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Metabolic imidacloprid resistance in the brown planthopper, Nilaparvata lugens, relies on multiple P450 enzymes
Insect Biochemistry and Molecular Biology 79 (2016) 50e56
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Metabolic imidacloprid resistance in the brown planthopper, Nilaparvata lugens, relies on multiple P450 enzymes Yixi Zhang, Yuanxue Yang, Huahua Sun, Zewen Liu* Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Weigang 1, Nanjing 210095, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 August 2016 Received in revised form 23 October 2016 Accepted 24 October 2016 Available online 26 October 2016
Target insensitivity contributing to imidacloprid resistance in Nilaparvata lugens has been reported to occur either through point mutations or quantitative change in nicotinic acetylcholine receptors (nAChRs). However, the metabolic resistance, especially the enhanced detoxification by P450 enzymes, is the major mechanism in fields. From one field-originated N. lugens population, an imidacloprid resistant strain G25 and a susceptible counterpart S25 were obtained to analyze putative roles of P450s in imidacloprid resistance. Compared to S25, over-expression of twelve P450 genes was observed in G25, with ratios above 5.0-fold for CYP6AY1, CYP6ER1, CYP6CS1, CYP6CW1, CYP4CE1 and CYP425B1. RNAi against these genes in vivo and recombinant tests on the corresponding proteins in vitro revealed that four P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, played important roles in imidacloprid resistance. The importance of the four P450s was not equal at different stages of resistance development based on their over-expression levels, among which CYP6ER1 was important at all stages, and that the others might only contribute at certain stages. The results indicated that, to completely reflect roles of P450s in insecticide resistances, their over-expression in resistant individuals, expression changes at the stages of resistance development, and catalytic activities against insecticides should be considered. In this study, multiple P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, have proven to be important in imidacloprid resistance. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Nilaparvata lugens Imidacloprid Metabolic resistance P450s
1. Introduction Cytochrome P450 monooxygenase is a multi-gene superfamily that has critical functions in the detoxification of xenobiotics including drugs, pesticides and plant toxins, as well as in the metabolism of endogenous compounds (Scott, 1999; Feyereisen, 2012). In insects, P450 monooxygenase-mediated detoxification is one of the major mechanisms of insecticide resistance. The whole genome sequence analysis and examining the expression of multiple genes simultaneously revealed that the over-expression of multiple P450 genes in insecticide resistant strains is a common phenomenon observed in many insect species (Liu et al., 2015). For example, the over-expression of two P450 genes CYP6G1 and CYP12D1 were found to be associated with DDT resistance in Drosophila melanogaster (Festucci-Buselli et al., 2005). Significant over-expression of multiple CYP genes (CYP4M6, CYP4M7,
* Corresponding author. E-mail address: [email protected] (Z. Liu). http://dx.doi.org/10.1016/j.ibmb.2016.10.009 0965-1748/© 2016 Elsevier Ltd. All rights reserved.
CYP6AE11, CYP9A12, CYP332A1 and CYP337B1) was also found in different deltamethrin-resistant strains of Helicoverpa armigera (Brun-Barale et al., 2010). In permethrin resistant house flies, Musca domestica, co-upregulation of three P450 genes, CYP4D4v2, CYP4G2, and CYP6A38, was reported (Zhu et al., 2008). The gene expression and induction by permethrin in different mosquito (Culex quinquefasciatus) populations indicated that multiple P450 genes were up-regulated in the case of insecticide resistance (Liu et al., 2011). Furthermore, the comparison of P450 expression profiles between permethrin susceptible and resistant mosquitos in the whole genome scale implied that not only multiple P450 genes were involved in insecticide resistance but up- or down-regulation of P450 genes might also be co-responsible for insecticide detoxification (Yang and Liu, 2011). Imidacloprid, the first commercial neonicotinoid insecticide, is used as a major insecticide to control many insect pests. Imidacloprid resistance has been reported in several species (Gorman et al., 2008; Wen et al., 2009; Matsumura et al., 2014; Garrood et al., 2016). Synergism studies, gene expression and enzyme activity assays proved that the enhanced detoxification of P450s was
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the major metabolic mechanism of imidacloprid resistance (Wen et al., 2009; Garrood et al., 2016). P450s were supposed to play important roles in imidacloprid resistance in several insect species, including D. melanogaster (CYP6G1), M. domestica (CYP6D1), Bemisia tabaci (CYP6CM1), C. pipiens pallens (CYP6F1), C. quinquefasciatus (CYP9M10), and Nilaparvata lugens (CYP6ER1, CYP6AY1) (Daborn et al., 2001; Liu and Scott, 1998; Karunker et al., 2008; Gong et al., 2005; Hardstone et al., 2010; Bass et al., 2011; Ding et al., 2013). Until now, however, these studies mainly focused on single P450 gene, and only three insect P450s, CYP6G1 (D. melanogaster), CYP6CM1 (B. tabaci) and CYP6AY1 (N. lugens) have been confirmed in the imidacloprid metabolism (Daborn et al., 2001; Karunker et al., 2008; Ding et al., 2013). The brown planthopper, Nilaparvata lugens, is a major rice pest throughout Asia which causes a dramatic reduction in rice yield and great economic losses (Gorman et al., 2008; Sogawa and Cheng, 1979; Wang et al., 2008). Previous studies showed that the target insensitivity caused by key amino acid substitution or quantitative change of nicotinic acetylcholine receptors (nAChRs) led to high resistance in N. lugens against imidacloprid (Liu et al., 2005; Zhang et al., 2015). However, the metabolic resistance, especially the enhanced detoxification of P450s, might play a superior role in the field population of N. lugens (Wen et al., 2009; Bass et al., 2011; Ding et al., 2013; Bao et al., 2016). The genome sequencing of N. lugens provided an opportunity to identify potential resistance-related P450 genes at the whole genome scale (Xue et al., 2014; Zhang et al., 2016). In the present study, two strains were obtained from one field-originated population by the successive selection for 25 generations with or without imidacloprid (Zhang et al., 2015). Comparing the expression profiles of fifty-four P450 genes in these two strains, four genes were supposed to importantly contribute to imidacloprid resistance, and their roles in different stages of resistance development were analyzed. 2. Materials and methods
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2.3. qRT-PCR validation of P450 genes expression Fifty-four P450 gene sequences were retrieved from the N. lugens genome (GenBank accession number AOSB00000000.1, BioProject PRJNA177647, provided by Prof. Zhang Chuanxi from Zhejiang University, Hangzhou, China; Xue et al., 2014). Total RNA was extracted using TRlzol® reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription to cDNA was performed using the PrimeScript™ RT reagent Kit and gDNA Eraser (Takara, Tokyo, Japan). The mRNA levels of the P450 genes were measured by quantitative realtime PCR (qRT-PCR) with the SYBR® Premix Ex Taq™ (Takara) in a 20 mL total reaction volume containing 200 ng of cDNA, 10 mL reaction buffer (SYBR Premix Ex Taq), 0.4 mL each of forward and reverse gene specific primer (Zhang et al., 2016) and 0.4 mL 50 Rox Reference Dye II, following the manufacturer instruction. Every qRT-PCR experiment was performed with at least three biological replicates. Gene expression difference was calculated with the 2DDCT method. Two reference genes (b-actin and GAPDH) were validated experimentally for each generation and treatment, and used for normalization according to the strategy described previously (Vandesompele et al., 2002). 2.4. RNA interference RNAi was performed as previously described (Liu et al., 2010). The dsRNAs of the targeted P450 genes and the bacterial Lac-Z (GenBank accession number: AJ308295), as a control, were synthesized using the T7 Ribomax Express RNAi System (Promega, Madison, WI, USA) and precipitated with isopropanol. Primers used to synthesize the dsRNA were summarized in Table S1. The dsRNA was resuspended in ultra-pure water, quantified spectrophotometrically at 260 nm and verified with agarose gel electrophoresis for its purity and integrity. It was then kept at 80 C for the subsequent application. 50 nL of dsRNA was microinjected at the conjunctive between prothorax and mesothorax of the 3rd instar N. lugens.
