Pesticide Biochemistry and Physiology xxx (2013) xxx–xxx
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Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China Xin Yang a, Wen Xie b, Shao-li Wang b, Qing-jun Wu b, Hui-peng Pan b, Ru-mei Li b, Ni-na Yang b, Bai-ming Liu b, Bao-yun Xu b, Xiaomao Zhou a, You-jun Zhang b,⇑ a b
Department of Pesticide Science, Hunan Agricultural University, Changsha 410128, PR China Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China
a r t i c l e
i n f o
Article history: Received 3 July 2013 Accepted 1 October 2013 Available online xxxx Keywords: Bemisia tabaci Insecticide resistance Imidacloprid qRT-PCR Cytochrome P450
a b s t r a c t The sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae), is an invasive and damaging pest of field crops worldwide. The neonicotinoid insecticide imidacloprid has been widely used to control this pest. We assessed the species composition (B vs. Q), imidacloprid resistance, and association between imidacloprid resistance and the expression of five P450 genes for 14–17 B. tabaci populations in 12 provinces in China. Fifteen of 17 populations contained only B. tabaci Q, and two populations contained both B and Q. Seven of 17 populations exhibited moderate to high resistance to imidacloprid, and eight populations exhibited low resistance to imidacloprid, compared with the most susceptible field WHHB population. In a study of 14 of the populations, resistance level was correlated with the expression of the P450 genes CYP6CM1 and CYP4C64 but not with the expression of CYP6CX1, CYP6CX4, or CYP6DZ7. This study indicates that B. tabaci Q has a wider distribution in China than previously reported. Resistance to imidacloprid in field populations of B. tabaci is associated with the increased expression of two cytochrome P450 genes (CYP6CM1 and CYP4C64). Ó 2013 Published by Elsevier Inc.
1. Introduction The sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae), is a destructive pest of numerous protected crops and field crops worldwide. This insect directly damages plants by feeding and indirectly damages plants by vectoring more than 100 plant viruses [1]. The B. tabaci species complex is composed of closely related sibling species. These species are morphologically indistinguishable but differ in host range, virus transmission, insecticide resistance, and endosymbionts [2]. Recent studies of mitochondrial cytochrome oxidase I (mtCOI) and inter-sibling species hybridization suggest that most of these B. tabaci variants represent genetically distinct cryptic species [3,4]. Among them, B. tabaci B (also known as ‘Middle East – Asia Minor
Abbreviations: PCR, polymerase chain reaction; qRT-PCR, quantitative real-time PCR; nAChRs, nicotinic acetylcholine receptors; B. tabaci, Bemisia tabaci. ⇑ Corresponding author. Address: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie, Haidian District, Beijing 100081, China. Fax: +86 10 82109518. E-mail addresses: [email protected] (X. Yang), [email protected] (W. Xie), [email protected] (S.-l. Wang), [email protected] (Q.-j. Wu), hppan0623@ sina.com (H.-p. Pan), [email protected] (R.-m. Li), [email protected] (N.-n. Yang), [email protected] (B.-m. Liu), [email protected] (B.-y. Xu), [email protected] (X. Zhou), [email protected] (Y.-j. Zhang).
1 species’) and Q (also known as ‘Mediterrenean species’) are the most invasive [2]. In China, B. tabaci was first recorded in the late 1940s [5]. The crop damage and economic losses caused by this pest, however, did not become serious until the introduction of B. tabaci B in the mid-1990s. Since then, B. tabaci B has rapidly spread across the entire country and has caused serious losses to many crops [6]. In 2003, B. tabaci Q was first found on poinsettia in Yunnan Province and was subsequently found in Beijing, Henan, and Shandong provinces [7]. During the past several years, B. tabaci Q has gradually displaced earlier, well-established populations of B. tabaci B and has become the dominant of B. tabaci in field agricultural systems in most parts of China [8,9]. Control of B. tabaci in crop systems worldwide has largely depended on chemical insecticides, but B. tabaci populations have developed resistance to a wide range of insecticides, including organophosphates, pyrethroids, insect growth regulators (IGRs), and neonicotinoid insecticides [10–12]. Especially during the past two decades, neonicotinoid insecticides have been widely used to control whiteflies in many countries. The major commercial neonicotinoid pesticide, imidacloprid, was introduced in 1991 and is widely used in China to control sucking and biting insect pests. High levels of resistance to neonicotinoid insecticides, however, have been recently reported for B. tabaci populations in America,
0048-3575/$ - see front matter Ó 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.pestbp.2013.10.002
Please cite this article in press as: X. Yang et al., Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China, Pestic. Biochem. Physiol. (2013), http://dx.doi.org/10.1016/j.pestbp.2013.10.002
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X. Yang et al. / Pesticide Biochemistry and Physiology xxx (2013) xxx–xxx
Spain, Crete, and Cyprus [13–16]. In China, imidacloprid resistance in B. tabaci was first detected in 2007; populations in Beijing and Xinjiang were susceptible to the insecticide but populations in Zhejiang, Jiangsu, and Hubei exhibited moderate to high resistance; the RF values (RF = LC50 of a resistant population/LC50 of a susceptible reference population) ranged from 23 to 84 [17]. In 2009, a B. tabaci Q population (YC-Q) with extremely high resistance to imidacloprid (RF = 1900) was found in Jiangsu province [18], and then two other resistant populations (RF = 64 and 57) were found in Shanghai and Jiangsu provinces [19]. The most significant mechanisms of resistance to neonicotinoid insecticides are decreased target-site sensitivity and increased metabolic detoxification by cytochrome P450 monooxygenases. A target site mutation of the nicotinic acetylcholine receptors (nAChR) (Y151S) was identified as the cause of target-site insensitivity to imidacloprid in Nilaparvata lugens in the laboratory [20]. However, this example of target-site resistance has yet to be detected in any field-collected insect population. The discovery of a single mutation (R81T) in Myzus persicae and its association with the reduced affinity of the nAChR for imidacloprid is the first example of field-evolved target-site resistance to neonicotinoid insecticides [21]. The cytochrome P450s constitute a multigenic superfamily of enzymes and play dominant roles in the metabolism of a wide variety of both endogenous and xenobiotic substances. In insects, more than 600 cytochrome P450s have been identified in 17 CYP families and subfamilies, and those in families 4, 6, 9, and 12 have been linked to resistance, i.e., constitutive transcriptional over-expression of CYP genes in these families causes enhanced metabolic detoxification of insecticides [22]. For example, overtranscription of a family CYP6 gene (CYP6G1) has been associated with resistance to DDT in the fruit fly, Drosophila melanogaster [23]. Recent evidence indicates that neonicotinoid resistance in numerous insects is based largely on P450s. Thus, CYP6D1, CYP6D2, and CYP6D3 are constitutively over-expressed in an imidacloprid-resistant strain of the house fly Musca domestica [24]. Enhanced oxidative detoxification of imidacloprid by P450s has also been documented in resistant B. tabaci. Karunker et al. [25,26] measured the expression of several P450 genes in the CYP4 and CYP6 families in resistant and susceptible strains; the mRNA levels of one of these genes, CYP6CM1, was strongly correlated with imidacloprid resistance in both B. tabaci B and Q. Moreover, according to molecular docking and dynamic simulations of interactions between imidacloprid and the CYP6CM1 allele (CYP6CM1vQ), the encoded enzyme catalyses the hydroxylation of imidacloprid to its less toxic 5-hydroxy form, indicating that
