Insect Science (2011) 18, 484494, DOI 10.1111/j.1744-7917.2010.01380.

ORIGINAL ARTICLE

Identication and characterization of a cytochrome P450 CYP6CX1 putatively associated with insecticide resistance in Bemisia tabaci
Hua-Mei Zhuang1 , Kuan-Fu Wang1 , Lin Zheng1 , Zu-Jian Wu1 , Tadashi Miyata2 and Gang Wu1
1

Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou, Fujian,

China, 2 Laboratory of Applied Entomology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

Abstract The novel full length of cytochrome P450 gene has been isolated in insecticideresistant (named CYP6CX1v1) and -susceptible (named CYP6CX1v2) Bemisia tabaci, which was identified as B biotype, in Shangjie, Fujian, China (Sj). CYP6CX1 (1 940 bp contained a 1 557 bp open reading frame) included conserved domains common to CYP6 members, such as heme-binding motif PFGEGPRFCIA, putative meander-binding sequence ETLR and PERF in helix-K, oxygen-binding motif AGLDPV and conserved sequence PEKFNP near the carboxyl end. There were four different replacements of amino acid residues between R and S B. tabaci (Thr300 Ala, Thr354Pro, Arg486His and Ile503Thr), among which the substitution Ile503Thr was located in the substrate recognition sites region. The mRNA transcription level of CYP6CX1v1 was 2.38-fold as high as that of CYP6CX1v2. The results indicated that the CYP6CX1 from the B biotype B. tabaci in Sj was one of the CYP6 members, and enhanced CYP6CX1 expression and substitute of amino acid residues might be involved in the resistance mechanisms in field B. tabaci. Key words Bemisia tabaci, biotype, cytochrome P450s monooxygenases, insecticide resistance

Introduction Bemisia tabaci (Gennadius) is a very important cosmopolitan insect pest of cruciferous crops and causes serious losses due to the direct (phloem feeding) or indirect damage (transmitting plant viruses and excreting honeydew) (G cmen & Devran, 2002). It is considered o to be a highly cryptic species complex, and more than 24 biotypes of B. tabaci have been identified by various techniques (Roditakis et al., 2005). Different B. tabaci vary in species of host plant, spread ability, adaptability to different habitats, transmitting plant viruses and resistance

Correspondence: Gang Wu, Department of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China. Tel & fax: +86 591 87646115; email: newugan@ 163.com

to insecticides, depending on different biotypes. Among the 24 biotypes, B-biotype of B. tabaci, due to its wide spread ability, high fecundity, polyphagous nature, fast adaptability to insecticides and being a vector of many geminiviruses, is one of the most damaging biotypes in numerous crops world-wide (Karunker et al., 2008). Further, B-, A-, Q-, Cv- and No-B-biotype have been found in China to-date, based on the sequence comparison of mitochondrial cytochrome oxidase I (mt-COI) gene sequences as markers, and B biotype was the most widespread and damaging biotype (Liu et al., 2007; Qiu et al., 2009). Because of the indiscriminate application of insecticides, B. tabaci had developed significant resistance to numerous insecticide classes (Prabhaker et al., 2008; Fern ndez et al., 2009). Resistance to approximately a 35 active ingredients had been reported for B tabaci in at least 20 countries world-wide (Roditakis et al., 2005). Among the detoxification systems, it is known

C 2010 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences

484

A novel P450 gene isolated from Bemisia tabaci

485

that P450s (encoded by CYP genes) plays a dominant role in the metabolism of a wide variety of both endogenous and xenobiotic substances, thus contributing numerous functions, including growth, development, nutrition and xenobiotic detoxification in insects (Karunker et al., 2008; Bautista et al., 2007). Only monooxygenase activity correlates with imidacloprid, thiamethoxam, acetamiprid and neonicotinoid resistance, among metabolizing enzymes such as esterases, glutathione S-transferases and cytochrome P450-dependent monooxygenases (Rauch & Nauen, 2003). P450 and carboxylesterase (CarE) are involved in a highly resistant B. tabaci strain, while glutathione S-transferase (GST) is not different between resistant and susceptible B. tabaci (Roditakis et al., 2006). The metabolisms of P450s are critical in resistance to insecticides in the field populations of B. tabaci (Karunker et al., 2008, 2009; Kang et al., 2006). The total number of P450 genes, a superfamily, in insects registered in the GenBank, is over 1 000. The P450 genes of insects belong to CYP4, CYP6, CYP9, CYP12, CYP15, CYP18, CYP28, CYP49 and CYP308 families. Among these families, CYP4, CYP6, CYP9, CYP12, CYP18 and CYP28 are found only in insect species (Berge et al., 1998; Amenya et al., 2008; Guo et al., 2009). The total 256 full-length sequences of P450 genes in insects are registered. Among them, 20% are CYP6 members and 45% CYP4 members (Guo et al., 2009). CYP4 and CYP6 members are thought to be involved in the resistance to insecticides in insect species because of significant over-expression. A complementary DNA (cDNA) microarray from B. tabaci was used to monitor changes in gene expression in a resistant B. tabaci population. One hundred and eleven expressed sequence tags (ESTs) were identified that are differentially up-regulated in the resistant strain after pyriproxyfen treatment. Many of the up-regulated ESTs belong to families usually associated with resistance and xenobiotic detoxification, and some ESTs belong to P450 families (Ghanim & Kontsedalov, 2007). Eleven distinct P450 cDNA sequences from Band Q-biotype B. tabaci were cloned and were classified as members of the CYP4 or CYP6 families. In addition, one full-length P450 gene CYP6CM1 was obtained, and constitutive over-expression, structural model and functional characterization of CYP6CM1, which was associated with imidacloprid resistance, have been reported (Karunker et al., 2008, 2009). However, there are few studies regarding P450 genes in B. tabaci. To study the potentially involved metabolic resistance by P450 gene, we investigated resistance levels and isolated a novel CYP6 P450 gene putatively associated with insecticide resistance in the resistant field, and susceptible insectarium, populations of B. tabaci.