2.1. Insect 2.5. Functional expression of P450s and insecticide metabolism The susceptible strain (Sus) of N. lugens was a laboratory strain originally collected from Nueva Ecija (Philippines) in 2004 and reared in a laboratory without contacting any insecticide for 12 years. A N. lugens field population was collected from a paddy field in Nueva Ecija (Philippines) in August 2011, and was separated into two strains (Zhang et al., 2015). One strain (G25) was continuously selected by imidacloprid in the laboratory for 25 generations, and the counterpart (S25) was reared as a laboratory strain without contacting any insecticide. During the selection procedure, 3000e5000 individuals were reproduced in each generation. Insects were kept indoor with rice seedlings at 25 ± 1 C, 70e80% relative humidity and 16-h light/8-h dark photoperiod. 2.2. Bioassay and synergistic experiment Bioassay was performed according to the rice seedling dip method (Wang et al., 2008). Rice seedlings were immersed in imidacloprid solution at a series of concentrations for 15s. After airdrying, the seedlings were placed in plastic cups with 1.5% agar to maintain the moist conditions. In the bioassay, thirty insects (the 5th instar nymphs) were inoculated to the seedlings treated with imidacloprid as mentioned above in three replications and the LC50 of imidacloprid was determine. In the synergism experiment, 2 mg synergist (triphenyl phosphate, diethyl maleate or piperonyl butoxide) in 0.08 mL acetone was topically delivered onto the prothorax notum of the test insects (the 5th instar nymphs) 1 h before the insecticide application (Tang et al., 2010).
P450 genes and the house fly P450 reductase cDNA sequences were subcloned into the expression vector pCWori at NdeI and HindIII or SpeI sites. The primers for the vector construction were summarized in Table S2. The plasmids were transformed into E. coli DH5a and transformants were selected with ampicillin. The cells were treated with lysozyme solution and the resulting membrane pellet was suspended in sodium phosphate buffer for enzymatic characterization (Ding et al., 2013). Enzymatic activities of the recombinant P450s were determined as described previously (Ding et al., 2013; Bao et al., 2016). In brief, the reaction mixture was contained 0.2 mmol P450 protein, 2 mmol house fly P450 reductase, NADPH-regenerating system and 3 mg/L imidacloprid. Instead of the recombinant P450s, the membrane fraction of untransformed E. coli cells was used as control. 200 mL solution was extracted from the reaction mixture at an interval of 20 min, and the imidacloprid concentration was determined by reversed-phase HPLC on a 5-mm SupelCosil LC18-DB column (4.6 cm 250 cm, Supelco) using a solvent mixture composed of 20% CH3CN in H2O, which was isocratic for 5 min, followed by a linear gradient up to 80% CH3CN in H2O for over 40 min (flow rate 1 mL/min). Imidacloprid elution was monitored by measuring the absorption at 270 nm using Spectroflow UV/Vis Detector (Kratos Analytical Instruments, USA). The main metabolites of imidacloprid by recombinant protein were identified by UPLC-MS (Ding et al., 2013; Yang et al., 2016). For enzymatic kinetic analysis, a series of concentrations (0.1e5 mg/L) were used. Rates of substrate turnover
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were plotted versus substrate concentration. Km and Kcat were determined in 20 min fitting to the MichaeliseMenten equation using non-linear regression. 2.6. Statistical analyses Data analyses were carried out using Data Processing System (DPS) software v9.50 (Tang and Zhang, 2013). Significant differences were analyzed by one-way ANOVA with at least three repeats. Multiple comparisons between the groups were performed using Student-Newman-Keuls (S-N-K) method. The level of significance for results was set at p < 0.05. 3. Results 3.1. Bioassays and synergism analyses A resistant strain G25 and a relatively susceptible strain S25 of N. lugens were obtained from one field-originated population from Nueva Ecija (Philippines) after the continuous selection for 25 generations with or without imidacloprid (Zhang et al., 2015). Comparing with the susceptible strain Sus (LC50 ¼ 0.14 mg/L), the resistance ratio (RR) in G25 was 434-fold, whereas the RR in S25 was 2 being relatively susceptible (Table 1). The three important synergists tested here did not show any effects on imidacloprid in the Sus and S25 strains. However, PBO (piperonyl butoxide) and TPP (triphenyl phosphate) significantly synergized imidacloprid in G25 with the synergism ratio (SR) of 5.88- and 1.27-fold, respectively, indicating that P450s played dominant roles in the metabolic imidacloprid resistance (Table 1). 3.2. Comparison of the expression levels of P450 genes between G25 and S25 To investigate the potential P450 genes associated with imidacloprid resistance, the expression levels of all P450 genes identified from N. lugens genome database were determined in S25 and G25 by qRT-PCR. Compared to S25, the expression levels of fifteen P450 genes were significantly different in G25, including seven in CYP4 Clade (Fig. 1A), six in CYP3 Clade (Fig. 1B), and two in Mito Clade (Fig. 1C). In these genes, seven P450 genes were up-regulated to more than 2-fold. The three most significantly up-regulated P450 genes were CYP6ER1, CYP6CS1 and CYP6AY1 in CYP3 Clade with 22.62-, 13.10- and 11.84-fold, respectively. In addition, the mRNA levels of CYP6FB2 and CYP408A1 were significantly lower in G25 than that in S25.
Table 1 Bioassays and synergism analysis of different N. lugens strains. Strain
Treatment
LC50 (mg$L1)
Slope
Sus
Imidacloprid þPBO þTPP þDEM Imidacloprid þPBO þTPP þDEM Imidacloprid þPBO þTPP þDEM
0.14 (0.13e0.15) a 0.13 (0.12e0.14) a 0.15 (0.14e0.17) a 0.15 (0.13e0.17) a 0.30 (0.27e0.34) b 0.27 (0.24e0.31) b 0.29 (0.26e0.33) b 0.30 (0.26e0.34) b 61.59 (55.43e68.64) e 10.47 (8.95e12.38) c 48.69 (43.25e55.59) d 59.13 (52.12e67.93) e
3.17 2.44 2.75 2.76 2.88 2.02 2.43 2.16 2.64 1.80 2.34 2.25
S25
G25
± ± ± ± ± ± ± ± ± ± ± ±
RR 0.25 0.32 0.32 0.25 0.20 0.34 0.26 0.24 0.39 0.32 0.22 0.28
1 1 1 1 2 2 2 2 434 74 343 416
SR 1.11 0.93 0.96 1.13 1.06 1.02 5.88 1.27 1.04
Data were presented as mean (95% confidence interval) in LC50 column and mean ± SEM. Different lowercases in LC50 column showed the significant differences at 0.05 level. RR, resistance ratio. SR, synergism ratio.
