Identification and functional analysis of a cytochrome P450 gene CYP9AQ2 involved in deltamethrin detoxification from Locusta migratoria

Accepted Manuscript Title: Identification and functional analysis of a cytochrome P450 gene CYP9AQ2 involved in deltamethrin detoxification from Locusta migratoria Author: Yanqiong Guo, Xueyao Zhang, Haihua Wu, Rongrong Yu, Jianzhen Zhang, Kun Yan Zhu, Yaping Guo, Enbo Ma PII: DOI: Reference:

S0048-3575(15)00004-8 http://dx.doi.org/doi: 10.1016/j.pestbp.2015.01.003 YPEST 3776

To appear in:

Pesticide Biochemistry and Physiology

Received date: Accepted date:

25-6-2014 5-1-2015

Please cite this article as: Yanqiong Guo, Xueyao Zhang, Haihua Wu, Rongrong Yu, Jianzhen Zhang, Kun Yan Zhu, Yaping Guo, Enbo Ma, Identification and functional analysis of a cytochrome P450 gene CYP9AQ2 involved in deltamethrin detoxification from Locusta migratoria, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/doi: 10.1016/j.pestbp.2015.01.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Identification and functional analysis of a cytochrome P450 gene CYP9AQ2

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involved in deltamethrin detoxification from Locusta migratoria

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Yanqiong Guo,a,b Xueyao Zhang,a Haihua Wu,a Rongrong Yu,a Jianzhen Zhang,a Kun Yan

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Zhu,c* Yaping Guo,a and Enbo Maa*

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a

Institute of Applied Biology, Shanxi University, Taiyuan, Shanxi 030006, China

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b

College of Agriculture, Shanxi Agricultural University, Taigu, Shanxi 030801, China

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c

Department of Entomology, 123 Waters Hall, Kansas State University, Manhattan, KS

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66506, USA

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Running title: Guo et al. a cytochrome P450 gene CYP9AQ2 in locust

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Corresponding authors Addresses: E. Ma, Institute of Applied Biology, Shanxi University,

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Taiyuan, Shanxi 030006, China, Tel: 86-351-701-8871, Fax: 86-351-701-8871; K.Y. Zhu,

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Department of Entomology, 123 Waters Hall, Kansas State University, Manhattan, KS 66506,

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USA, Tel: 1-785-532-4721, Fax: 1-785-532-6232.

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E-mail addresses: [email protected] (E. Ma); [email protected] (K.Y. Zhu)

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Research Highlight:

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►We identified a cytochrome P450 gene, CYP9AQ2, from locust. ► The expression of CYP9AQ2 was

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relatively higher in nymphal stages than in egg and adult stages. ► High expression of CYP9AQ2 was

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observed in detoxification tissues. ► Deltamethrin could induce the expression of CYP9AQ2.►RNAi

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revealed the detoxification function of CYP9AQ2 for deltamethrin.

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Graphical Abstract

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ABSTRACT

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A 1578-bp cDNA of a cytochrome P450 gene (CYP9AQ2) was sequenced from the migratory

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locust, Locusta migratoria. It contains an open reading frame (ORF) of 1557 bp that encodes

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519 amino acid residues. As compared with other known insect cytochrome P450 enzymes,

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the overall structure of its deduced protein is highly conserved. The expression of CYP9AQ2

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was relatively higher in nymphal stages than in egg and adult stages, and the highest

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expression was found in fourth-instar nymphs, which was 8.7-fold higher than that of eggs.

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High expression of CYP9AQ2 was observed in foregut, followed by hindgut, Malpighian

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tubules, brain and fat bodies, which were 75~142-fold higher than that in hemolymph. Low

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expression was found in midgut, gastric caecum and hemolymph. The expression of

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CYP9AQ2 was up-regulated by deltamethrin at the concentrations of 0.04, 0.08, and 0.12

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µg/mL and the maximal up-regulation was 2.6-fold at LD10 (0.04 µg/mL). RNA interference-

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mediated silencing of CYP9AQ2 led to an increased mortality of 25.3% when the nymphs

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were exposed to deltamethrin, suggesting that CYP9AQ2 plays an important role in

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deltamethrin detoxification in L. migratoria. Computational docking studies suggested that

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hydroxylation of the phenoxybenzyl moiety might be one of the deltamethrin metabolic

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pathways by CYP9AQ2.