CYP6CM1 is associated with reduced susceptibility to imidacloprid. Another P450 gene of B. tabaci, CYP6CX1, is putatively involved in the imidacloprid resistance of a field population [27]. In addition, to identify genes involved in various toxicological processes, researchers subjected an invasive B. tabaci B to pyrosequencingbased transcriptome analysis; the EST sequences of annotated cytochrome P450s P4506a8, CYP6v5, and CYP4v2, whose full-length sequences are CYP6CX4, CYP6DZ7, and CYP4C64, respectively, were significantly over-expressed in a thiamethoxam-resistant strain that was selected for in the laboratory [28]. Most research concerning the association between cytochrome P450s and imidacloprid resistance in insects has been conducted with laboratory populations. The general goals of the current research were to increase our understanding of imidacloprid resistance in field populations of B. tabaci. The specific objectives were to evaluate the current field status of B. tabaci species composition (B vs. Q biotypes) and imidacloprid resistance. We also determined whether the level of imidacloprid resistance in field populations was correlated with the expression of five P450 genes, which were reported to involve in imidacloprid resistance in laboratorial B. tabaci. 2. Materials and methods 2.1. Insect populations In 2011, adult B. tabaci were collected from different host plants at 17 locations in China (Table 1, Fig. 1). A total of approximately 1300 adults were collected per location, 300 of them were snap frozen in liquid nitrogen for 10 min and stored at 80 °C for subsequent analysis. The rest of the adults were used immediately in the bioassays. However, the population of SH, HZZJ, LYHN, and YLSX were maintained on cotton (Gossypium herbaceum L., cv. DP99B) in screen cages in a glasshouse at 25 °C for one generation to detected the resistance level. 2.2. Insecticide and bioassays A bioassay was conducted with formulated imidacloprid (700 g kg1 WG, Bayer Hangzhou Crop Science China Co. Ltd.). The bioassay used the living adults from the 17 populations and was conducted according to the leaf dipping method described by Feng et al. [29]. The insecticide was dissolved and diluted with distilled water containing Triton X-100 (0.1‰) to obtain imidacloprid concentrations of 25, 50, 100, 200, 400, 800, and 1600 mg L1.
Table 1 Background information and species composition (Q vs. B) for Bemisia tabaci collected from 17 locations in China. Populations 1–17 were collected in the current study. Population 13 was used as an imidacloprid-susceptible reference. Population code
Population name
Sampling location
Sampling date
Host plant
B. tabaci composition
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
HDBJ XTSBJ DXBJ NKBJ TZBJ TJ YLQTJ LFHB JZSX JYSD LYHN YLSX WHHB YZJS SH HZZJ CSHN
Haidian, Beijing (39°570 N, 116°190 E) Xiaotangshan, Beijing (40°100 N, 116°240 E) Daxing, Beijing (39°370 N, 116°210 E) Nankou, Beijing (40°130 N, 116°050 E) Tongzhou, Beijing (39°530 N, 116°440 E) Tianjing (39°080 N, 117°110 E) Yangliuqing, Tianjing (39°090 N, 117°000 E) Langfang, Hebei (39°360 N, 116°350 E) Jinzhong, Shanxi (37°250 N, 112°340 E) Jiyang, Shandong (39°490 N, 117°040 E) Luoyang, Henan (34°170 N, 108°040 E) Yangling, Shanxi (34°170 N, 108°040 E) Wuhan, Hubei (30°280 N, 114°200 E) Yangzhou, Jiangsu (32°210 N, 119°200 E) Shanghai (31°130 N, 121°190 E) Hangzhou, Zhejiang (30°180 N, 12°110 E) Changsha, Hunan (28°120 N, 113°050 E)
2011.06 2011.07 2011.08 2011.08 2011.09 2011.06 2011.09 2011.08 2011.10 2011.09 2011.08 2011.08 2011.10 2011.10 2011.10 2011.08 2011.10
Eggplant Eggplant Tomato Tomato Tomato Poinsettia Eggplant Cotton Tomato Eggplant and Pepper Eggplant and Cotton Pepper Cotton Cucumber Eggplant Melon Cucumber
Q Q Q Q Q Q Q Q Q Q B&Q B&Q Q Q Q Q Q
Please cite this article in press as: X. Yang et al., Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China, Pestic. Biochem. Physiol. (2013), http://dx.doi.org/10.1016/j.pestbp.2013.10.002
X. Yang et al. / Pesticide Biochemistry and Physiology xxx (2013) xxx–xxx
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Fig. 1. Sites where Bemisia tabaci was collected in China. Adults of B. tabaci were collected from 17 field sites in 2011. Inset: Map of China showing the provinces (shaded grey) where B. tabaci was collected.
Leaf discs (22 mm diameter) from cotton plants were dipped for 10 s in the insecticide solution or in distilled water (containing 0.1‰ triton X-100) as a control. After they were air dried, the leaf discs were placed with their adaxial surface downwards on agar (2 mL of 15 g/L) in a flat-bottomed glass tube (78 mm long). Adult whiteflies were collected by inverting the tubes above the leaves of the glasshouse cultures so that adults (mixed sex) would fly into the tube. The open end of the tube was then sealed with a cotton plug. Each tube contained 15–30 adults and was kept in an incubator at 25 °C with a 14:10 (L:D) photoperiod. Mortality was recorded after 48 h, and each combination of population and imidacloprid concentration was represented by four replicate tubes. 2.3. DNA/RNA extraction and cDNA synthesis Genomic DNA was extracted from individuals according to Frohlich [3] and was stored at 20 °C until analyzed. Total RNAs from field samples were extracted from 100 B. tabaci adults (mixed sex) using a Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. The resulting total RNA was resuspended in nuclease-free water and quantified with the spectrophotometer (Thermo Scientific Nanodrop 2000). Then, 1.0 lg of RNA of each sample was used to synthesize the firststrand cDNA using the PrimeScriptÒRT reagent Kit (Takara Bio, Tokyo, Japan) with gDNA Eraser (Perfect Real Time) (TaKara, Shiga, Japan) according to the manufacturer’s protocol. 2.4. B. tabaci B and Q determination B. tabaci individuals were identified based on specific mitochondrial cytochrome oxidase I (mtCOI) primers for B. tabaci B (forward primer, 50 -CTAGGGTTTATTGTTTGAGGTCATCATATATTC-30 and reverse primer, 50 -AATATCGACGAGGCATTCCCCCT-30 ) and for B. tabaci Q (forward primer, 50 -CTTGGTAACTCTTCTGTAGATGTGTGTT-30 and reverse primer, 50 -CCTTCCCGCAGAAGAAATTTTGTTC-30 ) [30]. The PCR parameters were as follows: denaturation at 94 °C for 5 min, followed by 35 cycles (94 °C for 30 s, 64 °C for 1 min, 72 °C for 1 min), and a final extension at 72 °C for 10 min.