3.3. RNA interference and effects on insecticide resistance In order to evaluate the roles of P450 genes in imidacloprid resistance, the P450 genes that up-regulated above 2-fold in G25 were knocked down by RNAi and the insecticide sensitivities of the survivals were determined. When dsRNA of different P450 genes was applied to individuals from G25 through microinjection, the mRNA level of the corresponding P450 genes were reduced to less than 50%. In G25, RNAi against CYP6AY1 and CYP6ER1 decreased the imidacloprid resistance most effectively with RR from 434- to 155- and 137-fold, respectively (Table 2). RNAi against CYP4CE1 and CYP6CW1 also significantly declined the imidacloprid resistance, from 434- to 220- and 278-fold, respectively. However, after treated with dsRNA of CYP425B1, CYP439A1 or CYP6CS1, G25 had no obvious changes in terms of the resistance ratio against imidacloprid. These results indicated that the four P450 genes, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, might importantly contribute to imidacloprid resistance. 3.4. Insecticide metabolism by recombinant P450 proteins To evaluate the roles of putative P450s in imidacloprid resistance in vitro, the enzymatic activities of five recombinant P450s (CYP6AY1, CYP6ER1, CYP6CS1, CYP6CW1 and CYP4CE1) for imidacloprid metabolism were determined. The total contents for different P450s were ranged from 360 to 440 nmol P450 per liter of cell lysate based on the reduced CO-reduced difference spectrum. All solutions were diluted to 350 nmol P450 per liter of cell lysate for further imidacloprid metabolism experiments. The concentration of imidacloprid in reaction mixtures was analyzed with the reversed-phase HPLC. In the course of a reaction, the remaining concentration of imidacloprid declined when the reaction was incubated with one of CYP6AY1, CYP6ER1, CYP6CW1 or CYP4CE1, but not with CYP6CS1 (Fig. 2A). Imidacloprid concentration changes in the reaction mixture were considerably different with CYP6AY1, CYP6ER1, CYP6CW1 or CYP4CE1. The biggest decrease of imidacloprid concentration was observed in the reaction with CYP6CW1 and followed by CYP6AY1, CYP6ER1 and CYP4CE1. The imidacloprid metabolite (5-hydroxy-imidacloprid or 4-hydroxy-imidacloprid, Ding et al., 2013) formation was also analyzed and compared between these four P450 proteins (Fig. 2B). No metabolite was observed in the incubation with CYP6CS1. Similar to the trend in enzymatic activity, the fastest metabolite formation was observed in the incubation with CYP6CW1 and in the same order (CYP6CW1 > CYP6AY1 > CYP6ER1 > CYP4CE1). Then key parameters were calculated for the four P450 proteins (Table 3), among which CYP6CW1 showed the highest affinity with imidacloprid (Km ¼ 25.03 ± 3.82 mM) and the biggest Kcat value (5.35 ± 0.71 pmol/min/pmol P450). In the negative control, the membrane fraction of untransformed E. coli cells was used instead of the recombinant P450s, the depletion of imidacloprid and the formation of main metabolite were not obviously observed. 3.5. Expression levels of the four P450 genes in 25 generations during selection In order to analyze the roles of the four P450 genes (CYP4CE1, CYP6CW1, CYP6AY1 and CYP6ER1) in imidacloprid resistance at different resistance development stages, their expression levels in 25 successive selection generations were detected and normalized to that of S25 (Fig. 3). The over-expression of CYP6ER1 was first observed in the second selection generation (G2), and with quick increase from G7 to G22, spanning almost all stages of imidacloprid resistance development. The obvious change in CYP6AY1 expression initially occurred in G8, and with a sharp increase from G14 to
Fig. 1. Fold change of G25 vs S25 in the expression levels of fifty-four P450 genes in N. lugens. The significant differences were marked by stars. *, significant at 0.05 level; **, significant at 0.01 level. (A) Fold change of G25 vs S25 in the expression level of P450 genes in CYP4 Clade, (B) Fold change of G25 vs S25 in the expression level of P450 genes in CYP3 Clade, and (C) Fold change of G25 vs S25 in the expression level of P450 genes in Mito Clade and CYP2 Clade.
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Table 2 Reduction of P450 genes mRNA levels by RNAi and effects of RNAi on insecticide resistance. Gene
Expression before RNAi
Expression after RNAi
RNAi Efficiency
LC50 (mg$L1) (95% FC)
RRa
Control CYP4CE1 CYP425B1 CYP439A1 CYP6AY1 CYP6CS1 CYP6CW1 CYP6ER1
e 7.57 ± 1.08 8.33 ± 1.59 2.39 ± 0.41 11.84 ± 1.82 13.10 ± 4.24 5.43 ± 1.12 22.62 ± 1.97
e 3.29 3.06 1.16 4.17 3.42 2.10 6.54
% 56.54 63.27 51.46 64.78 73.89 61.33 71.09
61.59 31.23 59.76 56.22 22.02 55.17 39.49 19.50
434 220 421 396 155 389 278 137
± ± ± ± ± ± ±
0.56 0.72 0.33 1.05 1.26 0.57 1.78
(55.43e68.64) (26.54e37.16) (51.99e69.92) (50.04e64.10) (19.15e25.76) (48.55e63.45) (34.75e45.02) (16.38e23.40)
a b a a c a b c
Data were presented as mean ± SEM. Different lowercases in the same column showed significant differences at 0.05 level. a RR, resistance ratio.
Table 3 The calculated parameters for recombinant P450s. P450
Km (mM)
CYP6AY1 CYP6ER1 CYP6CW1 CYP4CE1 CYP6CS1
46.36 33.22 25.03 58.53 e
± ± ± ±
6.14 4.64 3.82 8.34
Kcat (pmol/min/pmol) c b a c
3.66 2.85 5.35 1.97 e
± ± ± ±
0.39 0.33 0.71 0.32
b c a d
Data were presented as mean ± SEM. Different lowercases in the same column showed significant differences at 0.05 level.
Fig. 2. Time course of imidacloprid depletion and the main metabolite formation. (A) The remaining imidacloprid in incubation solution with imidacloprid and recombinant P450 protein at different time points; (B) The main metabolite formation of imidacloprid at different time points. Values were plotted as means ± SEM of at least three independent tests.
G17, during which imidacloprid resistance increased enormously (Zhang et al., 2015). The same changes occurred to CYP4CE1 as well. In contrast, CYP6CW1 levels increased significantly only at later stage, from G18 and G21.
4. Discussion The metabolic resistance caused by enhanced detoxification of xenobiotic metabolizing enzymes, such as carboxylesterases (CarEs), glutathione transferases (GSTs) and cytochrome P450s, has proven to be an important mechanism of insecticide resistance (Cui et al., 2015; Enayati et al., 2005; Feyereisen, 1999). In the current study, one imidacloprid resistant strain (G25) and its susceptible counterpart (S25) were separated from one field-originated population of N. lugens by the successive selection for 25 generations with or without imidacloprid. The strain G25 showed super high level of resistance to imidacloprid (434-fold compared with the
susceptible strain), whereas S25 was relatively susceptible (2-fold). Although TPP could significantly synergize imidacloprid in G25 (1.27-fold), the greatest synergistic effect was observed with the use of PBO, indicating the major role of P450s in metabolic resistance to imidacloprid. The enhanced detoxification of P450s has reported to be involved in the resistance to neonicotinoids, especially to imidacloprid, in insects, such as CYP6ER1 and CYP6AY1 in N. lugens, CYP6G1 in D. melanogaster and CYP6CM1vQ in B. tabaci (Bass et al., 2011; Ding et al., 2013; Joussen et al., 2008; Karunker et al., 2009). However, the finding of single or double P450s related to neonicotinoid resistance in insects might not reflect the overall perspective of metabolic resistance mechanisms of insect P450s, because P450s constitute a multigenic superfamily to metabolize a wide range of endogenous and exogenous compounds (Feyereisen, 1999; Guittard et al., 2011; Qiu et al., 2012). The over-expression of multiple P50s is probably a general case, and that over-expression of a single P450 is rather an exception. As a classical example, CYP6G1 was identified as a single insecticide-resistance allele in 20 DDT-resistant strains of D. melanogaster (Daborn et al., 2002). However, more reports have been demonstrated that multiple over-expressed P450 genes would be co-responsible for the high levels of insecticide resistance in insects, such as co-overexpressed CYP6G1 and CYP12D1 in DDT resistance in D. melanogaster (Festucci-Buselli et al., 2005) and co-upregulation of multiple P450 genes in permethrin resistant house flies (Zhu et al., 2008) and Culex mosquito (Liu et al., 2011; Yang and Liu, 2011). Recently, a genome sequencing of N. lugens provides an opportunity to find out all potential P450 genes associated with imidacloprid resistance (Xue et al., 2014). By comparing the expression levels of all P450 genes between G25 and S25 strains, and RNAi with dsRNA, four P450 genes (CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1) were supposed to be involved in imidacloprid resistance in N. lugens. The higher expression in resistant strain G25 was only a prerequisite for the importance of these P450s in imidacloprid resistance, and the contribution of a P450 was not just decided by its expression levels. In previous studies, although CYP6ER1 had higher expression levels in some resistant populations than that of CYP6AY1, the higher
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Fig. 3. Relative levels of four P450 genes in G and S insects for 25 successive generations in N. lugens. The expression profile of each generation in G insect was compared to S25 (G1, G2, G3 … G25 always compared to S25). Values were plotted as means ± SEM of at least three independent tests.