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Keywords：Cytochrome P450; Locusta migratoria; insecticide; deltamethrin; RNA

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interference

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1. Introduction Cytochrome P450 monooxygenases (CYPs) belong to a superfamily of diverse

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multifunctional enzymes that are ubiquitously distributed in all living organisms from

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bacteria to human [1]. CYPs are responsible for the oxidative metabolism of aerobic

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eukaryotes in the endoplasmic reticulum and release of water through an electron-transport

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system that involves cytochrome P450 reductase and cytochrome b5 [2]. In insects, CYPs are

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known to metabolize important endogenous compounds for growth and development, which

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include ecdysteroids, juvenile hormones, and fatty acids [3,4]. However, many CYPs play a

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central role in the metabolism of xenobiotics such as drugs, pesticides, and plant toxins [5-7].

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These enzymes are considered to be phase I detoxification enzymes important for converting

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xenobiotics to more hydrophilic metabolites that can be excreted either directly or after

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conjugation reactions mediated by phase II detoxification enzymes [1,3,4].

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Four major clans have been found in insect CYPs, which include CYP2, CYP3, CYP4

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and mitochondrial CYP clans [1]. The first three clans are named after the founding family in

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vertebrates and the last one is named by their subcellular location. Every clan has been

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further subdivided into different CYP families, based in the degree of amino acid sequence

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similarity [5,6]. The CYP2 clan comprises vertebrate CYP1 and CYP2 families, insect

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CYP15 and CYP18 families, and several families in the CYP300 series. Clan 3 is a large

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group of insect CYPs and comprises CYP6, CYP9, CYP28 and CYP308-310 families, which

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participate primarily in xenobiotic metabolism [1,3,4]. Clan 4 includes over 20 families, but

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mainly CYP4 family. The mitochondrial CYP clan contains insect CYPs which exist in the

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mitochondria, such as CYP12 family.

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With rapidly increased number of insect genomes sequenced, more CYP genes have been identified [8]. The number of CYP genes ranges from 37 in the body louse Pediculus 4 Page 4 of 24

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humanus humanus (Anoplura) to 170 in Culex quinquefasciatus in sequenced insect genomes

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[9,10]. For example, the four dipteran species Drosophila melanogaster, Aedes aegypti,

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Anopheles gambiae and Culex quinquefasciatus possess 91, 158, 102 and 170 CYP genes,

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respectively. Tribolium castaneum (Coleoptera), Bombyx mori (Lepidoptera), Apis mellifera

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(Hymenoptera) and Pediculus humanus humanus (Anoplura) possess 144, 84, 48 and 37 CYP

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genes, respectively [11,12].

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The migratory locust, Locusta migratoria is a typical orthopteran insect and one of the

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most destructive agricultural pests in the world [13]. Locust plagues are a significant problem

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in coastal low lands throughout East and SE Asia. Synthetic insecticides are often used to

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control the locust in pest management programs. Our previous studies have shown that

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deltamethrin can enhance cytochrome P450 monooxygenase activity of 7-EC-O-deethylation,

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which may be due to increased transcription of multiple CYP genes in response to

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deltamethrin exposures [14]. The insect CYP9 family has been known to play important roles

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in xenobiotic metabolism and insecticide resistance. However, little is known about the roles

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of CYP9 genes in locust.

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Locust genomic database established by Kang’s laboratory [15] and transcriptome database

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of our laboratory have provided an excellent platform and great opportunity for

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systematically identifying CYP and CYP-like genes and assessing their roles in insecticide

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metabolism in the locust. In this paper, we report: 1) cloning and sequencing of a cDNA

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putatively derived from CYP9AQ2 in L. migratoria; 2) developmental stage and tissue

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specific expression pattern of CYP9AQ2; 3) dose-dependent up-regulation of CYP9AQ2 by

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deltamethrin in nymphal stages; 4) roles of CYP9AQ2 in detoxification of insecticides

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revealed by RNA interference (RNAi); and 5) homology modeling of CYP9AQ2 and

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computational docking to explore possible deltamethrin metabolic pathways by CYP9AQ2.

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Results from this study are expected to help us better understand the molecular interactions 5 Page 5 of 24

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between insecticides and cytochrome P450s and the role of these genes in insecticide

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detoxification in L. migratoria.