2.5. Quantitative real-time PCR A total of 300 B. tabaci adults (three biological replicates, n = 100) from each of 14 of the 17 locations were subjected to qRT-PCR analysis. The following three populations were not included because of rearing difficulties or because they contained mixtures of B. tabaci B and Q: NKBJ, LYHN, and YLSX. Five cytochrome P450 genes, CYP6CM1, CYP6CX1, CYP6CX4, CYP6DZ7, and CYP4C64, were reported to correlated with neonicotinoid insecticides in B. tabaci, in which CYP6CM1 is strongly correlated with imidacloprid resistance in both B. tabaci B and Q [25,26], and CYP6CX1 is putatively involved in the resistance of imidacloprid [27], and the other three genes were significantly over-expressed in a thiamethoxam-resistant strain that was selected for in the laboratory [28]. Primers were designed to amplify a 90- to 200-bp fragment as listed in Table 3. The 25-ll reaction system consisted of 1 ll of diluted cDNA, 11.25 ll of SYBRÒ Green Real-time PCR Master Mix (TIANGEN, Corp, Beijing, China), and 0.5 ll of each primer. Real-time PCR was conducted with the ABI 7500 system using the following protocol: 3 min of activation at 95 °C followed by 40 cycles of 40 s at 95 °C, 40 s at 60 °C, and 45 s at 72 °C. A 2-fold dilution series of cDNA was used to construct a relative standard curve, which was used to convert threshold cycle (Ct-values) into raw data (relative quantities). For quantification of gene expression, the Ct values from the ABI 7500 software were imported directly into Microsoft Excel. The fold-changes in CYP450 gene expression, normalized to Actin, which is expressed at similar levels across all strains and life stages [31], which were found to be expressed at comparable levels in all populations analyzed, were calculated using the 244Ct method [32]. All samples were determined from triplicate samples. 2.6. Data analysis For determination of LC50 values, bioassay data were analyzed by probit analysis using the POLO program PC PoloPlus (Leora Software, Berkeley, CA). Mortality was corrected using Abbott’s formula for each probit analysis. The resistance factor (RF) for each
Please cite this article in press as: X. Yang et al., Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China, Pestic. Biochem. Physiol. (2013), http://dx.doi.org/10.1016/j.pestbp.2013.10.002
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Table 2 Responses of field populations of Bemisia tabaci from China to imidacloprid in the laboratory bioassay. Population
Na
Slope (±SE)
dfb
LC50 (mg L1) (95%FLc)
RRd
Resistance ratioe
HDBJ XTSBJ DXBJ NKBJ TZBJ TJ YLQTJ LFHB JZSX JYSD LYHN YLSX WHHB YZJS SH HZZJ CSHN
763 793 365 472 293 806 499 456 348 413 252 304 341 326 294 424 461
1.39 2.16 0.81 0.78 1.45 1.79 1.55 1.63 2.89 0.89 1.18 0.90 1.15 2.16 1.34 1.56 1.13
4 4 4 4 4 4 4 3 4 4 4 4 5 4 4 4 4
134 (105–171) 280 (234–336) 1889 (735–4852) 25,511 (717–908,070) 1488 (706–3133) 245 (199–302) 2129 (1053–4307) 948 (598–1501) 367 (254–531) 523 (255–1069) 320 (165–618) 2098 (505–8705) 82 (45–147) 448 (326–614) 192 (115–321 2790 (638–3125) 287 (138–597)
1.6 3.4 23.0 311.1 18.1 3.0 26.0 11.6 4.5 6.4 3.9 25.6 1 5.5 2.3 34.0 3.5
sus low mod hig mod low mod mod low low low mod – low low hig low
(±0.11) (±0.14) (±0.16) (±0.20) (±0.26) (±0.13) (±0.24) (±0.18) (±0.36) (±0.14) (±0.22) (±0.23) (±0.15) (±0.25) (±0.22) (±0.19) (±0.18)
a
N = numbers of B. tabaci in each bioassay. df = Degree of freedom. c FL = fiducial limit. d RR (resistance ratio) = LC50 of the sample population)/LC50 of population WHHB. e Insecticide resistance ratio was classified by using RFs as reported by Torres-vila. Susceptibility (sus, RF = 1), Low resistance (low, RF = 2–10), Moderate resistance (mod, RF = 11–30), High resistance (hig, RF > 30). b
population was calculated as the LC50 value of the population/LC50 value of the susceptible population (WHHB). Resistance was classified as low, moderate, or high according to RF values as reported by Torres-vila et al. [33]. Linear Model II regression analysis was used to test for functional relationship between the resistance level of the strains and the mean normalized expression value of each gene in each strain by using SPSS (SPSS for Windows, Rel. 17.0.0 2009. Chicago: SPSS Inc.) [25].
HDBJ, but not substantially greater in any of the 12 populations than in the susceptible reference population (Fig. 2D). Relative expression of CYP4C64 was very high in five populations (Fig. 2E). The correlation between relative expression values and resistance (RF values) was significant (P 6 0.05) and positive for CYP6CM1 (P = 0.026) and CYP6C64 (P = 0.002) but was not significant (P > 0.05) for CYP6CX1 (P = 0.265), CYP6CX4 (P = 0.335), or CYP6DZ7 (P = 0.600) (Fig. 3).