Fig. 4. Venn diagram analysis of the numbers of inducible P450 genes by imidacloprid (orange), the over-expressed P450 genes in G25 (gray) and P450s contributing to imidacloprid resistance (yellow) in N. lugens. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
catalytic activity of CYP6AY1 to metabolize imidacloprid endowed the comparable importance of two P450s in imidacloprid resistance (Bass et al., 2011; Ding et al., 2013; Bao et al., 2016). Here in the present study, CYP6CW1 showed the highest catalytic activity to metabolize imidacloprid with Kcat value of 5.35 ± 0.71 pmol/min/ pmol P450 among all test P450s, although the over-expression ratio for CYP6CW1 was 5.43-fold and less than that of CYP6AY1, CYP6ER1 and CYP4CE1 genes. The high catalytic activity of CYP6CW1 might compensate for its lower overexpression and endowed the comparable importance with other three P450s. Another typical example was CYP6CS1, which had no catalytic activity to metabolize imidacloprid at all, although its expression level in G25 was 7.57-fold of that in S25. These results indicated that the catalytic activity of P450s was as important as the expression level, in the context of evaluating their roles in insecticide resistance. From the point of view of the final results tested in G25, four P450s were important to imidacloprid resistance. Nevertheless, their roles were not equal among different stages of resistance development. From the over-expression of these P450 genes during resistance development, it was suggestive that CYP6ER1 would
play important roles at all stages, CYP6AY1 at middle to later stages with the sharp increase of imidacloprid resistance, while CYP4CE1 and CYP6CW1 only in few generations. So, when analyzing P450s in insects, the expression differences between strains, expression changes during resistance development, and catalytic activity of multiple P450s were key factors to build up their importance in insecticide resistance. In our previous study, screening the induction of P450 genes by imidacloprid in N. lugens showed that eighteen candidate P450 genes were up-regulated after imidacloprid application (Zhang et al., 2016). Here the detection of fifty-four P450 genes in N. lugens showed that twelve P450 genes were significantly overexpressed in G25 strain when compared to S25, among which five P450 genes in CYP4 Clade (CYP4CE1, CYP4DE1, CYP417A3, CYP425B1 and CYP439A1) and four in CYP3 Clade (CYP6AY1, CYP6CS1, CYP6CW1 and CYP6ER1) were up-regulated both in the previous induction study (Zhang et al., 2016) and in this study. However, the over-expression in G25 and induction up-regulation in Sus strain were not always coincident (Fig. 4). Six P450 genes in Clade4, four in Clade3, and three in Clade2 could be induced by imidacloprid (including up-regulation and down-regulation) (Zhang et al., 2016), but not differently expressed in G25. In contrast, though two P450 genes (CYP4FB2 and CYP439B1) in Clade4 and one (CYP6BD12) in Clade3 were not induced by imidacloprid, the over-expression was found in G25. The induction by xenobiotic, such as imidacloprid, offered a broader targets and detoxification enzymes that might be more likely to be involved in resistance (Le Goff et al., 2006). The induction by imidacloprid application and over-expression in resistant populations might provide more capacity for insect P450s such as CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1 in this study, to adapt to the stress from insecticide application. Therefore, xenobiotic inducibility of detoxification enzymes might represent risk factors for the development of resistance. In conclusion, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1 were supposed to play important roles, although might not equally, in imidacloprid resistance in N. lugens, based on the information from the comparison of the expression levels of all P450 genes between imidacloprid resistant and susceptible strains, their expression levels at different stages of resistance development, and their catalytic activities. The induction of these P450 genes by imidacloprid
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suggested their potentials in imidacloprid metabolism, especially when imidacloprid resistance was not high. Acknowledgement We would like to thank Dr. Na Yu from Ghent University (Belgium) for her help in improving the English language. This work was supported by National Natural Science Foundation of China (31322045, 31130045 and 31601656). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ibmb.2016.10.009. References Bao, H., Gao, H., Zhang, Y., Fan, D., Fang, J., Liu, Z., 2016. The roles of CYP6AY1 and CYP6ER1 in imidacloprid resistance in the brown planthopper: expression levels and detoxification efficiency. Pestic. Biochem. Physiol. 129, 70e74. Bass, C., Carvalho, R.A., Oliphant, L., Puinean, A.M., Field, L.M., Nauen, R., Williamson, M.S., Moores, G., Gorman, K., 2011. Overexpression of a cytochrome P450 monooxygenase, CYP6ER1, is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens. Insect Mol. Biol. 20, 763e773. Brun-Barale, A., Hema, O., Martin, T., Suraporn, S., Audant, P., Sezutsu, H., Feyereisen, R., 2010. Multiple P450 genes overexpressed in deltamethrinresistant strains of Helicoverpa armigera. Pest Manag. Sci. 66, 900e909. Cui, F., Li, M.X., Chang, H.J., Mao, Y., Zhang, H.Y., Lu, L.X., Yan, S.G., Lang, M.L., Liu, L., Qiao, C.L., 2015. Carboxylesterase-mediated insecticide resistance: quantitative increase induces broader metabolic resistance than qualitative change. Pestic. Biochem. Physiol. 121, 88e96. Daborn, P., Boundy, S., Yen, J., Pittendrigh, B., 2001. DDT resistance in Drosophila correlates with Cyp6g1 over-expression and confers cross-resistance to the neonicotinoid imidacloprid. Mol. Genet. Genomics 266, 556e563. Daborn, P.J., Yen, J.L., Bogwitz, M.R., Le Goff, G., Feil, E., Jeffers, S., Tijet, N., Perry, T., Heckel, D., Batterham, P., Feyereisen, R., Wilson, T.G., ffrench-Constant, R.H., 2002. A single P450 allele associated with insecticide resistance in Drosophila. Science 297, 2253e2256. Ding, Z., Wen, Y., Yang, B., Zhang, Y., Liu, S., Liu, Z., Han, Z., 2013. Biochemical mechanisms of imidacloprid resistance in Nilaparvata lugens: over-expression of cytochrome P450 CYP6AY1. Insect Biochem. Mol. Biol. 43, 1021e1027. Enayati, A.A., Ranson, H., Hemingway, J., 2005. Insect glutathione transferases and insecticide resistance. Insect Mol. Biol. 14, 3e8. Festucci-Buselli, R.A., Carvalho-Dias, A.S., de Oliveira-Andrade, M., CaixetaNunes, C., Li, H.M., Stuart, J.J., Muir, W., Scharf, M.E., Pittendrigh, B.R., 2005. Expression of Cyp6g1 and Cyp12d1 in DDT resistant and susceptible strains of Drosophila melanogaster. Insect Mol. Biol. 14, 69e77. Feyereisen, R., 1999. Insect P450 enzymes. Annu. Rev. Entomol. 44, 507e533. Feyereisen, R., 2012. Insect CYP Genes and P450 Enzymes. In: Gilbert, L.I. (Ed.), Insect Molecular Biology and Biochemistry. Academic Press, San Diego, pp. 236e316. Garrood, W.T., Zimmer, C.T., Gorman, K.J., Nauen, R., Bass, C., Davies, T.G.E., 2016. Field-evolved resistance to imidacloprid and ethiprole in populations of brown planthopper Nilaparvata lugens collected from across South and East Asia. Pest Manag. Sci. 72, 140e149. Gong, M.Q., Gu, Y., Hu, X.B., Sun, Y., Ma, L., Li, X.L., Sun, L.X., Sun, J., Qian, J., Zhu, C.-L., 2005. Cloning and overexpression of CYP6F1, a cytochrome P450 gene, from deltamethrin-resistant Culex pipiens pallens. Acta Biochim. Biophys. Sin. 37, 317e326. Gorman, K., Liu, Z., Denholm, I., Brüggen, K.-U., Nauen, R., 2008. Neonicotinoid resistance in rice brown planthopper, Nilaparvata lugens. Pest Manag. Sci. 64, 1122e1125. Guittard, E., Blais, C., Maria, A., Parvy, J.P., Pasricha, S., Lumb, C., Lafont, R., Daborn, P.J., Dauphin-Villemant, C., 2011. CYP18A1, a key enzyme of Drosophila steroid hormone inactivation, is essential for metamorphosis. Dev. Biol. 349, 35e45. Hardstone, M., Komagata, O., Kasai, S., Tomita, T., Scott, J., 2010. Use of isogenic strains indicates CYP9M10 is linked to permethrin resistance in Culex pipiens quinquefasciatus. Insect Mol. Biol. 19, 717e726. Joussen, N., Heckel, D.G., Haas, M., Schuphan, I., Schmidt, B., 2008. Metabolism of imidacloprid and DDT by P450 GYP6G1 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. 64, 65e73. Karunker, I., Benting, J., Lueke, B., Ponge, T., Nauen, R., Roditakis, E., Vontas, J., Gorman, K., Denholm, I., Morin, S., 2008. 