Materials and methods Sources of insects A field population of the subnymph of B. tabaci was collected from commercial crucifer (Brassica oleracea var. italica L.) vegetable fields in Shangjie, Minhou, Fujian, China (Sj), and introduced into an insectarium under field conditions in September 2006. B. tabaci in the insectarium was fed on cauliflower. The insectarium was constructed with a stainless-steel net and a glass roof at the Fujian Agriculture and Forestry University (FAFU), Fuzhou, China. The insectarium excluded external B. tabaci and was free from insecticides. The insectarium population of B. tabaci was collected from the insectarium in September 2009, and tentatively used as a related susceptible population (named as insectarium B. tabaci) in this study. Meanwhile, a field population of the subnymph of B. tabaci was collected from Sj in September 2009, and was used as the insecticide-resistant population (named as field B. tabaci). Insecticides were not applied in the fields for at least 1 week before the subnymphs of the field B. tabaci were collected. The subnymphs of B. tabaci collected from the field and the insectarium were then put into large vials in an environment chamber at 25 1 C for a photoperiod of 16 : 8 h L : D, and provided with 15% honey solution. The newly emerged adults of B. tabaci were used for experiments. The history of the insecticide application in Sj was the same as that described by Kang et al. (2006). To control B. tabaci, fenvalerate and chlopyrifos had been used in Sj for more than 30 years, and avermectin more than 10 years. The experiments to study the optimal reaction conditions for cloning the CYP6 gene and messenger RNA (mRNA) transcription was conducted from September 2008 by using the B. tabaci which were collected from the insectarium in FAFU and the fields in Sj. However, the B. tabaci collected from the insectarium and the field in Sj in September and October 2009 were used as insecticide-susceptible and -resistant populations of B. tabaci, respectively, to compare the differences in insecticide susceptibility and CYP6 gene between the insectarium and field populations of B. tabaci.

Insecticides Chlorpyrifos (technical grade, 95% pure) from Jinbo Pesticide Co., Ltd., Zhibo, Shangdong, China; fenvalerate (technical grade, 96% pure) from Sumitomo Chemical Co., Ltd., Osaka, Japan; avermectin (technical grade, 95.7% pure) from North China Pharmaceutical Group Corporation Aino Co., Ltd., Hebei, China, were used.

2010 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 484494
C

486

H. M. Zhuang et al.

Table 1 Oligonucleotide primers used in this study. Primers C1-J-2195 L2-N-3014 AP AUAP GDP-F GDP-R GSP1 GSP2 UPM NUP GSP3 GSP4 CYP6CX1v2-F CYP6CX1v2-R qF qR -actin -F -actin-R Sequences (5 to 3 ) 5 -TTGATTTTTTGGTCATCCAGAAGT-3 5 -TCCAATGCA CTAATCTGCCATATTA-3 5 -GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTT-3 5 -GGCCACGCGTCGACTAGT-3 5 -CGGA(A/G)AC(A/G/C/T)(A/C/T)(C/T)(A/G/C/T)(A/C)G(A/G/C/T)AA(A/G)TA(T/C)CC-3 5 -CGGG(A/G/C/T)CC(A/G/C/T)(G/T) (A/G/C/T)CC(A/G)AA(A/G/C/T)GG-3 5 -GCCGCTGGAATCATAAGACC-3 5 -TACTTTCCCGACCCAGAG-3 5 -CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3 5 -AAGCAGTGGTATCAACGCAGAGT-3 5 -GGTATGTAGGACCCAGGCAC-3 5 -TTTCATTGACGACCTGCTCC-3 5 -CGGGTATCCTAAAAAAATGG-3 5 -TGGTTATCAGTCCGAGGGCTT-3 5 -CGAACTGGCGTATCACC-3 5 -CCGCTGGAATCATAAGACC-3 5 -GCTGCCTCCACCTCATT-3 5 -ACCGCAAGATTCCATACCC-3 Product size (bp) 844

250

519 436

1 211 1 559 248 129

Bioassays The bioassay for adult B. tabaci was conducted by the dry film method (Kang et al., 2006). Briefly, 2 mL acetone solution of insecticide were poured into a glass vial (1.2 cm diameter, 10 cm length), and capped with a rubber plug. The solution in the vial was swirled for 10 s. Then, the excess solution was poured off, and the vial was placed on a wire rack upside down. Control vials were treated with acetone only in the same manner. Adult insects were introduced into the vial and left in contact with the insecticide in an environment chamber at 25 1 C at a photoperiod of 16 : 8 h L : D, and provided with 15% honey. Insect mortality was recorded 12 h after their introduction. Each lethal concentration at 50% (LC50 ) was calculated with five concentration levels and corresponding mortalities. No mortality was observed in the control during the bioassays. Adults, which did not respond to pencil tip prodding, were judged to be dead.