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2. Materials and methods

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2.1. Insect

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The eggs of L. migratoria were purchased from the Insect Protein Co., Ltd. Cangzhou,

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China and were incubated in a growth chamber at 28C and 50% relative humidity (RH) with

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a 14:10-h light: dark photoperiod. After hatching, locust nymphs were reared on fresh wheat

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sprouts under the same temperature and light conditions.

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2.2. Sequencing of cDNA

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CYP-like genes from L. migratoria were revealed by searching the sequences in the

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non-redundant locust transcriptome database using the keyword CYP or cytochrome P450. A

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cDNA sequence (consencus15972), putatively encoding an enzyme of CYP9 family, was

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selected for subsequent work. The translated amino acid sequence was compared with other

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insect CYP's of CYP9 family and searched using BLASTP against the non-redundant

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database at the National Center for Biotechnology Information (NCBI,

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http://www.ncbi.nlm.nih.gov/) to further reveal its identity.

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To sequence the full-length cDNA of the CYP gene, the gastric caeca of fifth-instar

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nymphs were used to extract total RNA by using RNAisoTM Plus (Takara, Dalian, China).

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The first-strand cDNA was synthesized from 1.5 μg of total RNA using the SMARTTM RACE

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cDNA amplification kit (Clontech, Mountain View, CA, USA). The CYP cDNA sequence

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was amplified using gene-specific primers complementary to the 5’- and 3’- ends of the

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cDNA using first-strand cDNA as a template. A pair of specific primers includes 5′-

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CATTGGAAGAAGAAAGGTG-3′ (forward) and 5′-TCAAGCTGACATGGGACAT-3′

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(reverse). PCR was performed based on the following parameters: 94 C for 1 min, followed 6 Page 6 of 24

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by 35 cycles of amplification: 94 C for 30 s, 55 C for 30 s and 72 C for 2 min, and

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followed by a final extension step of 72 C for 7 min. PCR products were separated by 1%

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agarose gel electrophoresis and stained with ethidium bromide (EB). A single band

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corresponding to approximately 1607 bp was excised and the fragment was recovered with

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the Gel Extraction Mini Kit (Tiangen, China). The purified fragment was subcloned into

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pGEM-T easy vector (Promega, Madison, USA) and then sequenced in both directions by

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Beijing AuGCT Biotechnology Co., Ltd (Beijing, China).

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2.3. Sequence analysis

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The prediction of the open reading frame (ORF) and the translation of the cDNA

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sequence into amino acid sequence were performed using the translation tool in ExPaSy

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(http://www.expasy.org/tools/dna.html). The molecular mass and isoelectric point (pI) were

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predicted based on their amino acid sequences. Signal peptide was predicted with the SignalP

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3.0 program using Neural networks method (http://www.cbs.dtu.dk/services/SignalP/).

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The locust cDNA sequence was compared with CYP3A4 (GenBank accession number:

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51247719) from human and those of CYP9A subfamily members from other insects

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deposited in GenBank by using GENEDOC software [16]. The locust CYP gene was finally

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named CYP9AQ2.

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2.4. Analysis of tissue- and stage-dependent expression patterns

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For analyses of the CYP9AQ2 expression patterns at different developmental stages of

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the locust, total RNA samples were prepared from 20 eggs, 20 nymphs of each of five instars

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and five adults by using RNAisoTM Plus (TaKaRa, China). For analyses of the CYP9AQ2

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expression patterns in different tissue types, total RNA samples were prepared from the

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foregut, midgut, gastric caeca, hindgut, Malpighian tubules, fat bodies, muscles, hemolymph

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and brains from fifth-instar nymphs. At least three independent biological replicates were 7 Page 7 of 24

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prepared for expression analysis. To remove potential genomic DNA contaminations, total

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RNA samples were treated with RNase-free DNase I (TaKaRa, China). Subsequently, first-

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strand cDNA was synthesized from 4 μg of each total RNA sample with an oligo(dT) primer

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using MLV reverse transcriptase (Fermentas, New York, USA).

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2.5. Treatment of insects with deltamethrin

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Five different concentrations (0.01, 0.02, 0.04, 0.08 and 0.12 μg/mL) of deltamethrin

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(Sigma, St Louis, USA) were used. In each treatment, 15-20 third-instar nymphs were

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topically applied with 3 μL of each deltamethrin solution or acetone (control) to the abdomen

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between the second and third sterna. Each treatment was repeated four times. After 12 h,

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surviving locusts were quickly frozen in liquid nitrogen for subsequent experiments.