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. 38, 634e644. Karunker, I., Morou, E., Nikou, D., Nauen, R., Sertchook, R., Stevenson, B.J., Paine, M.J., Morin, S., Vontas, J., 2009. Structural model and functional characterization of the Bemisia tabaci CYP6CM1vQ, a cytochrome P450 associated with high levels of imidacloprid resistance. Insect Biochem. Mol. Biol. 39, 697e706. Le Goff, G., Hilliou, F., Siegfried, B.D., Boundy, S., Wajnberg, E., Sofer, L., Audant, P., ffrench-Constant, R.H., Feyereisen, R., 2006. Xenobiotic response in Drosophila melanogaster: sex dependence of P450 and GST gene induction. Insect Biochem. Mol. Biol. 36, 674e682. Liu, N., Li, M., Gong, Y., Liu, F., Li, T., 2015. Cytochrome P450s-Their expression, regulation, and role in insecticide resistance. Pestic. Biochem. Physiol. 120, 77e81. Liu, N., Li, T., Reid, W.R., Yang, T., Zhang, L., 2011. Multiple Cytochrome P450 Genes: their constitutive overexpression and permethrin induction in insecticide resistant mosquitoes, Culex quinquefasciatus. PLoS One 6, e23403. Liu, N., Scott, J.G., 1998. Increased transcription of CYP6D1 causes cytochrome P450mediated insecticide resistance in house fly. Insect Biochem. Mol. Biol. 28, 531e535. Liu, S., Ding, Z., Zhang, C., Yang, B., Liu, Z., 2010. Gene knockdown by intro-thoracic injection of double-stranded RNA in the brown planthopper, Nilaparvata lugens. Insect Biochem. Mol. Biol. 40, 666e671. Liu, Z., Williamson, M.S., Lansdell, S.J., Denholm, I., Han, Z., Millar, N.S., 2005. A nicotinic acetylcholine receptor mutation conferring target-site resistance to imidacloprid in Nilaparvata lugens (brown planthopper). Proc. Natl. Acad. Sci. U. S. A. 102, 6. Matsumura, M., Sanada-Morimura, S., Otuka, A., Ohtsu, R., Sakumoto, S., Takeuchi, H., Satoh, M., 2014. Insecticide susceptibilities in populations of two rice planthoppers, Nilaparvata lugens and Sogatella furcifera, immigrating into Japan in the period 2005-2012. Pest Manag. Sci. 70, 615e622. Qiu, Y., Tittiger, C., Wicker-Thomas, C., Le Goff, G., Young, S., Wajnberg, E., Fricaux, T., Taquet, N., Blomquist, G.J., Feyereisen, R., 2012. An insect-specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 109, 14858e14863. Scott, J.G., 1999. Cytochromes P450 and insecticide resistance. Insect Biochem. Mol. Biol. 29, 757e777. Sogawa, K., Cheng, C., 1979. Economic Thresholds, Nature of Damage, and Losses Caused by the Brown Planthopper. Brown Planthopper: Threat to Rice Production in Asia. International Rice Res. Institute, Manila, pp. 125e142. Tang, J., Li, J., Shao, Y., Yang, B., Liu, Z., 2010. Fipronil resistance in the whitebacked planthopper (Sogatella furcifera): possible resistance mechanisms and crossresistance. Pest Manag. Sci. 66, 121e125. Tang, Q.Y., Zhang, C.X., 2013. Data Processing System (DPS) software with experimental design, statistical analysis and data mining developed for use in entomological research. Insect Sci. 20, 254e260. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, 12. http://dx.doi.org/10.1186/gb-2002-3-7-research0034. Wang, Y., Chen, J., Zhu, Y.C., Ma, C., Huang, Y., Shen, J., 2008. Susceptibility to neonicotinoids and risk of resistance development in the brown planthopper, Nilaparvata lugens (Stål) (Homoptera: Delphacidae). Pest Manag. Sci. 64, 1278e1284. Wen, Y., Liu, Z., Bao, H., Han, Z., 2009. Imidacloprid resistance and its mechanisms in field populations of brown planthopper, Nilaparvata lugens Stål in China. Pestic. Biochem. Physiol. 94, 36e42. Xue, J., Zhou, X., Zhang, C.X., Yu, L.L., Fan, H.W., Wang, Z., Xu, H.J., Xi, Y., Zhu, Z.R., Zhou, W.W., Pan, P.L., Li, B.L., Colbourne, J.K., Noda, H., Suetsugu, Y., Kobayashi, T., Zheng, Y., Liu, S., Zhang, R., Liu, Y., Luo, Y.D., Fang, D.M., Chen, Y., Zhan, D.L., Lv, X.D., Cai, Y., Wang, Z.B., Huang, H.J., Cheng, R.L., Zhang, X.C., Lou, Y.H., Yu, B., Zhuo, J.C., Ye, Y.X., Zhang, W.Q., Shen, Z.C., Yang, H.M., Wang, J., Wang, J., Bao, Y.Y., Cheng, J.A., 2014. Genomes of the rice pest brown planthopper and its endosymbionts reveal complex complementary contributions for host adaptation. Genome Biol. 15, 12. http://dx.doi.org/10.1186/s13059-0140521-0. Yang, T., Liu, N., 2011. Genome analysis of cytochrome p450s and their expression profiles in insecticide resistant mosquitoes, Culex quinquefasciatus. PLoS One 6, e29418. Yang, Y., Huang, L., Wang, Y., Zhang, Y., Fang, S., Liu, Z., 2016. No cross-resistance between imidacloprid and pymetrozine in the brown planthopper: status and mechanisms. Pestic. Biochem. Physiol. 130, 79e83. Zhang, J., Zhang, Y., Wang, Y., Yang, Y., Cang, X., Liu, Z., 2016. Expression induction of P450 genes by imidacloprid in Nilaparvata lugens: a genome-scale analysis. Pestic. Biochem. Physiol. 132, 59e64. Zhang, Y., Wang, X., Yang, B., Hu, Y., Huang, L., Bass, C., Liu, Z., 2015. Reduction in mRNA and protein expression of a nicotinic acetylcholine receptor 8 subunit is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens. J. Neurochem. 135, 686e694. Zhu, F., Li, T., Zhang, L., Liu, N., 2008. Co-up-regulation of three P450 genes in response to permethrin exposure in permethrin resistant house flies, Musca domestica. BMC Physiol. 8, 18. http://dx.doi.org/10.1186/1472-6793-8-18.
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Metabolic imidacloprid resistance in the brown planthopper, Nilaparvata lugens, relies on multiple P450 enzymes Yixi Zhang, Yuanxue Yang, Huahua Sun, Zewen Liu* Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Weigang 1, Nanjing 210095, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 August 2016 Received in revised form 23 October 2016 Accepted 24 October 2016 Available online 26 October 2016
Target insensitivity contributing to imidacloprid resistance in Nilaparvata lugens has been reported to occur either through point mutations or quantitative change in nicotinic acetylcholine receptors (nAChRs). However, the metabolic resistance, especially the enhanced detoxification by P450 enzymes, is the major mechanism in fields. From one field-originated N. lugens population, an imidacloprid resistant strain G25 and a susceptible counterpart S25 were obtained to analyze putative roles of P450s in imidacloprid resistance. Compared to S25, over-expression of twelve P450 genes was observed in G25, with ratios above 5.0-fold for CYP6AY1, CYP6ER1, CYP6CS1, CYP6CW1, CYP4CE1 and CYP425B1. RNAi against these genes in vivo and recombinant tests on the corresponding proteins in vitro revealed that four P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, played important roles in imidacloprid resistance. The importance of the four P450s was not equal at different stages of resistance development based on their over-expression levels, among which CYP6ER1 was important at all stages, and that the others might only contribute at certain stages. The results indicated that, to completely reflect roles of P450s in insecticide resistances, their over-expression in resistant individuals, expression changes at the stages of resistance development, and catalytic activities against insecticides should be considered. In this study, multiple P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, have proven to be important in imidacloprid resistance. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Nilaparvata lugens Imidacloprid Metabolic resistance P450s
1. Introduction Cytochrome P450 monooxygenase is a multi-gene superfamily that has critical functions in the detoxification of xenobiotics including drugs, pesticides and plant toxins, as well as in the metabolism of endogenous compounds (Scott, 1999; Feyereisen, 2012). In insects, P450 monooxygenase-mediated detoxification is one of the major mechanisms of insecticide resistance. The whole genome sequence analysis and examining the expression of multiple genes simultaneously revealed that the over-expression of multiple P450 genes in insecticide resistant strains is a common phenomenon observed in many insect species (Liu et al., 2015). For example, the over-expression of two P450 genes CYP6G1 and CYP12D1 were found to be associated with DDT resistance in Drosophila melanogaster (Festucci-Buselli et al., 2005). Significant over-expression of multiple CYP genes (CYP4M6, CYP4M7,
* Corresponding author. E-mail address: [email protected] (Z. Liu). http://dx.doi.org/10.1016/j.ibmb.2016.10.009 0965-1748/© 2016 Elsevier Ltd. All rights reserved.