Biotype identification Single adults of B. tabaci was used for DNA extraction according to the method of Luo et al. (2002). Mi-

tochondrial cytochrome oxidase I (mt COI) gene sequences were selected as a marker gene to identify the biotype of B. tabaci of Sj in our study. The universal primers, C1-J-2195 and L2-N-3014 (Table 1), were used for the amplification of mt-COI gene. Polymerase chain reaction (PCR) was done at a 25 L volume, and the DNA was first denatured for 5 min at 94 C; followed by 35 cycles at 94 C for 30 s, 50 C for 30 s and 72 C for 60 s; and a final extension for 7 min at 72 C. PCR products were electrophoresed as above, and the sequencing was carried out for five adult individuals of B. tabaci. The fragment was named Origin-FJ. The pure sample (Origin-FJ) was sequenced by Guangzhou Office, Shanghai Invitrogen Biotechnology Co., Ltd, China (Shanghai Invitrogen), and analyzed by Clustalw2 (http://www.ebi.ac.uk/Tools/clustalw2/). The nucleotide sequences of mt-COI of B. tabaci were compared with those of Texas-B-biotype, Argentina-B-biotype, India-Bbiotype and Israel-B-biotype. The sequencing result of mt-COI of Origin-FJ was 844 bp. Intercepting 720 bp of Origin-FJ corresponding to the sequence of United States Texas-B, homology to other four sequences of B-biotypes of B. tabaci, were analyzed by the Clustalw2 (Fig. 1). The results indicated that there was a very small difference among Origin-FJ and the other four B-biotypes of B. tabaci. The internal consistency was higher than 99.7%.

Journal compilation

2010 The Authors Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 484494
C

A novel P450 gene isolated from Bemisia tabaci

487

Fig. 1 The comparison of mitochondrial cytochrome oxidase I sequence among Origin-FJ, Texas-B, Argentina-B, India-B and Israel-B. Identical residues were designated by dashes. The different nucleic acids are indicated by a dark background. The consistency of the sequence of Origin-FJ (the same pair of comparison sites divided by the total sites) was 99.7% (with Texas-B, GenBank AF164675), 100% (with Argentina-B, GenBank AF340216), 99.7% (with India-B, GenBank AF321927) and 99.7% (with Israel-B, GenBank AF418671), respectively.

Therefore, Origin-FJ in this study was identified as Bbiotype. RNA extraction and cDNA synthesis RNA for subsequent P450 gene amplification and cloning was prepared from both insectarium and field populations of B. tabaci; 150 to 200 adults of the field

B. tabaci adults were homogenized in 1 mL Trizol Reagent (Invitrogen, Shanghai, China). Each RNA sample was treated with RNase-free Dnase (TaKaRa, Shanghai, China). Reverse transcription was then performed on 3 g of each RNA sample in a 20-L reaction using Superscript III Reverse Transcriptase Kit (Invitrogen) and Adapter primer (AP, Table 1), following the suppliers instructions.

2010 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 484494
C

488

H. M. Zhuang et al.

PCR amplification of P450 cDNA fragments, cloning and sequence analysis For the amplification of P450s gene, degenerate primers GDP-F and GDP-R (Table 1) were used. These primers were designed based on sequences surrounding hemebinding regions of CYP6 families. Thermocycling conditions consisted of an initial denaturation step at 94 C for 4 min, followed by 35 cycles (94 C for 30 s, 45 C for 30 s, 72 C for 30 s) and a final extension at 72 C for 7 min. PCR products of the expected size were purified from 1% (w/v) agarose/TAE gel using TaKaRa Agarose Gel DNA Purification Kit and cloned using the pMDTM 18-T Vector (TaKaRa). Plasmids were sent to Shanghai Invitrogen, and sequenced by automated DNA sequencer ABI model 3700. The fragment 1 with 250 bp was obtained. Based on fragment 1, we designed GSP1 (Table 1) as reverse primer, together with Abridge Universal Amplification primers (AUAP, Table 1) as forward primer, to amplify a fragment by PCR, consisting of denaturation at 94 C for 5 min followed by 35 cycles of 94 C for 30 s, 50 C for 30 s and 72 C for 2 min and final extension at 72 C for 10 min. The fragment 2 with 519 bp was obtained.

initial CYP6CX1 cDNA fragment and the 3 - and 5 -RACE (fragments 3 and 4) in the insectarium population of B. tabaci were carried out in the same way as in the field B. tabaci. The primers for cloning the internal fragments in the insectarium B. tabaci were designed based on the sequence of CYP6CX1v1. The primers used in 3 - and 5 -RACE in the insectarium B. tabaci were the same as those in the field B. tabaci. The full length of CYP6CX1 in the insectarium B. tabaci (named CYP6CX1v2) was edited and assembled. The full-length nucleotide sequence of the open reading frame (ORF) in the insectarium B. tabaci was confirmed by PCR amplification using primers CYP6CX1v2-F (at positions 4968) and CYP6CX1v2-R (reverse complementary to nucleotides at 1 6231 643) (Table 1). The RT-PCR products were purified directly from bands excised from agarose gels and cloned into pMDTM 18-T Vector (TaKaRa). Positive clones were sent to Shanghai Invitrogen to be sequenced. Software including DNAMAN (http://www.lynnon.com/) and ClustalW2 were used to analyze the gene sequences.