3. Results
4. Discussion
3.1. B. tabaci determination
B. tabaci B and Q are two invasive and globally important pests of agricultural and ornamental crops [2]. After B. tabaci B was first found in China in the mid-1990s, it rapidly spread and caused serious damage to many crops [6]. B. tabaci Q, in contrast, was first recorded in China on poinsettias in Yunnan Province in 2003; it then rapidly spread into many regions, including Beijing, Shandong, Henan, and Zhejiang provinces [7]. In 2007, B. tabaci Q was found in field crops in 13 provinces of China and was determined to be the dominant species [8]. In 2009, Pan et al. [9] reported that B. tabaci Q represented 100.0% of 44 populations from 14 provinces. In the current study, B. tabaci Q was detected in all 17 locations sampled in 2011 while B. tabaci B was found in only two locations and in much smaller numbers than B. tabaci Q. This study therefore confirms that B. tabaci Q has become the dominant B. tabaci species in most parts of China. As the earliest commercial neonicotinoid insecticide, imidacloprid has been extensively used for control of B. tabaci in horticultural and other cropping systems [34]. In the past decade, however, B. tabaci B and Q have developed high degrees of resistance to imidacloprid. After about 30 generations of selective pressure under laboratory conditions, a population of B. tabaci B evolved a high degree of imidacloprid resistance, i.e., its RF value was 490 [35]. Previous studies have also documented a high level of neonicotinoid resistance for B. tabaci Q in America, Spain, Crete, and Cyprus [13–16]. In this study, all 17 populations exhibited resistance to imidacloprid; five populations exhibited moderate resistance, and two exhibited high resistance compared with the most susceptible population of WHHB. One population, NKBJ, exhibited very high resistance in that its RF was 311. A previous study reported that a population of B. tabaci Q (YC-Q) in the field
DNA was isolated from 20 to 30 individual whiteflies from each of the 17 populations. PCR amplification was conducted with primers that amplified a single DNA product with an expected size of 478 bp for B. tabaci B and 303 bp for B. tabaci Q. PCR results indicated that 15 populations (across 10 provinces) contained only B. tabaci Q (Table 1). The other two populations contained both B and Q; Q represented 70.0% of the B. tabaci in population LYHN and 84.2% in population YLSX. 3.2. Bioassay One of the 17 B. tabaci populations was susceptible to imidacloprid (RF values of 1.6) (Table 2). Eight populations had low resistance (RF values of 2.3–6.4), five had moderate resistance (RF values of 11.6–26), and two had high resistance (RF values of 34– 311.1). 3.3. CYP450 gene expression qRT-PCR was used to compare the expression of five CYP450 genes in 14 B. tabaci Q populations vs. in the susceptible reference population (WHHB). Relative expression of CYP6CM1 was high in populations TZBJ and HZZJ and moderately high in populations YLQTJ, JZSX, YZJS, and CSHN (Fig. 2A). Relative expression of CYP6CX1 was high in populations HDBJ and TJ and moderatedly high in populations TZBJ, JZSX and YZJS (Fig. 2B). Expression of CYP6CX4 was moderately high in populations YLQTJ, JZSX and YZJS (Fig. 2C). Relative expression of CYP6DZ7 was high in population
Please cite this article in press as: X. Yang et al., Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China, Pestic. Biochem. Physiol. (2013), http://dx.doi.org/10.1016/j.pestbp.2013.10.002
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X. Yang et al. / Pesticide Biochemistry and Physiology xxx (2013) xxx–xxx Table 3 Primers used in quantitative real-time PCR. Putative gene
Primers 50 –30 a
Annealing temperature (°C)
Product (bp)
CYP6CM1 (GenBank GQ214539)
F-CACTCTTTTGGATTTACTGC R-GTGAAGCTGCCTCTTTAATG F-GTGCCCTACATCTCGCCTATC R-CATTTCTTTCGTCGTCTCCAAC F-TTGACAAACTTGCGGGGAACCTC R-CACAGTCTTTCAGCGTCTCGT F-CTGTCTACGGTCTCCATC R-CCGAAAGGCAGATACGAT F-TCGGATTACGTCAGAGCTATTTAC R-GTGGAGCACGCTTAGACA F-ACCGCAAGATTCCATACCC R-CGCTGCCTCCACCTCATT
60
110
60
90
60
126
60
118
60
138
60
174
CYP6CX1 (GenBank GQ292715) CYP6CX4 (GenBank JN165265) CYP6DZ7 (GenBank JX144365) CYP4C64 (GenBank JX144366) Actin (GenBank AF071908) a
F, forward primer; R, reverse primer.
Fig. 2. Expression profiles of five CYP450 genes from B. tabaci representing 14 populations (populations codes listed along the X-axes) in China. (A) CYP6CM1, (B) CYP6CX1, (C) CYP6CX4, (D) CYP6DZ7, and (E) CYP4C64. Relative gene expression was measured by qRT-PCR. The Ct value for tested genes were normalized to the Ct value for Actin and calculated relative to a calibrator using the formula 2DDCt. Values represent means ± SE for three independent replicates.
Please cite this article in press as: X. Yang et al., Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China, Pestic. Biochem. Physiol. (2013), http://dx.doi.org/10.1016/j.pestbp.2013.10.002
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Fig. 3. Linear regression analysis between resistance level and relative gene expression. (A) CYP6CM1, (B) CYP6CX1, (C) CYP6CX4, (D) CYP6DZ7, and (E) CYP4C64. Linear regression analysis was used to test for functional relationship between the resistance level of the strains and the mean normalized expression value of each gene in each strain. Significant regression lines (P 6 0.05) are marked with an asterisk.
of Jiangsu province in China had an imidacloprid RF of 1900 [36]. These results demonstrate that B. tabaci has developed substantial resistance to imidacloprid in most parts of China. Previous studies suggested that the enhancement of metabolic detoxification resulting from an increase in the expression of cytochrome P450 genes leads to resistance to neonicotinoid insecticides [22]. In B. tabaci, expression of the cytochrome P450 gene CYP6CM1 was strongly correlated with neonicotinoid resistance in laboratory populations of B. tabaci B and Q; in addition, the heterologous, in vitro expression of the B. tabaci Q CYP6CM1 allele (CYP6CM1vQ) resulted in rapid detoxification of imidacloprid [13,14], and assessment of the cross-metabolism potential of CYP6CM1vQ against additional neonicotinoid molecules showed that clothianidin and thiacloprid are metabolized by the recombi-
nant enzyme [36]. In the current study, the expression of CYP6CM1 was positively correlated with imidacloprid resistance in 14 field populations. This result suggests that CYP6CM1 is involved in imidacloprid resistance not only in laboratory populations but also in field populations in China. In contrast to Zhuang et al. [27], who reported that an increase in CYP6CX1 expression might be involved in imidacloprid resistance in a field population, the correlation between CYP6CX1 expression and imidacloprid resistance was not significant in the current study. Perhaps over-expression of CYP6CX1 contributes to imidacloprid resistance in some field populations but not others. The expression patterns of CYP6CX4 and CYP6DZ7 were similar to that of CYP6CX1. These two genes were also over-expressed in a few populations but the expression of CYP6CX4 and CYP6DZ7 was
Please cite this article in press as: X. Yang et al., Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China, Pestic. Biochem. Physiol. (2013), http://dx.doi.org/10.1016/j.pestbp.2013.10.002
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not correlated with imidacloprid resistance. These results indicate that these genes do not contribute to imidacloprid resistance in most field populations of B. tabaci Q. However, these two genes were over-expressed in a laboratory B. tabaci B population (TH2000) that had a thiamethoxam RF of approximately 70 [28]. Expression of the P450 gene CYP4C64 was high in eight populations in the current study, and CYP4C64 expression was positively correlated with imidacloprid resistance. A previous paper reported that this gene was also over-expressed in a laboratory population of B. tabaci B that had been selected for thiamethoxam resistance [28]. Another study found that five CYP4 genes (CYP4C67, CYP4DA1, CYP4C68, CYP4DB1, and CYP4G70) in Diaphorina citri are induced by exposure to imidacloprid [37]. Based on the current and previous studies, we suspect that CYP4C64 may be as important as CYP6CM1 for imidacloprid resistance. Although these results demonstrate a strong association between imidacloprid resistance and CYP4C64 expression in field populations of B. tabaci Q, further studies of the gene and the encoded protein are needed to confirm its role. In summary, this paper provides evidence that B. tabaci Q has continued to spread into most parts of China and has gradually replaced B. tabaci B as the dominant species. The imidacloprid resistance levels in field populations were correlated with overexpression of two P450 genes (CYP6CM1 and CYP4C64). Although this indicates that resistance involves detoxification by the two P450 monooxygenases, our findings do not exclude the possibility that the other P450 monooxygenases and target-site resistance to imidacloprid may also be important.
Conflict of interests The authors have declared that no conflict of interest exists.
Acknowledgments This research was supported by the National Science Fund for Distinguished Young Scholars (31025020); the 863 program (2012AA101502); the National Science &Technology pillar program (2012BAD19B01); the Special Fund for Agro-scientific Research in the Public Interest (201203038); and the Beijing Key Laboratory for Pest Control and Sustainable Cultivation of Vegetables.