CYP6AE11, CYP9A12, CYP332A1 and CYP337B1) was also found in different deltamethrin-resistant strains of Helicoverpa armigera (Brun-Barale et al., 2010). In permethrin resistant house flies, Musca domestica, co-upregulation of three P450 genes, CYP4D4v2, CYP4G2, and CYP6A38, was reported (Zhu et al., 2008). The gene expression and induction by permethrin in different mosquito (Culex quinquefasciatus) populations indicated that multiple P450 genes were up-regulated in the case of insecticide resistance (Liu et al., 2011). Furthermore, the comparison of P450 expression profiles between permethrin susceptible and resistant mosquitos in the whole genome scale implied that not only multiple P450 genes were involved in insecticide resistance but up- or down-regulation of P450 genes might also be co-responsible for insecticide detoxification (Yang and Liu, 2011). Imidacloprid, the first commercial neonicotinoid insecticide, is used as a major insecticide to control many insect pests. Imidacloprid resistance has been reported in several species (Gorman et al., 2008; Wen et al., 2009; Matsumura et al., 2014; Garrood et al., 2016). Synergism studies, gene expression and enzyme activity assays proved that the enhanced detoxification of P450s was
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the major metabolic mechanism of imidacloprid resistance (Wen et al., 2009; Garrood et al., 2016). P450s were supposed to play important roles in imidacloprid resistance in several insect species, including D. melanogaster (CYP6G1), M. domestica (CYP6D1), Bemisia tabaci (CYP6CM1), C. pipiens pallens (CYP6F1), C. quinquefasciatus (CYP9M10), and Nilaparvata lugens (CYP6ER1, CYP6AY1) (Daborn et al., 2001; Liu and Scott, 1998; Karunker et al., 2008; Gong et al., 2005; Hardstone et al., 2010; Bass et al., 2011; Ding et al., 2013). Until now, however, these studies mainly focused on single P450 gene, and only three insect P450s, CYP6G1 (D. melanogaster), CYP6CM1 (B. tabaci) and CYP6AY1 (N. lugens) have been confirmed in the imidacloprid metabolism (Daborn et al., 2001; Karunker et al., 2008; Ding et al., 2013). The brown planthopper, Nilaparvata lugens, is a major rice pest throughout Asia which causes a dramatic reduction in rice yield and great economic losses (Gorman et al., 2008; Sogawa and Cheng, 1979; Wang et al., 2008). Previous studies showed that the target insensitivity caused by key amino acid substitution or quantitative change of nicotinic acetylcholine receptors (nAChRs) led to high resistance in N. lugens against imidacloprid (Liu et al., 2005; Zhang et al., 2015). However, the metabolic resistance, especially the enhanced detoxification of P450s, might play a superior role in the field population of N. lugens (Wen et al., 2009; Bass et al., 2011; Ding et al., 2013; Bao et al., 2016). The genome sequencing of N. lugens provided an opportunity to identify potential resistance-related P450 genes at the whole genome scale (Xue et al., 2014; Zhang et al., 2016). In the present study, two strains were obtained from one field-originated population by the successive selection for 25 generations with or without imidacloprid (Zhang et al., 2015). Comparing the expression profiles of fifty-four P450 genes in these two strains, four genes were supposed to importantly contribute to imidacloprid resistance, and their roles in different stages of resistance development were analyzed. 2. Materials and methods
51
2.3. qRT-PCR validation of P450 genes expression Fifty-four P450 gene sequences were retrieved from the N. lugens genome (GenBank accession number AOSB00000000.1, BioProject PRJNA177647, provided by Prof. Zhang Chuanxi from Zhejiang University, Hangzhou, China; Xue et al., 2014). Total RNA was extracted using TRlzol® reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription to cDNA was performed using the PrimeScript™ RT reagent Kit and gDNA Eraser (Takara, Tokyo, Japan). The mRNA levels of the P450 genes were measured by quantitative realtime PCR (qRT-PCR) with the SYBR® Premix Ex Taq™ (Takara) in a 20 mL total reaction volume containing 200 ng of cDNA, 10 mL reaction buffer (SYBR Premix Ex Taq), 0.4 mL each of forward and reverse gene specific primer (Zhang et al., 2016) and 0.4 mL 50 Rox Reference Dye II, following the manufacturer instruction. Every qRT-PCR experiment was performed with at least three biological replicates. Gene expression difference was calculated with the 2DDCT method. Two reference genes (b-actin and GAPDH) were validated experimentally for each generation and treatment, and used for normalization according to the strategy described previously (Vandesompele et al., 2002). 2.4. RNA interference RNAi was performed as previously described (Liu et al., 2010). The dsRNAs of the targeted P450 genes and the bacterial Lac-Z (GenBank accession number: AJ308295), as a control, were synthesized using the T7 Ribomax Express RNAi System (Promega, Madison, WI, USA) and precipitated with isopropanol. Primers used to synthesize the dsRNA were summarized in Table S1. The dsRNA was resuspended in ultra-pure water, quantified spectrophotometrically at 260 nm and verified with agarose gel electrophoresis for its purity and integrity. It was then kept at 80 C for the subsequent application. 50 nL of dsRNA was microinjected at the conjunctive between prothorax and mesothorax of the 3rd instar N. lugens.
2.1. Insect 2.5. Functional expression of P450s and insecticide metabolism The susceptible strain (Sus) of N. lugens was a laboratory strain originally collected from Nueva Ecija (Philippines) in 2004 and reared in a laboratory without contacting any insecticide for 12 years. A N. lugens field population was collected from a paddy field in Nueva Ecija (Philippines) in August 2011, and was separated into two strains (Zhang et al., 2015). One strain (G25) was continuously selected by imidacloprid in the laboratory for 25 generations, and the counterpart (S25) was reared as a laboratory strain without contacting any insecticide. During the selection procedure, 3000e5000 individuals were reproduced in each generation. Insects were kept indoor with rice seedlings at 25 ± 1 C, 70e80% relative humidity and 16-h light/8-h dark photoperiod. 2.2. Bioassay and synergistic experiment Bioassay was performed according to the rice seedling dip method (Wang et al., 2008). Rice seedlings were immersed in imidacloprid solution at a series of concentrations for 15s. After airdrying, the seedlings were placed in plastic cups with 1.5% agar to maintain the moist conditions. In the bioassay, thirty insects (the 5th instar nymphs) were inoculated to the seedlings treated with imidacloprid as mentioned above in three replications and the LC50 of imidacloprid was determine. In the synergism experiment, 2 mg synergist (triphenyl phosphate, diethyl maleate or piperonyl butoxide) in 0.08 mL acetone was topically delivered onto the prothorax notum of the test insects (the 5th instar nymphs) 1 h before the insecticide application (Tang et al., 2010).