Real-time quantitative PCR (qPCR) in adult B. tabaci Quantitative PCR was conducted using an MiniOpticon System for real-time PCR Detection (Bio-Rad, Hercules, CA, USA) with the SYBR Premix Ex Taq (TaKaRa) kit. Reaction mixtures (final volume 20 L) contained 2 SYBR Premix Ex Taq, 200 nmol/L of each primer, 2 L cDNA and 7.2 L Rnase-free water. PCR conditions were 95 C for 10 s, followed by 40 cycles of 95 C for 6 s and 55 C for 25 s. Fluorescence was measured after each cycle. Relative mRNA expression of CYP6CX1v1 and CYP6CX1v2 was measured in reference to the actin gene. Based on the sequence of CYP6CX1v1 and CYP6CX1v2, the same region of nuclotide acid sequence was selected to design the primers qF and qR, for qPCR. In addition, the primers, -actin-F and -actin-R, were used for amplifying the -actin gene (Table 1). Standard curves of target gene and reference gene were made, using triplicate serial dilutions with six different cDNA concentrations covering a 3 125-fold concentration range. The homogeneity of the PCR products was confirmed by melting curve analysis. The mean normalized expression value of P450 gene was calculated by comparing the threshold cycle (Ct) of the gene to that of -actin gene according to the equations of standard curves of target gene and reference gene (-actin gene), respectively (Larionov et al., 2005). In brief, the equation of standard curve of -actin gene was y = 0.295x + 6.78 (r2 = 0.999), and the equation of standard curve of CYP6CX1 gene was y = 0.322x + 8.89
C

Rapid amplification of cDNA ends (RACE) 3 - and 5 - RACE reactions were performed to complete the cDNA sequence of CYP6CX1 gene of the field B. tabaci. 3 -RACE was performed by using GSP2 and AUAP (Table 1), and PCR conditions were 94 C for 5 min, followed by 35 cycles of 94 C for 30 s, 50 C for 30 s, 72 C for 1 min and a final extension step of 72 C for 10 min. The fragment 3 was obtained. For 5 -RACE amplification, the first-strand cDNA was synthesized from RNA using the SMARTTM race cDNA amplification kit (Clontech, Takara, Japan). 5 -RACE used Universal Primer A Mix (UPM) and GSP3 (Table 1), and rounds of PCR (35 cycles) consisted of denaturation at 94 C for 5 min followed by 35 cycles of 94 C for 30 s, 61 C for 30 s and 72 C for 2 min and a final extension at 72 C for 10 min. The PCR product was used for re-amplification using Nested Universal Primer (NUP) and GSP4 (Table 1), and rounds of PCR (35 cycles) consisting of denaturation at 94 C for 5 min followed by 35 cycles of 94 C for 30 s, 62 C for 30 s and 72 C for 2 min with a final extension at 72 C for 10 min. The fragment 4 was obtained. The initial CYP6CX1 cDNA fragment (fragments 1 and 2) and cDNA ends obtained by 3 -RACE and 5 -RACE (fragments 3 and 4) were edited and assembled for full-length cDNA of the field B. tabaci (named CYP6CX1v1). The clones of the

Journal compilation

2010 The Authors Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 484494

A novel P450 gene isolated from Bemisia tabaci Table 2 Comparisons on susceptibility to insecticides in the field and insectarium populations of Bemisia tabaci. Insectarium population Insecticides LC50 (95% CI) (mg/L) (12 h) 88.0 (75.4103) 43.9 (24.778.9) 0.16 (0.140.18) Slope SE 2.78 0.22 4.18 0.96 2.23 0.14 Field population LC50 (95% CI) (mg/L) (12 h) 3 940 (3 4774 464) 1 019 (8991 155) 5.76 (3.718.93) Slope SE 3.17 0.21 2.84 0.18 5.08 1.09

489

Resistance ratio

Fenvarelate Chlorpyrifos Avermectin

44.8 23.2 36.0

LC50 , lethal concentration at 50%.

(r2 = 0.995); X = Ct value. The relative expression was CYP6CX1 = 100.322Ct1+8.89 /100.295Ct2+6.78 , where Ct1 = threshold cycle of target gene and Ct2 = threshold cycle of reference gene. Each reaction was performed in triplicate to minimize variation within experiments, and the mean of at least three independent biological replicates was calculated. Statistical analysis The bioassay data were analyzed for x2 , LC50 values, and their 95% confidence intervals (95% CI) by probit analysis using a data processing system (Tang & Feng, 1997). t-tests were also calculated using the data processing system (Tang & Feng, 1997).