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2.6. Reverse transcription quantitative PCR (RT-qPCR) analysis

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The expression levels of CYP9AQ2 were quantified by RT-qPCR using a Biosystems

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7300 Real-time PCR System (Applied Biosystems Inc, Foster, USA) and SYBR® Premix Ex

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Taq TM II kit (Takara, China). Each 20 μL reaction contained 10 μL SYBR Green, 0.8 μL

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each primer, 0.4 μL ROX, 2 μL cDNA template, and double distilled water. The cycling

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parameters consisted of initial step at 95 C for 10 s, followed by 40 cycles of 95 C for 5 s

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and 60 C for 31 s. Each RT-qPCR experiment consisted of three independent biological

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replicates, each with two technical replicates. For RT-qPCR analysis, specific primers of

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CYP9AQ2 (forward 5′-GGAGAACAAGCACCTCATCAA-3′ and reverse 5′-

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ACCACCTTCGCTTCCATCA-3′) were designed. A β-actin gene, shown a stable expression

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as described previously [17], was used as a reference gene to normalize the expression levels

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of CYP9AQ2 among the samples. The β-actin sequence-specific primers (forward 5’-

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CGAAGCACAGTCAAAGA

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GAGGTA and reverse 5’- GCTTCAGTCAAGAGAACAGGATG) were specific to one (EST 8 Page 8 of 24

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accession: LMC_004540) of the seven closely related actin genes based on the alignments of

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their nucleotide sequences identified from LocustDB

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database (http://locustdb.genomics.org.cn/) using GENEDOC.

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A sample cDNA was serially diluted (1, 1/2, 1/4, 1/8, and 1/16) and used to construct the

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standard curve. Primer pairs were only used that the standard curve is linear and had R2≥0.98.

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The threshold cycle (Ct) value for each dilution was then plotted against the log of its

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concentration. Target quantities were calculated from separate standard curves generated for

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each experiment. Results of three replications were averaged to give the final relative

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transcription levels of CYP9AQ2.

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2.7. Functional analysis of CYP9AQ2 by RNAi

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The primers used to synthesize dsRNA for CYP9AQ2 were designed based on the

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sequence domains of CYP9AQ2. The sequences of T7_CYP9AQ2 forward and reverse

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primers were 5′-taatacgactcactatagggAGGATTTCGACCACTTCACG and 5′-

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taatacgactcactatagggGTCGTACACCGTGGACTTGA, respectively. A cDNA template

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containing 500-bp CYP9AQ2 fragment (272–771) was generated to synthesize dsRNA by

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using T7 RiboMAX™ Express RNAi System (Promega). The synthesized dsRNA was

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dissolved in nuclease-free water, and examined by 1.5% agarose gel. The final concentration

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of dsRNA was adjusted to 1.5 μg/μL and stored at −20 °C until use.

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Second-instar nymphs of 2-day old were used for dsRNA injection experiments. In each

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treatment or control, each of 15 nymphs was injected with 2 μL (3 μg/locust) dsCYP9AQ2 or

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dsGFP into the abdomen between the second and third abdominal segments using a

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microinjector (Ningbo, China). Each treatment or control was repeated three times. To assess

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the transcript levels of CYP9AQ2 at different times following injections, the whole body of

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the nymphs was used for subsequent RNA extraction. The first-strand cDNA was reversed-

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transcribed as previously described. For each group, a pool of five nymphs was used for 9 Page 9 of 24

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RNAi efficiency test at three time points (12, 24 and 48 h) after the injections by using RT-

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qPCR as described in Section 2.6. The experiment was repeated three times.

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For insecticide treatments following dsRNA injections, 60 nymphs from dsRNA-

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treated or control group were separated into three subgroups as replicates. At the time point

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showing the highest RNAi efficiency, 3 μL of each of four insecticide solutions, including

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DDT (120 μg/mL), carbaryl (8 μg/mL), deltamethrin (0.03 μg/mL) and malathion (70

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μg/mL), was topically applied onto the abdomen between the second and third sterna of each

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nymph. The mortalities of the treated and control nymphs were assessed at 24 h after the

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insecticide treatment.