P450 genes and the house fly P450 reductase cDNA sequences were subcloned into the expression vector pCWori at NdeI and HindIII or SpeI sites. The primers for the vector construction were summarized in Table S2. The plasmids were transformed into E. coli DH5a and transformants were selected with ampicillin. The cells were treated with lysozyme solution and the resulting membrane pellet was suspended in sodium phosphate buffer for enzymatic characterization (Ding et al., 2013). Enzymatic activities of the recombinant P450s were determined as described previously (Ding et al., 2013; Bao et al., 2016). In brief, the reaction mixture was contained 0.2 mmol P450 protein, 2 mmol house fly P450 reductase, NADPH-regenerating system and 3 mg/L imidacloprid. Instead of the recombinant P450s, the membrane fraction of untransformed E. coli cells was used as control. 200 mL solution was extracted from the reaction mixture at an interval of 20 min, and the imidacloprid concentration was determined by reversed-phase HPLC on a 5-mm SupelCosil LC18-DB column (4.6 cm 250 cm, Supelco) using a solvent mixture composed of 20% CH3CN in H2O, which was isocratic for 5 min, followed by a linear gradient up to 80% CH3CN in H2O for over 40 min (flow rate 1 mL/min). Imidacloprid elution was monitored by measuring the absorption at 270 nm using Spectroflow UV/Vis Detector (Kratos Analytical Instruments, USA). The main metabolites of imidacloprid by recombinant protein were identified by UPLC-MS (Ding et al., 2013; Yang et al., 2016). For enzymatic kinetic analysis, a series of concentrations (0.1e5 mg/L) were used. Rates of substrate turnover
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were plotted versus substrate concentration. Km and Kcat were determined in 20 min fitting to the MichaeliseMenten equation using non-linear regression. 2.6. Statistical analyses Data analyses were carried out using Data Processing System (DPS) software v9.50 (Tang and Zhang, 2013). Significant differences were analyzed by one-way ANOVA with at least three repeats. Multiple comparisons between the groups were performed using Student-Newman-Keuls (S-N-K) method. The level of significance for results was set at p < 0.05. 3. Results 3.1. Bioassays and synergism analyses A resistant strain G25 and a relatively susceptible strain S25 of N. lugens were obtained from one field-originated population from Nueva Ecija (Philippines) after the continuous selection for 25 generations with or without imidacloprid (Zhang et al., 2015). Comparing with the susceptible strain Sus (LC50 ¼ 0.14 mg/L), the resistance ratio (RR) in G25 was 434-fold, whereas the RR in S25 was 2 being relatively susceptible (Table 1). The three important synergists tested here did not show any effects on imidacloprid in the Sus and S25 strains. However, PBO (piperonyl butoxide) and TPP (triphenyl phosphate) significantly synergized imidacloprid in G25 with the synergism ratio (SR) of 5.88- and 1.27-fold, respectively, indicating that P450s played dominant roles in the metabolic imidacloprid resistance (Table 1). 3.2. Comparison of the expression levels of P450 genes between G25 and S25 To investigate the potential P450 genes associated with imidacloprid resistance, the expression levels of all P450 genes identified from N. lugens genome database were determined in S25 and G25 by qRT-PCR. Compared to S25, the expression levels of fifteen P450 genes were significantly different in G25, including seven in CYP4 Clade (Fig. 1A), six in CYP3 Clade (Fig. 1B), and two in Mito Clade (Fig. 1C). In these genes, seven P450 genes were up-regulated to more than 2-fold. The three most significantly up-regulated P450 genes were CYP6ER1, CYP6CS1 and CYP6AY1 in CYP3 Clade with 22.62-, 13.10- and 11.84-fold, respectively. In addition, the mRNA levels of CYP6FB2 and CYP408A1 were significantly lower in G25 than that in S25.
Table 1 Bioassays and synergism analysis of different N. lugens strains. Strain
Treatment
LC50 (mg$L1)
Slope
Sus
Imidacloprid þPBO þTPP þDEM Imidacloprid þPBO þTPP þDEM Imidacloprid þPBO þTPP þDEM
0.14 (0.13e0.15) a 0.13 (0.12e0.14) a 0.15 (0.14e0.17) a 0.15 (0.13e0.17) a 0.30 (0.27e0.34) b 0.27 (0.24e0.31) b 0.29 (0.26e0.33) b 0.30 (0.26e0.34) b 61.59 (55.43e68.64) e 10.47 (8.95e12.38) c 48.69 (43.25e55.59) d 59.13 (52.12e67.93) e
3.17 2.44 2.75 2.76 2.88 2.02 2.43 2.16 2.64 1.80 2.34 2.25
S25
G25
± ± ± ± ± ± ± ± ± ± ± ±
RR 0.25 0.32 0.32 0.25 0.20 0.34 0.26 0.24 0.39 0.32 0.22 0.28
1 1 1 1 2 2 2 2 434 74 343 416
SR 1.11 0.93 0.96 1.13 1.06 1.02 5.88 1.27 1.04
Data were presented as mean (95% confidence interval) in LC50 column and mean ± SEM. Different lowercases in LC50 column showed the significant differences at 0.05 level. RR, resistance ratio. SR, synergism ratio.
3.3. RNA interference and effects on insecticide resistance In order to evaluate the roles of P450 genes in imidacloprid resistance, the P450 genes that up-regulated above 2-fold in G25 were knocked down by RNAi and the insecticide sensitivities of the survivals were determined. When dsRNA of different P450 genes was applied to individuals from G25 through microinjection, the mRNA level of the corresponding P450 genes were reduced to less than 50%. In G25, RNAi against CYP6AY1 and CYP6ER1 decreased the imidacloprid resistance most effectively with RR from 434- to 155- and 137-fold, respectively (Table 2). RNAi against CYP4CE1 and CYP6CW1 also significantly declined the imidacloprid resistance, from 434- to 220- and 278-fold, respectively. However, after treated with dsRNA of CYP425B1, CYP439A1 or CYP6CS1, G25 had no obvious changes in terms of the resistance ratio against imidacloprid. These results indicated that the four P450 genes, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, might importantly contribute to imidacloprid resistance. 3.4. Insecticide metabolism by recombinant P450 proteins To evaluate the roles of putative P450s in imidacloprid resistance in vitro, the enzymatic activities of five recombinant P450s (CYP6AY1, CYP6ER1, CYP6CS1, CYP6CW1 and CYP4CE1) for imidacloprid metabolism were determined. The total contents for different P450s were ranged from 360 to 440 nmol P450 per liter of cell lysate based on the reduced CO-reduced difference spectrum. All solutions were diluted to 350 nmol P450 per liter of cell lysate for further imidacloprid metabolism experiments. The concentration of imidacloprid in reaction mixtures was analyzed with the reversed-phase HPLC. In the course of a reaction, the remaining concentration of imidacloprid declined when the reaction was incubated with one of CYP6AY1, CYP6ER1, CYP6CW1 or CYP4CE1, but not with CYP6CS1 (Fig. 2A). Imidacloprid concentration changes in the reaction mixture were considerably different with CYP6AY1, CYP6ER1, CYP6CW1 or CYP4CE1. The biggest decrease of imidacloprid concentration was observed in the reaction with CYP6CW1 and followed by CYP6AY1, CYP6ER1 and CYP4CE1. The imidacloprid metabolite (5-hydroxy-imidacloprid or 4-hydroxy-imidacloprid, Ding et al., 2013) formation was also analyzed and compared between these four P450 proteins (Fig. 2B). No metabolite was observed in the incubation with CYP6CS1. Similar to the trend in enzymatic activity, the fastest metabolite formation was observed in the incubation with CYP6CW1 and in the same order (CYP6CW1 > CYP6AY1 > CYP6ER1 > CYP4CE1). Then key parameters were calculated for the four P450 proteins (Table 3), among which CYP6CW1 showed the highest affinity with imidacloprid (Km ¼ 25.03 ± 3.82 mM) and the biggest Kcat value (5.35 ± 0.71 pmol/min/pmol P450). In the negative control, the membrane fraction of untransformed E. coli cells was used instead of the recombinant P450s, the depletion of imidacloprid and the formation of main metabolite were not obviously observed. 3.5. Expression levels of the four P450 genes in 25 generations during selection In order to analyze the roles of the four P450 genes (CYP4CE1, CYP6CW1, CYP6AY1 and CYP6ER1) in imidacloprid resistance at different resistance development stages, their expression levels in 25 successive selection generations were detected and normalized to that of S25 (Fig. 3). The over-expression of CYP6ER1 was first observed in the second selection generation (G2), and with quick increase from G7 to G22, spanning almost all stages of imidacloprid resistance development. The obvious change in CYP6AY1 expression initially occurred in G8, and with a sharp increase from G14 to
Fig. 1. Fold change of G25 vs S25 in the expression levels of fifty-four P450 genes in N. lugens. The significant differences were marked by stars. *, significant at 0.05 level; **, significant at 0.01 level. (A) Fold change of G25 vs S25 in the expression level of P450 genes in CYP4 Clade, (B) Fold change of G25 vs S25 in the expression level of P450 genes in CYP3 Clade, and (C) Fold change of G25 vs S25 in the expression level of P450 genes in Mito Clade and CYP2 Clade.