Results Determination of resistance levels to insecticides in B. tabaci Significant resistances to fenvalerate, chlorpyrifos and avermectin were found in the field population of B. tabaci, as compared to those in the insectarium population of B. tabaci. The resistance ratios were 44.8 for fenvalerate, 23.2 for chlopyrifos and 36 for avermectin, respectively. The resistances to fenvalerate and avermectin were high (Table 2). Cloning and sequencing analyses of CYP6CX1 Based on sequences of the fragment 1 (250 bp), fragment 2 (519 bp), fragment 3 (436 bp) and fragment 4 (1 211 bp), a novel complete sequence P450 cDNA (CYP6CX1v1) with 1 940 bp in the field B. tabaci was obtained by overlaying the cloned sequences. The CYP6CX1v1 contained a 1 557 bp ORF encoding 518 amino acid residues. The predicted isoelectric point of

the cDNA-deduced protein was 8.56 and the molecular weight (MW) was 58 784 Da, within the range (46 60 kDa) of other reported cytochrome P450s (Nelson et al., 1993). The deduced amino acid sequence contains important conserved domains common to CYP6 members, such as the oxygen- binding motif AGXXPX (i.e., AGLDPV at position 322327), the heme-binding decapeptide PFXXGXXXCXA (i.e., PFGEGPRFCIA at position 454464) and the putative meander-binding sequences EXXR and PXRF (ETLR at 380383 and PXRF at 436439) in helix-K. In addition, a conserved domain (FPDP, at 428431) common to another CYP6 member (Y/FPD/EP) and a PXXFXP near the carboxyl end (i.e., PEKFNP at position 431436) were found (Figs. 2 and 3) (Nelson et al., 1993; Karunker et al., 2008). A BLAST (www.ncbi.ie) search indicated that CYP6CX1 amino acid sequence was identical to CYP6 families that shared the highest homology. A phylogenetic tree of B. tabaci (Bt) P450s deduced amino acid sequences and selected P450s from another eight insect species is shown in Fig. 4. The protein encoded by CYP6CX1 had the highest amino acid identity with CYP6CM1vB from B. tabaci (GenBank EU642555, 43%) and CYP6CM1vQ from B. tabaci (GenBank EU344879, 43%). The identity of CYP6CX1 to the CYP6 subfamilies from other insect species, including Tribolium castaneum (Ta), Anopheles funestus (Af), Culex quinquefasciatus (Cq), Nilaparvata lugens (Nl), Blattella germanica (Bg), Anopheles gambiae (Ag), Anopheles minimus (Am) and Lygus lineolaris (Ll), were from 32% to 34%. Therefore, CYP6CX1v1 was a typical cytochrome P450 gene. The phylogenetic analysis placed CYP6XC1v1 and CYP6XC1v2 in the CYP6 family and CYP6C subfamily. Comparison of CYP6CX1 between R and S B. tabaci To analyze the differences between CYP6CX1v1 from the field B. tabaci and CYP6CX1v2 from the insectarium B. tabaci, alignment of CYP6CX1v1 and CYP6CX1v2 nucleotide sequences (Fig. 2) and their putative CYP amino

2010 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 484494
C

490

H. M. Zhuang et al.

Fig. 2 Alignment of complementary DNA (cDNA) sequences of CYP6CX1v1 and CYP6CX1v2 from Bemisia tabaci. Identical residues are designated by dashes. The different nucleic acids are indicated by a dark background.

acid sequences were studied (Fig. 3). Figure 3 shows that there were four different positions between the field and insectarium B. tabaci at 300 position (Thr to Ala), 354 position (Thr to Pro), 486 position (Arg to His) and 503 position (Ile to Thr), respectively, among which the substitution Ile503Thr was located in the substrate recognition sites region (DRETFTLNP).

mRNA expression of CYP6CX1 in R and S of adult B. tabaci The mRNA transcription level of CYP6CX1v1 was significantly higher than that of CYP6CX1v2 (t-test, P < 0.05). The mRNA transcription level of CYP6CX1v1 was 2.38-fold as high as that of CYP6CX1v2 (Fig. 5).

Journal compilation

2010 The Authors Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 484494
C

A novel P450 gene isolated from Bemisia tabaci

491

Fig. 3 Alignment of deduced amino acid sequences of CYP6CX1v1 and CYP6CX1v2. Identical residues are designated by dashes. The four amino acid substitutions at positions 300, 354, 486 and 503 are indicated by a dark background. Conserved domains common to CYP6 members are boxed, such as the heme-binding decapeptide PFXXGXXXCXA (position 454464), the oxygen-binding domain AGXXPX (position 322327), a conserved amino acid sequence PXXFXP (position 431436), a putative meander-binding sequence EXXR (position 380383) and PXRF (position 436439) in helix-K.