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Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China Xin Yang a, Wen Xie b, Shao-li Wang b, Qing-jun Wu b, Hui-peng Pan b, Ru-mei Li b, Ni-na Yang b, Bai-ming Liu b, Bao-yun Xu b, Xiaomao Zhou a, You-jun Zhang b,⇑ a b
Department of Pesticide Science, Hunan Agricultural University, Changsha 410128, PR China Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China
a r t i c l e
i n f o
Article history: Received 3 July 2013 Accepted 1 October 2013 Available online xxxx Keywords: Bemisia tabaci Insecticide resistance Imidacloprid qRT-PCR Cytochrome P450
a b s t r a c t The sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae), is an invasive and damaging pest of field crops worldwide. The neonicotinoid insecticide imidacloprid has been widely used to control this pest. We assessed the species composition (B vs. Q), imidacloprid resistance, and association between imidacloprid resistance and the expression of five P450 genes for 14–17 B. tabaci populations in 12 provinces in China. Fifteen of 17 populations contained only B. tabaci Q, and two populations contained both B and Q. Seven of 17 populations exhibited moderate to high resistance to imidacloprid, and eight populations exhibited low resistance to imidacloprid, compared with the most susceptible field WHHB population. In a study of 14 of the populations, resistance level was correlated with the expression of the P450 genes CYP6CM1 and CYP4C64 but not with the expression of CYP6CX1, CYP6CX4, or CYP6DZ7. This study indicates that B. tabaci Q has a wider distribution in China than previously reported. Resistance to imidacloprid in field populations of B. tabaci is associated with the increased expression of two cytochrome P450 genes (CYP6CM1 and CYP4C64). Ó 2013 Published by Elsevier Inc.
1. Introduction The sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae), is a destructive pest of numerous protected crops and field crops worldwide. This insect directly damages plants by feeding and indirectly damages plants by vectoring more than 100 plant viruses [1]. The B. tabaci species complex is composed of closely related sibling species. These species are morphologically indistinguishable but differ in host range, virus transmission, insecticide resistance, and endosymbionts [2]. Recent studies of mitochondrial cytochrome oxidase I (mtCOI) and inter-sibling species hybridization suggest that most of these B. tabaci variants represent genetically distinct cryptic species [3,4]. Among them, B. tabaci B (also known as ‘Middle East – Asia Minor
Abbreviations: PCR, polymerase chain reaction; qRT-PCR, quantitative real-time PCR; nAChRs, nicotinic acetylcholine receptors; B. tabaci, Bemisia tabaci. ⇑ Corresponding author. Address: Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie, Haidian District, Beijing 100081, China. Fax: +86 10 82109518. E-mail addresses: [email protected] (X. Yang), [email protected] (W. Xie), [email protected] (S.-l. Wang), [email protected] (Q.-j. Wu), hppan0623@ sina.com (H.-p. Pan), [email protected] (R.-m. Li), [email protected] (N.-n. Yang), [email protected] (B.-m. Liu), [email protected] (B.-y. Xu), [email protected] (X. Zhou), [email protected] (Y.-j. Zhang).
1 species’) and Q (also known as ‘Mediterrenean species’) are the most invasive [2]. In China, B. tabaci was first recorded in the late 1940s [5]. The crop damage and economic losses caused by this pest, however, did not become serious until the introduction of B. tabaci B in the mid-1990s. Since then, B. tabaci B has rapidly spread across the entire country and has caused serious losses to many crops [6]. In 2003, B. tabaci Q was first found on poinsettia in Yunnan Province and was subsequently found in Beijing, Henan, and Shandong provinces [7]. During the past several years, B. tabaci Q has gradually displaced earlier, well-established populations of B. tabaci B and has become the dominant of B. tabaci in field agricultural systems in most parts of China [8,9]. Control of B. tabaci in crop systems worldwide has largely depended on chemical insecticides, but B. tabaci populations have developed resistance to a wide range of insecticides, including organophosphates, pyrethroids, insect growth regulators (IGRs), and neonicotinoid insecticides [10–12]. Especially during the past two decades, neonicotinoid insecticides have been widely used to control whiteflies in many countries. The major commercial neonicotinoid pesticide, imidacloprid, was introduced in 1991 and is widely used in China to control sucking and biting insect pests. High levels of resistance to neonicotinoid insecticides, however, have been recently reported for B. tabaci populations in America,
0048-3575/$ - see front matter Ó 2013 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.pestbp.2013.10.002
Please cite this article in press as: X. Yang et al., Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China, Pestic. Biochem. Physiol. (2013), http://dx.doi.org/10.1016/j.pestbp.2013.10.002
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Spain, Crete, and Cyprus [13–16]. In China, imidacloprid resistance in B. tabaci was first detected in 2007; populations in Beijing and Xinjiang were susceptible to the insecticide but populations in Zhejiang, Jiangsu, and Hubei exhibited moderate to high resistance; the RF values (RF = LC50 of a resistant population/LC50 of a susceptible reference population) ranged from 23 to 84 [17]. In 2009, a B. tabaci Q population (YC-Q) with extremely high resistance to imidacloprid (RF = 1900) was found in Jiangsu province [18], and then two other resistant populations (RF = 64 and 57) were found in Shanghai and Jiangsu provinces [19]. The most significant mechanisms of resistance to neonicotinoid insecticides are decreased target-site sensitivity and increased metabolic detoxification by cytochrome P450 monooxygenases. A target site mutation of the nicotinic acetylcholine receptors (nAChR) (Y151S) was identified as the cause of target-site insensitivity to imidacloprid in Nilaparvata lugens in the laboratory [20]. However, this example of target-site resistance has yet to be detected in any field-collected insect population. The discovery of a single mutation (R81T) in Myzus persicae and its association with the reduced affinity of the nAChR for imidacloprid is the first example of field-evolved target-site resistance to neonicotinoid insecticides [21]. The cytochrome P450s constitute a multigenic superfamily of enzymes and play dominant roles in the metabolism of a wide variety of both endogenous and xenobiotic substances. In insects, more than 600 cytochrome P450s have been identified in 17 CYP families and subfamilies, and those in families 4, 6, 9, and 12 have been linked to resistance, i.e., constitutive transcriptional over-expression of CYP genes in these families causes enhanced metabolic detoxification of insecticides [22]. For example, overtranscription of a family CYP6 gene (CYP6G1) has been associated with resistance to DDT in the fruit fly, Drosophila melanogaster [23]. Recent evidence indicates that neonicotinoid resistance in numerous insects is based largely on P450s. Thus, CYP6D1, CYP6D2, and CYP6D3 are constitutively over-expressed in an imidacloprid-resistant strain of the house fly Musca domestica [24]. Enhanced oxidative detoxification of imidacloprid by P450s has also been documented in resistant B. tabaci. Karunker et al. [25,26] measured the expression of several P450 genes in the CYP4 and CYP6 families in resistant and susceptible strains; the mRNA levels of one of these genes, CYP6CM1, was strongly correlated with imidacloprid resistance in both B. tabaci B and Q. Moreover, according to molecular docking and dynamic simulations of interactions between imidacloprid and the CYP6CM1 allele (CYP6CM1vQ), the encoded enzyme catalyses the hydroxylation of imidacloprid to its less toxic 5-hydroxy form, indicating that
