of P450 gene transcripts by RNAi, resulting in reduced resistance, is widely employed to study the roles of P450s in insecticide resistance. For example, ingestion of dsRNA was shown to successfully silence CYP4Q3 in resistant Leptinotarsa decemlineata, increasing its susceptibility to imidacloprid [33]. Additionally, knockdown of CYP6BG1 by RNAi has been observed to increase the

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toxicity of chlorantraniliprole in P. xylostella [34]. RNAi via the dsRNA oral feeding method [26,27] has also been employed to clarify the specific influence of the P450

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genes on the susceptibility to thiamethoxam in A. gossypii. Among the over-expressed P450 genes in CYP3 clade, successful suppression of the transcriptional levels of

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CYP6CY14 through RNAi can significantly increase thiamethoxam susceptibility in resistant cotton aphids (Figure 6). This finding indicates that the enzymes encoded by

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the CYP6CY14 gene may contribute to the detoxification of thiamethoxam in A. gossypii. Due to the limitation of RNAi (such as CYP6CY9 and CYP6CZ1 were not

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able to silence by dsRNA in our preliminary experiment), the other over-expressed P450 genes were not able to testify their associations with resistance in this study.

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The results of present study showed that the P450 genes of CYP3 clade may play main roles in thiamethoxam resistance. Our finding may facilitate further

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heterologous expression analyses of P450 functions in thiamethoxam resistance. The present study also revealed the changes in the expression of P450 genes in the ecdysone biosynthesis pathway in the resistant strain. These results will be useful for

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understanding the resistance mechanism of thiamethoxam and for the management of insecticide-resistant cotton aphids in China.

Acknowledgements

This work was sponsored by the National Natural Science Foundation of China (31772188, 31301728).

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biological actions. Vitam. Horm. 60 (2000) 1-73. [33] E. Kaplanoglu, P. Chapman, I.M. Scott, C. Donly, Overexpression of a

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cytochrome P450 and a UDP-glycosyltransferase is associated with imidacloprid resistance in the Colorado potato beetle, Leptinotarsa decemlineata. Sci Rep. 7

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(2017) 1762.

[34] X. Li, R. Li, B. Zhu, X. Gao, P. Liang, Overexpression of cytochrome P450 CYP6BG1 may contribute to chlorantraniliprole resistance in Plutella xylostella

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(L.). Pest Manag Sci. (2017) doi: 10.1002/ps.4816.

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Figure and Table Legends Table 1. Synergistic effects of PBO on the toxicity of thiamethoxam in the SS and ThR strains *Data taken from Wei et al. (2017). [21] Probit model fitted using POLO-PC (LeOra Software, 1987)

b

Confidence limits.

c

SR (synergism ratio) = LC50 of thiamethoxam /LC50 of thiamethoxam with synergist.

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a

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PBO: final concentration of 80-mg L-1 .

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Figure 1. Transcription levels of P450s in the SS and ThR strains determined via real-time PCR.

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GAPDH and EF1a were employed as internal reference genes. Error bars indicate 95% confidence intervals (n=3). * Significant difference as determined by Student’s t-test (P < 0.05). **Significant difference as determined by Student’s t-test (P <

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0.01).

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Figure 2. P450 genes in the ecdysone synthesis pathway. Halloween genes, including CYP302A1 (Disembodied, Dib), CYP306A1 (Phantom,

Sad).

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Phm), CYP307A1 (Spook, Spo), CYP314A1 (Shade, Shd), and CYP315A1 (Shadow,

Figure 3. Transcription levels of ecdysone response genes (EcR, USP, Br-C and E74) in the SS and ThR strains as determined via real time PCR. GAPDH and EF1a were used as internal reference genes. Error bars indicate 95% confidence intervals (n=3). * Significant difference as determined by Student’s t-test (P < 0.05). **Significant difference as determined by Student’s t-test (P < 0.01). Figure. 4. Alignme nt of amino acid sequences of five P450 genes of CYP3 clade from A. gossypii. Conserved domains common to the cytochrome P450s are boxed; these domains include the helix-C motif (WxxxR), the oxygen-binding motif (helix I)

ACCEPTED MANUSCRIPT ([A/G]GX[E/D]T[T/S]), the helix K motif (EXXRXXP), the heme-binding motif (PFXXGXXXCXG) and the conserved Meander motif (PXXFXP). Red arrows indicate the conservative amino acid for catalytic activity.

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Figure. 5. Alignment of amino acid sequences of ecdysone synthesis-related P450 genes from A. gossypii. Conserved domains include the helix-C motif (WxxxR), the oxygen-binding motif (helix I) ([A/G]GX[E/D]T[T/S]), the helix K motif (EXXRXXP), the heme-binding motif (PFXXGXXXCXG) and the conserved Meander motif (PXXFXP). Red arrows indicate the conservative amino acid for catalytic activity.

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Figure. 6. dsRNA-mediated suppression of CYP6CY14 transcripts and its effect

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on thiamethoxam toxicity in A. gossypii.

(A) dsRNA- mediated suppression of CYP6CY14 transcripts in resistant aphids fed an

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artificial diet with dsRNA (100 ng/μL). (B) Mean mortality ± SE (n=3) of resistant cotton aphids after they were fed thiamethoxam (1.0 mg/L) and a dsRNA mixture

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(100 ng/μL of dsRNA) for 48 h. Each treatment included three replicates, and eighty adults of resistant aphids were used in each replication. Error bars indicate 95% confidence intervals (n=3). * Significant difference as determined by Student’s t- test

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(P < 0.05). **Significant difference as determined by Student’s t-test (P < 0.01).

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Table S1. Primers used in the experiments. dsRNA, double-stranded RNA; EF1a, elongation factor 1-alpha; F, forward; GAPDH,

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glyceraldehyde-3-phosphate dehydrogenase; ORF, open reading frame; R, reverse. Lower-case letters indicate the T7 RNA polymerase promoter. Supporting information legends Supplementary data 1. GeneBank accession numbers of A. gossypii P450 genes.

Graphical Abstract

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Table 1. Synergistic effects of PBO on the toxicity of thiamethoxam in the SS and ThR strains *

Thiamethoxam / Strain

thiamethoxam

Fit of probit line a

LC50 ( 95% CLb) (mg -1

SRd

(at 95%

c

L )

CL )

Slope±SE

χ

df

+synergist Thiamethoxam

P

1.96±0.16

13.59

16

0.27

1.13 (0.97 -1.33)

-

Thiamethoxam+PBO

2.48±0.22

17.24

16

0.15

1.19 (1.02-1.40)

0.95

Thiamethoxam

3.20±0.35

25.31

16

0.10

15.58 (13.02-18.49)

-

Thiamethoxam+PBO

2.56±0.22

11.26

16

0.49

5.19 (4.50-5.96)

3.00

2

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SS

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ThR

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*Data taken from Wei et al. (2017).21 Probit model fitted using POLO-PC (LeOra Software, 1987)

b

Confidence limits.

c

SR (synergism ratio) = LC50 of thiamethoxam /LC50 of thiamethoxam with synergist.

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a

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PBO: final concentrations were 80-mg L-1.

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Nine P450 genes, especially CYP6CY14, in Clade3 were significantly overexpressed in resistant strain.



The transcripts of ecdysone synthesis-related P450 genes were up-regulated in resistant strain.

Ecdysone response genes, EcR, USP and Br-C were overexpressed in resistant

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

strain.

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Suppression of CYP6CY14 increased the sensitivity of resistant aphids to

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thiamethoxam.

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

Graphics Abstract

Figure 1

Figure 2

Figure 3

Figure 4

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Figure 6