Discussion Polymerase chain reaction-based cloning strategies with degenerate primers make it possible to rapidly amplify members of CYP superfamilies in many insects. In this study, we used a pair of degenerate primers (Table 1) based on sequences surrounding heme-binding regions of CYP6 to amplify a short P450s fragment, then according to the short fragment, we designed four gene-specific primers used for another fragment and 3 - and 5 -RACE reactions. Just as described in the Results section, six conserved domains, which were the conservative sequences of CYP6 family (Nelson et al., 1993; Karunker et al., 2008), existed in CYP6CX1. Because there were no differences in the base sequences corresponding to those of the primers used for 3 - and 5 -RACE between CYP6CX1v1 and CYP6CX1v2, the 3 - and 5 -RACE in both S and R B. tabaci could be carried out successfully by using the same primers. In accordance with the P450 nomenclature system (Nebert et al., 1991), the subsequent Arabic numeral after

CYP indicated the different gene families of CYP superfamilies. The subsequent capital letter after the Arabic numeral indicated different gene subfamilies. The amino acid sequence with identity higher than 40% belongs to the same gene family, such as CYP6. CYP6CX1v1 and CYP6CX1v2 obtained in this study showed higher than 40% identity to the CYP6 genes of other insect species, and belonged to CYP6. In our previous results, a fragment (1 002 bp from the field B. tabaci) were submitted and named as CYP6CX1v1 by GenBank Submissions Staff (GenBank GQ292715). CYP6CX1v1 had 49% identity to CYP6CM1 (GenBank EU642555) from B. tabaci reported by Karunker et al. (2008). Based on the fragment (GenBank GQ292715), the full-length CYP6 genes were isolated successfully in both field and insectarium B. tabaci in this study, and were then named as CYP6CX1v1 and CYP6CX1v2, respectively. CYP6CX1v1 and CYP6CX1v2 showed the highest identity to CYP6CM1 (43%), and could be identified as a new CYP6 family member. CYP6A2 containing three point mutations were constitutively over-expressed and involved in DDT metabolism

2010 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 484494
C

492

H. M. Zhuang et al.

Fig. 4 Phylogenetic tree of P450 deduced amino acid sequences of Bemisia tabaci (Bt) and selected P450 from other insect species. Tribolium castaneum (Ta), Anopheles funestus (Af), Culex quinquefasciatus (Cq), Nilaparvata lugens (Nl), Blattella germanica (Bg), Anopheles gambiae (Ag), Anopheles minimus (Am), Lygus lineolaris (Ll). The identify of CYP6CX1 to the other CYP6 subfamilies from other insect species and the GenBank accession number are shown in parenthesis after the CYP name: 6BK14-Ta (33%, EFA05731), 6BK17-Ta (33%, ABX64450), 6BK4-Ta (33%, NP_001123875), 6BK12-Ta (32%, EFA12529), 6BQ5-Ta (33%, EFA02819), 6BQ7-Ta (32%, EFA02821), 6BQ13-Ta (32%, EEZ99338), 6P9-Af (32%, ABC87786), 6P13-Af (33%, ABO77953), 6P3-Ag (33%, AF487534), 6P7-Am (34%, AAR88141), 6BB1v2-Cq (32%, XP_001847401), 6a8-Cq (34%, XP_001870174), 6X1v2-Ll (33%, AAL15174), 6X1v3Ll (33%, AAM94461), 6X1v1-Ll (33%, AAL15173), 6cm1-Bt (43%, ACS92724), 6CM1vB-Bt (43%, ACD84797), 6CM1vQ-Bt (43%, ACA51846), 6AX1-Nl (34%, CAH65681), 6J1-Bg (33%, AF281325).
3.0

2.5 Relative fold change

2.0

1.5

1.0

0.5

0.0 Insectarium Field

Fig. 5 Messenger RNA (mRNA) expression of CYP6CX1 gene in the insectarium (white) and field (black) adults of Bemisa tabaci. Relative quantities indicate that the levels of transcripts are normalized to the internal standard (-actin gene). The R mRNA expression in B. tabaci was expressed relative to that in insectarium B. tabaci, set at 1. Each bar represents the mean SD of three independent experiments.

of D. melanogaster. The location of the mutations in a model of the 3D structure of the CYP6A2 protein suggested that some of them may be important for enzyme activity of this molecule. This has been verified by heterologous expression (Berge et al., 1998). Two amino acid residue mutation of CYP gene, coupled with upregulation of mRNA expression (2.1-fold higher mRNA expression in pyrethroid-resistant strains), was possibly related to resistance development in the tarnished plant bug (Zhu & Snodgrass, 2003). There were six putative substrate recognition sites (SRS16) in CYP6CM1 in B. tabaci (Karunker et al., 2009). A total of four amino acid residue replacements were found in CYP6CX1v1. The amino acid replacements of CYP might be involved in insecticide resistance or result from polymorphism among the individuals. Because the substitution Ile503Thr was located in the SRS6 region (DRETFTLNP), the mutation might be important in the function of CYP6CX1v1. The other three amino acid replacements might result from polymorphism. Nevertheless, the functions of the four amino acid replacements should be verified by heterologous expression and metabolisms in vitro.