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2.8. Homology modeling

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The deduced protein sequence of CYP9AQ2 as a target sequence was submitted to

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BLASTP program (http://blast.ncbi.nlm.nih.gov/) to search for known protein structures

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against Protein Database (PDB). A series of candidate homology models was generated by

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MODELER 9v12 through comparative protein structure prediction. The most proper

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homology model of CYP9AQ2 was evaluated by MolPDF scores compared with homology

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templates. The generated model was further improved by using the DOPE-based loop

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modeling protocol [18-20].

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Docking of deltamethrin and CYP9AQ2 was performed with the AutoDock tools

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[21,22]. The molecule of deltamethrin used for docking to CYP9AQ2 was kept fixed during

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the docking.

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2.9. Statistical analysis

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All data were expressed as the mean ± S.E. Fold changes in gene expression between

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control and treated locusts, and differences among the tissues and development stages were

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subjected to Student's t-test and one-way analysis of variance (ANOVA) in combination with 10 Page 10 of 24

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a Fisher’s least significant difference (LSD) multiple comparison tests, respectively, by using

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the SPSS statistics program (Chicago, USA). Statistical differences were considered

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significant at P<0.05.

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3. Results and discussion

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3.1. Identification and characterization of CYP9AQ2

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After an initial search for the transcripts of CYP9 gene family from the locust

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transcriptome database, we identified a transcript putatively encoding a part of a protein

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sequence in CYP9 family. Further cloning and sequencing experiments resulted in a complete

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cDNA sequence consisting of 1578 bp with an open reading frame (ORF) of 1557 bp that

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encodes 519 amino acid residues. The predicted molecular mass and pI of its deduced protein

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are 58.79 kDa and 8.65, respectively. This new cytochrome P450 was named CYP9AQ2 by

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the P450 Nomenclature Committee (Dr. D. Nelson, personal communication) and given the

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GenBank accession number of HM131841.

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CYP9AQ2 appeared to be the first gene in the CYP9 family identified in L. migratoria.

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Alignments of the deduced amino acid sequence of CYP9AQ2 with the members of CYP9A

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subfamily from other insect species revealed a number of conserved motifs. Six approximate

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substrate recognition sites (SRS) regions (Fig. 1) were predicted on the basis of the secondary

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structural elements [23-25]. The oxygen binding motif of these conserved motifs deserves a

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special attention for the lack of the conserved residue threonine. Specifically, threonine of the

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oxygen binding motif is replaced by serine (Fig. 1). A similar replacement of threonine by a

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different amino acid residue has been reported in other CYP9 sequences [2]. However, it is

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unknown as to whether or not such a replacement will affect the catalytic function of the

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enzyme.

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3.2. Tissue-specific and developmental expression of CYP9AQ2 11 Page 11 of 24

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Because tissue-specific expression patterns of CYP genes in animals may be related to

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their roles in the particular tissues [26], the expression levels of CYP9AQ2 were determined

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in different tissues of the fifth-instar nymphs by using RT-qPCR (Fig. 2A). The highest

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expression of CYP9AQ2 was observed in foregut (142-fold) followed by hindgut (82-fold),

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Malpighian tubules (88-fold), brain (84-fold) and fat bodies (75-fold) whereas the lowest was

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in the hemolymph where the expression was arbitrarily set 1 in this study. The high

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expression of the gene in these tissues supported the notion that these tissues could play

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important roles in detoxification. However, further studies are needed to confirm its role in

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insecticide detoxification.

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The expression patterns of CYP9AQ2 in all seven development stages of the locust were

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examined in whole body by using RT-qPCR (Fig. 2B). The expression was 3.7~8.7 -fold

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higher in the nymphal stages and 2.6-fold higher in the adult stage than in the egg stage when

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the expression was arbitrarily set 1 for eggs in this study. The highest expression was found in

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fourth-instar nymphs (8.7-fold). Similarly, it has been reported that the expression level of

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CYP6F1 was the highest in fourth-instar larvae in Culex pipiens pallens [27]. It was

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speculated that the highest expression of CYP6F1 in fourth-instar larvae might be due to the

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adaptive regulation of the insect to metabolize xenobiotics upon exposures. Because of

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significantly increased food uptake in fourth-instar nymphs of the locust, up-regulation of

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CYP9AQ2 gene may help metabolize the plant chemicals in the locusts.