CYP6CM1 is associated with reduced susceptibility to imidacloprid. Another P450 gene of B. tabaci, CYP6CX1, is putatively involved in the imidacloprid resistance of a field population [27]. In addition, to identify genes involved in various toxicological processes, researchers subjected an invasive B. tabaci B to pyrosequencingbased transcriptome analysis; the EST sequences of annotated cytochrome P450s P4506a8, CYP6v5, and CYP4v2, whose full-length sequences are CYP6CX4, CYP6DZ7, and CYP4C64, respectively, were significantly over-expressed in a thiamethoxam-resistant strain that was selected for in the laboratory [28]. Most research concerning the association between cytochrome P450s and imidacloprid resistance in insects has been conducted with laboratory populations. The general goals of the current research were to increase our understanding of imidacloprid resistance in field populations of B. tabaci. The specific objectives were to evaluate the current field status of B. tabaci species composition (B vs. Q biotypes) and imidacloprid resistance. We also determined whether the level of imidacloprid resistance in field populations was correlated with the expression of five P450 genes, which were reported to involve in imidacloprid resistance in laboratorial B. tabaci. 2. Materials and methods 2.1. Insect populations In 2011, adult B. tabaci were collected from different host plants at 17 locations in China (Table 1, Fig. 1). A total of approximately 1300 adults were collected per location, 300 of them were snap frozen in liquid nitrogen for 10 min and stored at 80 °C for subsequent analysis. The rest of the adults were used immediately in the bioassays. However, the population of SH, HZZJ, LYHN, and YLSX were maintained on cotton (Gossypium herbaceum L., cv. DP99B) in screen cages in a glasshouse at 25 °C for one generation to detected the resistance level. 2.2. Insecticide and bioassays A bioassay was conducted with formulated imidacloprid (700 g kg1 WG, Bayer Hangzhou Crop Science China Co. Ltd.). The bioassay used the living adults from the 17 populations and was conducted according to the leaf dipping method described by Feng et al. [29]. The insecticide was dissolved and diluted with distilled water containing Triton X-100 (0.1‰) to obtain imidacloprid concentrations of 25, 50, 100, 200, 400, 800, and 1600 mg L1.
Table 1 Background information and species composition (Q vs. B) for Bemisia tabaci collected from 17 locations in China. Populations 1–17 were collected in the current study. Population 13 was used as an imidacloprid-susceptible reference. Population code
Population name
Sampling location
Sampling date
Host plant
B. tabaci composition
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
HDBJ XTSBJ DXBJ NKBJ TZBJ TJ YLQTJ LFHB JZSX JYSD LYHN YLSX WHHB YZJS SH HZZJ CSHN
Haidian, Beijing (39°570 N, 116°190 E) Xiaotangshan, Beijing (40°100 N, 116°240 E) Daxing, Beijing (39°370 N, 116°210 E) Nankou, Beijing (40°130 N, 116°050 E) Tongzhou, Beijing (39°530 N, 116°440 E) Tianjing (39°080 N, 117°110 E) Yangliuqing, Tianjing (39°090 N, 117°000 E) Langfang, Hebei (39°360 N, 116°350 E) Jinzhong, Shanxi (37°250 N, 112°340 E) Jiyang, Shandong (39°490 N, 117°040 E) Luoyang, Henan (34°170 N, 108°040 E) Yangling, Shanxi (34°170 N, 108°040 E) Wuhan, Hubei (30°280 N, 114°200 E) Yangzhou, Jiangsu (32°210 N, 119°200 E) Shanghai (31°130 N, 121°190 E) Hangzhou, Zhejiang (30°180 N, 12°110 E) Changsha, Hunan (28°120 N, 113°050 E)
2011.06 2011.07 2011.08 2011.08 2011.09 2011.06 2011.09 2011.08 2011.10 2011.09 2011.08 2011.08 2011.10 2011.10 2011.10 2011.08 2011.10
Eggplant Eggplant Tomato Tomato Tomato Poinsettia Eggplant Cotton Tomato Eggplant and Pepper Eggplant and Cotton Pepper Cotton Cucumber Eggplant Melon Cucumber
Q Q Q Q Q Q Q Q Q Q B&Q B&Q Q Q Q Q Q
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X. Yang et al. / Pesticide Biochemistry and Physiology xxx (2013) xxx–xxx
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Fig. 1. Sites where Bemisia tabaci was collected in China. Adults of B. tabaci were collected from 17 field sites in 2011. Inset: Map of China showing the provinces (shaded grey) where B. tabaci was collected.
Leaf discs (22 mm diameter) from cotton plants were dipped for 10 s in the insecticide solution or in distilled water (containing 0.1‰ triton X-100) as a control. After they were air dried, the leaf discs were placed with their adaxial surface downwards on agar (2 mL of 15 g/L) in a flat-bottomed glass tube (78 mm long). Adult whiteflies were collected by inverting the tubes above the leaves of the glasshouse cultures so that adults (mixed sex) would fly into the tube. The open end of the tube was then sealed with a cotton plug. Each tube contained 15–30 adults and was kept in an incubator at 25 °C with a 14:10 (L:D) photoperiod. Mortality was recorded after 48 h, and each combination of population and imidacloprid concentration was represented by four replicate tubes. 2.3. DNA/RNA extraction and cDNA synthesis Genomic DNA was extracted from individuals according to Frohlich [3] and was stored at 20 °C until analyzed. Total RNAs from field samples were extracted from 100 B. tabaci adults (mixed sex) using a Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. The resulting total RNA was resuspended in nuclease-free water and quantified with the spectrophotometer (Thermo Scientific Nanodrop 2000). Then, 1.0 lg of RNA of each sample was used to synthesize the firststrand cDNA using the PrimeScriptÒRT reagent Kit (Takara Bio, Tokyo, Japan) with gDNA Eraser (Perfect Real Time) (TaKara, Shiga, Japan) according to the manufacturer’s protocol. 2.4. B. tabaci B and Q determination B. tabaci individuals were identified based on specific mitochondrial cytochrome oxidase I (mtCOI) primers for B. tabaci B (forward primer, 50 -CTAGGGTTTATTGTTTGAGGTCATCATATATTC-30 and reverse primer, 50 -AATATCGACGAGGCATTCCCCCT-30 ) and for B. tabaci Q (forward primer, 50 -CTTGGTAACTCTTCTGTAGATGTGTGTT-30 and reverse primer, 50 -CCTTCCCGCAGAAGAAATTTTGTTC-30 ) [30]. The PCR parameters were as follows: denaturation at 94 °C for 5 min, followed by 35 cycles (94 °C for 30 s, 64 °C for 1 min, 72 °C for 1 min), and a final extension at 72 °C for 10 min.