Journal compilation

2010 The Authors Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 484494
C

A novel P450 gene isolated from Bemisia tabaci

493

Besides amino acid replacement, P450-mediated resistance to insects might be associated with another two mechanisms, P450 over-expression or the combined effects of amino acid replacement and P450 over-expression (Hemingway et al., 2004). The major mechanism in all samples investigated appeared to be enhanced detoxification by cytochrome P450 monooxygenases, based on the mRNA transcription of 11 distinct P450 cDNA sequences from B- and Q-biotype B. tabaci (Karunker et al., 2008). In this study, significantly higher expression of CYP6CX1 might be involved in the resistance to fenvalerate, chlorpyrifos and avermectin in the field B. tabaci collected from Sj. However, the evidence to correlate the relationship between the transcription level of mRNA and resistance mechanisms was weak because only 2.38-fold difference in CYP6CX1 transcription level between the insectarium and the field B. tabaci was found. However, it is known that P450-mediated oxidative degradation is the major mechanism of insecticide resistance in the field population of B. tabaci in Sj, based on the synergistic effects on the susceptibility of insecticides, including chlorpyrifos, fenvalerate, avermectin and imidaclorpid, by using enzyme inhibitors (Kang et al., 2006). Although the B. tabaci strains in this study were different from those used in Kang et al. (2006), the two field populations of B. tabaci used in this study and by Kang et al. (2006) were collected from Sj, and the collection times for the two insect populations were close (i.e., 2008 and 2006, respectively). Fenvalerate and chlopyrifos has been used in Sj for more than 30 years to control B. tabaci, and avermectin for more than 10 years. Because P450 gene was verified to be involved in the resistance to fenvalerate, chlopyrifos and avermectin in the Sj population of B. tabaci (Kang et al., 2006), and the insecticides used to control B. tabaci were similar in Sj during 2006 to 2009 according to our field investigation, the resistance mechanism to the three insecticides might be similar in the field B. tabaci used in this study and in Kang et al. (2006). Nevertheless, it was known that cytochrome P450s played a crucial role in insecticide resistance of insects through metabolic detoxification, and CYP6 family P450 was widely known to be involved in metabolic resistance to pyrethrins and organophosphates in some other insect species (Nikou et al., 2003; Bautista et al., 2007; Amenya et al., 2008; Zhou et al., 2010). It has been speculated that CYP6CX1 might be involved in resistances to the three insecticides in the field B. tabaci in Sj. In this study, a novel P450 gene putatively associated with insecticide resistance was identified from B. tabaci. Because the field B. tabaci showed significant resistance to three different classes of insecticides (i.e., fenvarerate, avermectin and chlorpyrifos), besides the P450 gene, such

as CYP6CX1v1, other detoxification enzymes and specific insensitive targets might be involved in the resistance in B. tabaci depending on the different insecticides. The direct relationships between the mutation or expression and the function of CYP6CX1 in insecticide resistance in B. tabaci should be conducted in our further research. The susceptible and the resistant populations in this study were collected from Sj in 2006 and 2009, respectively and therefore, did not share a common genetic background, although the insectarium and the field populations of B. tabaci originated from Sj. Strains with the same genetic background are needed for studying resistance mechanisms. In addition, insectarium B. tabaci was not really a susceptible strain (susceptible homozygote), but a related susceptible strain with some susceptible and resistant heterozygote; back-cross experiments should be conducted in the future to obtain a susceptible and resistant homozygote with the same genetic background. Acknowledgments We thank C. W. Li, graduate student at FAFU, for his help in collecting the insects from the field and rearing them. This work was financially supported by the National Natural Science Foundation of China (30771413), the National Natural Science Foundation of Fujian Province (009J01071) and the Key Program of Education Ministry, China (207055, JA06010). References
Amenya, D.A., Naguran, R., Lo, T.C., Ranson, H., Spillings, B.L., Wood, O.R., Brooke, B.D., Coetzee, M. and Koekemoer, L.L. (2008) Overexpression of a cytochrome P450 (CYP6P9) in a major African malaria vector, Anopheles funestus, resistant to pyrethroids. Insect Molecular Biology, 17, 1925. Bautista, M.A.M., Tanaka, T. and Miyata, T. (2007) Identification of permethrin-inducible cytochrome P450s from the diamondback moth, Plutella xylostella (L.) and the possibility of involvement in permethrin resistance. Pesticide Biochemistry and Physiology, 87, 8593. Berge, J.B., Feyereisen, R. and Amichot, M. (1998) Cytochrome P450 monooxygenases and insecticide resistance in insects. Philosophical Transactions of the Royal Society B, 353, 1701 1705. Fern ndez, E., Gr valos, C., Haro, P.J., Cifuentes, D. and Bielza, a a P. (2009) Insecticide resistance status of Bemisia tabaci Qbiotype in south-eastern Spain. Pest Management Science, 65, 885891. Ghanim, M. and Kontsedalov, S. (2007) Gene expression in pyriproxyfen- resistant Bemisia tabaci Q biotype. Pest Management Science, 63, 776783.