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Table 2 Reduction of P450 genes mRNA levels by RNAi and effects of RNAi on insecticide resistance. Gene
Expression before RNAi
Expression after RNAi
RNAi Efficiency
LC50 (mg$L1) (95% FC)
RRa
Control CYP4CE1 CYP425B1 CYP439A1 CYP6AY1 CYP6CS1 CYP6CW1 CYP6ER1
e 7.57 ± 1.08 8.33 ± 1.59 2.39 ± 0.41 11.84 ± 1.82 13.10 ± 4.24 5.43 ± 1.12 22.62 ± 1.97
e 3.29 3.06 1.16 4.17 3.42 2.10 6.54
% 56.54 63.27 51.46 64.78 73.89 61.33 71.09
61.59 31.23 59.76 56.22 22.02 55.17 39.49 19.50
434 220 421 396 155 389 278 137
± ± ± ± ± ± ±
0.56 0.72 0.33 1.05 1.26 0.57 1.78
(55.43e68.64) (26.54e37.16) (51.99e69.92) (50.04e64.10) (19.15e25.76) (48.55e63.45) (34.75e45.02) (16.38e23.40)
a b a a c a b c
Data were presented as mean ± SEM. Different lowercases in the same column showed significant differences at 0.05 level. a RR, resistance ratio.
Table 3 The calculated parameters for recombinant P450s. P450
Km (mM)
CYP6AY1 CYP6ER1 CYP6CW1 CYP4CE1 CYP6CS1
46.36 33.22 25.03 58.53 e
± ± ± ±
6.14 4.64 3.82 8.34
Kcat (pmol/min/pmol) c b a c
3.66 2.85 5.35 1.97 e
± ± ± ±
0.39 0.33 0.71 0.32
b c a d
Data were presented as mean ± SEM. Different lowercases in the same column showed significant differences at 0.05 level.
Fig. 2. Time course of imidacloprid depletion and the main metabolite formation. (A) The remaining imidacloprid in incubation solution with imidacloprid and recombinant P450 protein at different time points; (B) The main metabolite formation of imidacloprid at different time points. Values were plotted as means ± SEM of at least three independent tests.
G17, during which imidacloprid resistance increased enormously (Zhang et al., 2015). The same changes occurred to CYP4CE1 as well. In contrast, CYP6CW1 levels increased significantly only at later stage, from G18 and G21.
4. Discussion The metabolic resistance caused by enhanced detoxification of xenobiotic metabolizing enzymes, such as carboxylesterases (CarEs), glutathione transferases (GSTs) and cytochrome P450s, has proven to be an important mechanism of insecticide resistance (Cui et al., 2015; Enayati et al., 2005; Feyereisen, 1999). In the current study, one imidacloprid resistant strain (G25) and its susceptible counterpart (S25) were separated from one field-originated population of N. lugens by the successive selection for 25 generations with or without imidacloprid. The strain G25 showed super high level of resistance to imidacloprid (434-fold compared with the
susceptible strain), whereas S25 was relatively susceptible (2-fold). Although TPP could significantly synergize imidacloprid in G25 (1.27-fold), the greatest synergistic effect was observed with the use of PBO, indicating the major role of P450s in metabolic resistance to imidacloprid. The enhanced detoxification of P450s has reported to be involved in the resistance to neonicotinoids, especially to imidacloprid, in insects, such as CYP6ER1 and CYP6AY1 in N. lugens, CYP6G1 in D. melanogaster and CYP6CM1vQ in B. tabaci (Bass et al., 2011; Ding et al., 2013; Joussen et al., 2008; Karunker et al., 2009). However, the finding of single or double P450s related to neonicotinoid resistance in insects might not reflect the overall perspective of metabolic resistance mechanisms of insect P450s, because P450s constitute a multigenic superfamily to metabolize a wide range of endogenous and exogenous compounds (Feyereisen, 1999; Guittard et al., 2011; Qiu et al., 2012). The over-expression of multiple P50s is probably a general case, and that over-expression of a single P450 is rather an exception. As a classical example, CYP6G1 was identified as a single insecticide-resistance allele in 20 DDT-resistant strains of D. melanogaster (Daborn et al., 2002). However, more reports have been demonstrated that multiple over-expressed P450 genes would be co-responsible for the high levels of insecticide resistance in insects, such as co-overexpressed CYP6G1 and CYP12D1 in DDT resistance in D. melanogaster (Festucci-Buselli et al., 2005) and co-upregulation of multiple P450 genes in permethrin resistant house flies (Zhu et al., 2008) and Culex mosquito (Liu et al., 2011; Yang and Liu, 2011). Recently, a genome sequencing of N. lugens provides an opportunity to find out all potential P450 genes associated with imidacloprid resistance (Xue et al., 2014). By comparing the expression levels of all P450 genes between G25 and S25 strains, and RNAi with dsRNA, four P450 genes (CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1) were supposed to be involved in imidacloprid resistance in N. lugens. The higher expression in resistant strain G25 was only a prerequisite for the importance of these P450s in imidacloprid resistance, and the contribution of a P450 was not just decided by its expression levels. In previous studies, although CYP6ER1 had higher expression levels in some resistant populations than that of CYP6AY1, the higher
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Fig. 3. Relative levels of four P450 genes in G and S insects for 25 successive generations in N. lugens. The expression profile of each generation in G insect was compared to S25 (G1, G2, G3 … G25 always compared to S25). Values were plotted as means ± SEM of at least three independent tests.
Fig. 4. Venn diagram analysis of the numbers of inducible P450 genes by imidacloprid (orange), the over-expressed P450 genes in G25 (gray) and P450s contributing to imidacloprid resistance (yellow) in N. lugens. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
catalytic activity of CYP6AY1 to metabolize imidacloprid endowed the comparable importance of two P450s in imidacloprid resistance (Bass et al., 2011; Ding et al., 2013; Bao et al., 2016). Here in the present study, CYP6CW1 showed the highest catalytic activity to metabolize imidacloprid with Kcat value of 5.35 ± 0.71 pmol/min/ pmol P450 among all test P450s, although the over-expression ratio for CYP6CW1 was 5.43-fold and less than that of CYP6AY1, CYP6ER1 and CYP4CE1 genes. The high catalytic activity of CYP6CW1 might compensate for its lower overexpression and endowed the comparable importance with other three P450s. Another typical example was CYP6CS1, which had no catalytic activity to metabolize imidacloprid at all, although its expression level in G25 was 7.57-fold of that in S25. These results indicated that the catalytic activity of P450s was as important as the expression level, in the context of evaluating their roles in insecticide resistance. From the point of view of the final results tested in G25, four P450s were important to imidacloprid resistance. Nevertheless, their roles were not equal among different stages of resistance development. From the over-expression of these P450 genes during resistance development, it was suggestive that CYP6ER1 would
play important roles at all stages, CYP6AY1 at middle to later stages with the sharp increase of imidacloprid resistance, while CYP4CE1 and CYP6CW1 only in few generations. So, when analyzing P450s in insects, the expression differences between strains, expression changes during resistance development, and catalytic activity of multiple P450s were key factors to build up their importance in insecticide resistance. In our previous study, screening the induction of P450 genes by imidacloprid in N. lugens showed that eighteen candidate P450 genes were up-regulated after imidacloprid application (Zhang et al., 2016). Here the detection of fifty-four P450 genes in N. lugens showed that twelve P450 genes were significantly overexpressed in G25 strain when compared to S25, among which five P450 genes in CYP4 Clade (CYP4CE1, CYP4DE1, CYP417A3, CYP425B1 and CYP439A1) and four in CYP3 Clade (CYP6AY1, CYP6CS1, CYP6CW1 and CYP6ER1) were up-regulated both in the previous induction study (Zhang et al., 2016) and in this study. However, the over-expression in G25 and induction up-regulation in Sus strain were not always coincident (Fig. 4). Six P450 genes in Clade4, four in Clade3, and three in Clade2 could be induced by imidacloprid (including up-regulation and down-regulation) (Zhang et al., 2016), but not differently expressed in G25. In contrast, though two P450 genes (CYP4FB2 and CYP439B1) in Clade4 and one (CYP6BD12) in Clade3 were not induced by imidacloprid, the over-expression was found in G25. The induction by xenobiotic, such as imidacloprid, offered a broader targets and detoxification enzymes that might be more likely to be involved in resistance (Le Goff et al., 2006). The induction by imidacloprid application and over-expression in resistant populations might provide more capacity for insect P450s such as CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1 in this study, to adapt to the stress from insecticide application. Therefore, xenobiotic inducibility of detoxification enzymes might represent risk factors for the development of resistance. In conclusion, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1 were supposed to play important roles, although might not equally, in imidacloprid resistance in N. lugens, based on the information from the comparison of the expression levels of all P450 genes between imidacloprid resistant and susceptible strains, their expression levels at different stages of resistance development, and their catalytic activities. The induction of these P450 genes by imidacloprid
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