2.5. Quantitative real-time PCR A total of 300 B. tabaci adults (three biological replicates, n = 100) from each of 14 of the 17 locations were subjected to qRT-PCR analysis. The following three populations were not included because of rearing difficulties or because they contained mixtures of B. tabaci B and Q: NKBJ, LYHN, and YLSX. Five cytochrome P450 genes, CYP6CM1, CYP6CX1, CYP6CX4, CYP6DZ7, and CYP4C64, were reported to correlated with neonicotinoid insecticides in B. tabaci, in which CYP6CM1 is strongly correlated with imidacloprid resistance in both B. tabaci B and Q [25,26], and CYP6CX1 is putatively involved in the resistance of imidacloprid [27], and the other three genes were significantly over-expressed in a thiamethoxam-resistant strain that was selected for in the laboratory [28]. Primers were designed to amplify a 90- to 200-bp fragment as listed in Table 3. The 25-ll reaction system consisted of 1 ll of diluted cDNA, 11.25 ll of SYBRÒ Green Real-time PCR Master Mix (TIANGEN, Corp, Beijing, China), and 0.5 ll of each primer. Real-time PCR was conducted with the ABI 7500 system using the following protocol: 3 min of activation at 95 °C followed by 40 cycles of 40 s at 95 °C, 40 s at 60 °C, and 45 s at 72 °C. A 2-fold dilution series of cDNA was used to construct a relative standard curve, which was used to convert threshold cycle (Ct-values) into raw data (relative quantities). For quantification of gene expression, the Ct values from the ABI 7500 software were imported directly into Microsoft Excel. The fold-changes in CYP450 gene expression, normalized to Actin, which is expressed at similar levels across all strains and life stages [31], which were found to be expressed at comparable levels in all populations analyzed, were calculated using the 244Ct method [32]. All samples were determined from triplicate samples. 2.6. Data analysis For determination of LC50 values, bioassay data were analyzed by probit analysis using the POLO program PC PoloPlus (Leora Software, Berkeley, CA). Mortality was corrected using Abbott’s formula for each probit analysis. The resistance factor (RF) for each
Please cite this article in press as: X. Yang et al., Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China, Pestic. Biochem. Physiol. (2013), http://dx.doi.org/10.1016/j.pestbp.2013.10.002
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Table 2 Responses of field populations of Bemisia tabaci from China to imidacloprid in the laboratory bioassay. Population
Na
Slope (±SE)
dfb
LC50 (mg L1) (95%FLc)
RRd
Resistance ratioe
HDBJ XTSBJ DXBJ NKBJ TZBJ TJ YLQTJ LFHB JZSX JYSD LYHN YLSX WHHB YZJS SH HZZJ CSHN
763 793 365 472 293 806 499 456 348 413 252 304 341 326 294 424 461
1.39 2.16 0.81 0.78 1.45 1.79 1.55 1.63 2.89 0.89 1.18 0.90 1.15 2.16 1.34 1.56 1.13
4 4 4 4 4 4 4 3 4 4 4 4 5 4 4 4 4
134 (105–171) 280 (234–336) 1889 (735–4852) 25,511 (717–908,070) 1488 (706–3133) 245 (199–302) 2129 (1053–4307) 948 (598–1501) 367 (254–531) 523 (255–1069) 320 (165–618) 2098 (505–8705) 82 (45–147) 448 (326–614) 192 (115–321 2790 (638–3125) 287 (138–597)
1.6 3.4 23.0 311.1 18.1 3.0 26.0 11.6 4.5 6.4 3.9 25.6 1 5.5 2.3 34.0 3.5
sus low mod hig mod low mod mod low low low mod – low low hig low
(±0.11) (±0.14) (±0.16) (±0.20) (±0.26) (±0.13) (±0.24) (±0.18) (±0.36) (±0.14) (±0.22) (±0.23) (±0.15) (±0.25) (±0.22) (±0.19) (±0.18)
a
N = numbers of B. tabaci in each bioassay. df = Degree of freedom. c FL = fiducial limit. d RR (resistance ratio) = LC50 of the sample population)/LC50 of population WHHB. e Insecticide resistance ratio was classified by using RFs as reported by Torres-vila. Susceptibility (sus, RF = 1), Low resistance (low, RF = 2–10), Moderate resistance (mod, RF = 11–30), High resistance (hig, RF > 30). b
population was calculated as the LC50 value of the population/LC50 value of the susceptible population (WHHB). Resistance was classified as low, moderate, or high according to RF values as reported by Torres-vila et al. [33]. Linear Model II regression analysis was used to test for functional relationship between the resistance level of the strains and the mean normalized expression value of each gene in each strain by using SPSS (SPSS for Windows, Rel. 17.0.0 2009. Chicago: SPSS Inc.) [25].
HDBJ, but not substantially greater in any of the 12 populations than in the susceptible reference population (Fig. 2D). Relative expression of CYP4C64 was very high in five populations (Fig. 2E). The correlation between relative expression values and resistance (RF values) was significant (P 6 0.05) and positive for CYP6CM1 (P = 0.026) and CYP6C64 (P = 0.002) but was not significant (P > 0.05) for CYP6CX1 (P = 0.265), CYP6CX4 (P = 0.335), or CYP6DZ7 (P = 0.600) (Fig. 3).
3. Results
4. Discussion
3.1. B. tabaci determination
B. tabaci B and Q are two invasive and globally important pests of agricultural and ornamental crops [2]. After B. tabaci B was first found in China in the mid-1990s, it rapidly spread and caused serious damage to many crops [6]. B. tabaci Q, in contrast, was first recorded in China on poinsettias in Yunnan Province in 2003; it then rapidly spread into many regions, including Beijing, Shandong, Henan, and Zhejiang provinces [7]. In 2007, B. tabaci Q was found in field crops in 13 provinces of China and was determined to be the dominant species [8]. In 2009, Pan et al. [9] reported that B. tabaci Q represented 100.0% of 44 populations from 14 provinces. In the current study, B. tabaci Q was detected in all 17 locations sampled in 2011 while B. tabaci B was found in only two locations and in much smaller numbers than B. tabaci Q. This study therefore confirms that B. tabaci Q has become the dominant B. tabaci species in most parts of China. As the earliest commercial neonicotinoid insecticide, imidacloprid has been extensively used for control of B. tabaci in horticultural and other cropping systems [34]. In the past decade, however, B. tabaci B and Q have developed high degrees of resistance to imidacloprid. After about 30 generations of selective pressure under laboratory conditions, a population of B. tabaci B evolved a high degree of imidacloprid resistance, i.e., its RF value was 490 [35]. Previous studies have also documented a high level of neonicotinoid resistance for B. tabaci Q in America, Spain, Crete, and Cyprus [13–16]. In this study, all 17 populations exhibited resistance to imidacloprid; five populations exhibited moderate resistance, and two exhibited high resistance compared with the most susceptible population of WHHB. One population, NKBJ, exhibited very high resistance in that its RF was 311. A previous study reported that a population of B. tabaci Q (YC-Q) in the field