2010 The Authors Journal compilation C Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 484494
C

494

H. M. Zhuang et al. Nelson, D.R., Kamataki, T., Waxman, D.J., Guengerich, F.P., Estabrook, R.W., Feyereisen, R., Gonzalez, F.J., Coon, J.J., Gunsalus, I.C., Gotoh, O., Okuda, K. and Nebert, D.W. (1993) The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA Cell Biology, 12, 151. Nikou, D., Ranson, H. and Hemingway, J. (2003) An adultspecific CYP6 P450 gene is overexpressed in a pyrethroidresistant strain of the malaria vector, Anopheles gambiae. Gene, 318, 91102. Prabhaker, N., Castle, S.J., Buckelew, L. and Toscano, N.C. (2008) Baseline susceptibility of Bemisia tabaci B biotype (Hemiptera: Aleyrodidae) populations from California and Arizona to spiromesifen. Journal of Economic Entomology, 101, 174181. Qiu, B.L., Chen, Y., Liu, L., Peng, W., Li, X., Ahmed, M.Z, Mathur, V Du, Y. and Ren, S. (2009) Identification of three ., major Bemisia tabaci biotypes in China based on morphological and DNA polymorphisms. Progress in Natural Science, 19, 713718. Rauch, N. and Nauen, R. (2003) Identification of biochemical markers linked to neonicotinoid cross resistance in Bemisia tabaci (Hemiptera: Aleyrodidae). Archives of Insect Biochemistry and Physiology, 54, 165176. Roditakis, E., Roditakis, N.E. and Tsagkarakou, A. (2005) Insecticide resistance in Bemisia tabaci (Homoptera: Aleyrodidae) populations from Crete. Pest Management Science, 61, 577582. Roditakis, E., Tsagkarakou, A. and Vontas, J. (2006) Identification of mutations in the para sodium channel of Bemisia tabaci from Crete, associated with resistance to pyrethroids. Pesticide Biochemistry and Physiology, 85, 161166. Tang, Q.Y. and Feng, M.G. (1997) Practical statistics and DPS data processing system. DPS Data Processing System for Practical Statistics (eds. Q.Y. Tan & M.G. Feng), pp. 188 195. China Agricultural Press, Beijing, China. Zhou, X., Sheng, C., Li, M., Wan, H., Liu, D. and Qiu, X. (2010) Expression responses of nine cytochrome P450 genes to xenobiotics in the cotton bollworm Helicoverpa armigera. Pesticide Biochemistry and Physiology, 97, 209213. Zhu, Y.C. and Snodgrass, G.L. (2003) Cytochrome P450 CYP6X1 cDNAs and mRNA expression levels in three strains of the tarnished plant bug Lygus lineolaris (Heteroptera: Miridae) having different susceptibilities to pyrethroid insecticide. Insect Molecular Biology, 12, 3949. Accepted June 20, 2010

G cmen, H. and Devran, Z. (2002) Determination of geo netic variation in population of Bemisia tabaci in Antalya. Turkish Journal of Agriculture and Forestry, 26, 211216. Guo, T.T., Jiang, H. and Gao, X.W. (2009) Diversity, evolution and expression regulation of cytochrome P450 monooxygenase genes in insects. Acta Entomologica Sinica, 52, 301 311. Hemingway, J., Hawkes, N.J., McCarroll, L. and Ranso, N.H. (2004) The molecular basis of insecticide resistance in mosquitoes. Insect Biochemistry and Molecular Biology, 34, 653665. Kang, C.Y., Wu, G. and Miyata, T. (2006) Synergism of enzyme inhibitors and mechanisms of insecticide resistance in Bemissia tabaci (Gennadius) (Homoptera: Aleyrodidae). Journal of Applied Entomology, 130, 377385. Karunker, I., Benting, J., Lueke, B., Ponge, T., Nauen, R., Roditakis, E., Vontas, J., Gorman, K., Denholm, I. and 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 Biochemistry and Molecular Biology, 38, 634 644. Karunker, I., Morou, E., Nikou, D., Nauen, R., Sertchook, R., Stevensonc, R.J., Paine, M.J.I., Morin, S. and Vontas, J. (2009) Structural model and functional characterization of the Bemisia tabaci CYP6CM1vQ, a cytochrome P450 associated with high levels of imidacloprid resistance. Insect Biochemistry and Molecular Biology, 39, 697706. Larionov, A., Krause, A. and Millerm, W. (2005) A standard curve based method for relative real time PCR data processing. BMC Bioinformatics, 6, 62. Liu, S.S., Barro, P.J.D., Xu, J., Luan, J.B., Zang, L.S., Ruan, Y.M. and Wan, F.H. (2007) Asymmetric mating interactions drive widespread invasion and displacement in a whitefly. Science, 318, 17691772. Luo, C., Yao, Y., Wang, R.J., Yan, F.M., Hu, D.X. and Hang, Z.L. (2002) The use of mitochondrial cytochrome oxidase I (mtCOI) gene sequences for the identification of biotypes of Bemisia tabaci (Gennadius) in China. Acta Entomologica Sinica, 45, 759763. Nebert, D.W., Nelson, D.R., Coon, M.J., Estabrook, R.W., Feyereisen, R., Fujii-Kuriyama, Y., Gonzalez, F.J., Guengerich, F.P., Gunsalus, I.C., Johnson, E.F., Loper, J.C., Sato, R., Waterman, M.R. and Waxman, D.J. (1991) The P450 superfamily: Update on new sequences, gene mapping, and recommended nomenclature. DNA Cell Biology, 10, 114.

Journal compilation

2010 The Authors Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 484494
C