DNA was isolated from 20 to 30 individual whiteflies from each of the 17 populations. PCR amplification was conducted with primers that amplified a single DNA product with an expected size of 478 bp for B. tabaci B and 303 bp for B. tabaci Q. PCR results indicated that 15 populations (across 10 provinces) contained only B. tabaci Q (Table 1). The other two populations contained both B and Q; Q represented 70.0% of the B. tabaci in population LYHN and 84.2% in population YLSX. 3.2. Bioassay One of the 17 B. tabaci populations was susceptible to imidacloprid (RF values of 1.6) (Table 2). Eight populations had low resistance (RF values of 2.3–6.4), five had moderate resistance (RF values of 11.6–26), and two had high resistance (RF values of 34– 311.1). 3.3. CYP450 gene expression qRT-PCR was used to compare the expression of five CYP450 genes in 14 B. tabaci Q populations vs. in the susceptible reference population (WHHB). Relative expression of CYP6CM1 was high in populations TZBJ and HZZJ and moderately high in populations YLQTJ, JZSX, YZJS, and CSHN (Fig. 2A). Relative expression of CYP6CX1 was high in populations HDBJ and TJ and moderatedly high in populations TZBJ, JZSX and YZJS (Fig. 2B). Expression of CYP6CX4 was moderately high in populations YLQTJ, JZSX and YZJS (Fig. 2C). Relative expression of CYP6DZ7 was high in population
Please cite this article in press as: X. Yang et al., Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China, Pestic. Biochem. Physiol. (2013), http://dx.doi.org/10.1016/j.pestbp.2013.10.002
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X. Yang et al. / Pesticide Biochemistry and Physiology xxx (2013) xxx–xxx Table 3 Primers used in quantitative real-time PCR. Putative gene
Primers 50 –30 a
Annealing temperature (°C)
Product (bp)
CYP6CM1 (GenBank GQ214539)
F-CACTCTTTTGGATTTACTGC R-GTGAAGCTGCCTCTTTAATG F-GTGCCCTACATCTCGCCTATC R-CATTTCTTTCGTCGTCTCCAAC F-TTGACAAACTTGCGGGGAACCTC R-CACAGTCTTTCAGCGTCTCGT F-CTGTCTACGGTCTCCATC R-CCGAAAGGCAGATACGAT F-TCGGATTACGTCAGAGCTATTTAC R-GTGGAGCACGCTTAGACA F-ACCGCAAGATTCCATACCC R-CGCTGCCTCCACCTCATT
60
110
60
90
60
126
60
118
60
138
60
174
CYP6CX1 (GenBank GQ292715) CYP6CX4 (GenBank JN165265) CYP6DZ7 (GenBank JX144365) CYP4C64 (GenBank JX144366) Actin (GenBank AF071908) a
F, forward primer; R, reverse primer.
Fig. 2. Expression profiles of five CYP450 genes from B. tabaci representing 14 populations (populations codes listed along the X-axes) in China. (A) CYP6CM1, (B) CYP6CX1, (C) CYP6CX4, (D) CYP6DZ7, and (E) CYP4C64. Relative gene expression was measured by qRT-PCR. The Ct value for tested genes were normalized to the Ct value for Actin and calculated relative to a calibrator using the formula 2DDCt. Values represent means ± SE for three independent replicates.
Please cite this article in press as: X. Yang et al., Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China, Pestic. Biochem. Physiol. (2013), http://dx.doi.org/10.1016/j.pestbp.2013.10.002
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X. Yang et al. / Pesticide Biochemistry and Physiology xxx (2013) xxx–xxx
Fig. 3. Linear regression analysis between resistance level and relative gene expression. (A) CYP6CM1, (B) CYP6CX1, (C) CYP6CX4, (D) CYP6DZ7, and (E) CYP4C64. Linear regression analysis was used to test for functional relationship between the resistance level of the strains and the mean normalized expression value of each gene in each strain. Significant regression lines (P 6 0.05) are marked with an asterisk.
of Jiangsu province in China had an imidacloprid RF of 1900 [36]. These results demonstrate that B. tabaci has developed substantial resistance to imidacloprid in most parts of China. Previous studies suggested that the enhancement of metabolic detoxification resulting from an increase in the expression of cytochrome P450 genes leads to resistance to neonicotinoid insecticides [22]. In B. tabaci, expression of the cytochrome P450 gene CYP6CM1 was strongly correlated with neonicotinoid resistance in laboratory populations of B. tabaci B and Q; in addition, the heterologous, in vitro expression of the B. tabaci Q CYP6CM1 allele (CYP6CM1vQ) resulted in rapid detoxification of imidacloprid [13,14], and assessment of the cross-metabolism potential of CYP6CM1vQ against additional neonicotinoid molecules showed that clothianidin and thiacloprid are metabolized by the recombi-
nant enzyme [36]. In the current study, the expression of CYP6CM1 was positively correlated with imidacloprid resistance in 14 field populations. This result suggests that CYP6CM1 is involved in imidacloprid resistance not only in laboratory populations but also in field populations in China. In contrast to Zhuang et al. [27], who reported that an increase in CYP6CX1 expression might be involved in imidacloprid resistance in a field population, the correlation between CYP6CX1 expression and imidacloprid resistance was not significant in the current study. Perhaps over-expression of CYP6CX1 contributes to imidacloprid resistance in some field populations but not others. The expression patterns of CYP6CX4 and CYP6DZ7 were similar to that of CYP6CX1. These two genes were also over-expressed in a few populations but the expression of CYP6CX4 and CYP6DZ7 was
Please cite this article in press as: X. Yang et al., Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China, Pestic. Biochem. Physiol. (2013), http://dx.doi.org/10.1016/j.pestbp.2013.10.002
X. Yang et al. / Pesticide Biochemistry and Physiology xxx (2013) xxx–xxx
not correlated with imidacloprid resistance. These results indicate that these genes do not contribute to imidacloprid resistance in most field populations of B. tabaci Q. However, these two genes were over-expressed in a laboratory B. tabaci B population (TH2000) that had a thiamethoxam RF of approximately 70 [28]. Expression of the P450 gene CYP4C64 was high in eight populations in the current study, and CYP4C64 expression was positively correlated with imidacloprid resistance. A previous paper reported that this gene was also over-expressed in a laboratory population of B. tabaci B that had been selected for thiamethoxam resistance [28]. Another study found that five CYP4 genes (CYP4C67, CYP4DA1, CYP4C68, CYP4DB1, and CYP4G70) in Diaphorina citri are induced by exposure to imidacloprid [37]. Based on the current and previous studies, we suspect that CYP4C64 may be as important as CYP6CM1 for imidacloprid resistance. Although these results demonstrate a strong association between imidacloprid resistance and CYP4C64 expression in field populations of B. tabaci Q, further studies of the gene and the encoded protein are needed to confirm its role. In summary, this paper provides evidence that B. tabaci Q has continued to spread into most parts of China and has gradually replaced B. tabaci B as the dominant species. The imidacloprid resistance levels in field populations were correlated with overexpression of two P450 genes (CYP6CM1 and CYP4C64). Although this indicates that resistance involves detoxification by the two P450 monooxygenases, our findings do not exclude the possibility that the other P450 monooxygenases and target-site resistance to imidacloprid may also be important.
Conflict of interests The authors have declared that no conflict of interest exists.
Acknowledgments This research was supported by the National Science Fund for Distinguished Young Scholars (31025020); the 863 program (2012AA101502); the National Science &Technology pillar program (2012BAD19B01); the Special Fund for Agro-scientific Research in the Public Interest (201203038); and the Beijing Key Laboratory for Pest Control and Sustainable Cultivation of Vegetables.
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