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3.3. Expression response of CYP9AQ2 to deltamethrin exposures

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To determine if the expression of CYP9AQ2 can be up-regulated by insecticides, we

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performed RT-qPCR to analyze the expression level of the nymphs treated with deltamethrin

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at different concentrations. Our studies showed no significant up-regulation of CYP9AQ2 by

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deltamethrin at two lower concentrations, probably due to rapid metabolism of deltamethin,

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which did not allow the insecticide to reach to the threshold for up-regulation of the gene 12 Page 12 of 24

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(Fig. 3). Similar results have also been reported in Musca domestica, where up-regulations of

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CYP4D4v2, CYP4G2, and CYP6A38 were not found in permethrin-resistant house flies after

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exposed to permethrin at lower doses [28]. In contrast, deltamethrin increased gene

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expression of CYP9AQ2 by 2.2- to 2.6-fold (P < 0.05) at three higher concentrations

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examined in this study. The maximum up-regulation of CYP9AQ2 by deltamethrin occurred

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at the concentration of 0.04 μg/mL (LD10) (Fig. 3).

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3.4. Functional analysis of CYP9AQ2 by RNAi

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RT-qPCR analyses at different time points (12, 24 and 48 h) after the injection of

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dsRNA showed decreased transcript levels of CYP9AQ2 at 12 h (about 54%), 24 h (about

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36%) and 48 h (about 57%) as compared with those in the controls (Fig. 4A), indicating a

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reasonably good silencing of CYP9AQ2 at 24 h after dsRNA injection. Such a suppression of

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the target gene was not restored diminished at 48 h. Thus, at 24 h after the injection of

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dsCYP9AQ2, we assessed the susceptibility of the dsRNA-injected locusts to different

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insecticides. The mortalities of the locusts injected with dsGFP and dsCYP9AQ2 after

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exposed to deltamethrin at the dose of 1.5 ng/g body weight were 22.9±2.2 and 48.2±7.4%,

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respectively (Fig. 4B). In contrast, similar treatments with malathion, DDT and carbaryl in

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the locusts after RNAi for CYP9AQ2 did not show significant effects on the susceptibility of

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the locusts to these insecticides.

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RNAi has been used to analyze the roles of CYP6AE14 in gossypol metabolism in

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Helicoverpa armigera [29], and CYP6G1 in DDT metabolism in D. melanogaster [30,31]. In

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a permethrin-resistant strain of the diamondback moth (Plutella xylostella), suppression of

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the overexpressed CYP6BG1 by RNAi led to significantly increased susceptibility of the

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insects the insecticide [32]. Thus, RNAi can be effectively used to assess the role of a CYP

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gene in insecticide detoxification, and our results support that CYP9AQ2 played an important

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role in detoxification of deltamethrin. 13 Page 13 of 24

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3.5. Homology modeling and deltamethrin docking of CYP9AQ2 The elucidation of three-dimensional structure of a protein can often help researchers

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better understand the protein function [25]. Our homology search of CYP9AQ2 protein

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sequence against the Protein Database (PDB) resulted in the highest sequence identity (34%)

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between a mammalian CYP (CYP3A4) and CYP9AQ2. According to template resolution and

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ligand molecular type, the X-ray structures of CYP3A4, 1TQN [33] and 3UA1 were the best

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templates. These two structures and their ligands (heme) were used as homology templates,

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simultaneously. The characteristic motif and SRSs (Substrate Recognition Sites) of CYPs

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were analyzed and labeled. Similarly, the CYP heme-binding domain signature and CYP

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oxygen binding sequence were signed (Fig. 5A). SRS1 positions in the loop, whereas SRS2

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and SRS3 are located between helices F and G. SRS4 is located in the helix I, whereas SRS5

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forms β-sheet. The SRS1, SRS4 and SRS5 above the heme-binding site form the bulk of the

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catalytic pocket.

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To help rationalize the experimental data, computational docking studies were

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performed using the homology model of CYP9AQ2. In keeping with the results of functional

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analysis, the deltamethrin molecule is not only located at the active site, but also the

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phenoxybenzyl moiety of the deltamethrin are closed to the heme of the homology model

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(Fig. 5B). It should be emphasized that the major site of ring hydroxylation of deltamethrin

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was the 4'-position and the 5'-position of the phenoxybenzyl moiety

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(http://www.inchem.org/documents/ehc/ehc/ehc97.htm). Analysis of deltamethrin metabolism

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by CYP6M2 suggested that 4'-hydroxylation was the major pathway of metabolism [34].

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Indeed, 4'-hydroxylation at this position has been reported as a major site of hydroxylation by

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most insect CYPs [35]. Furthermore, the 4'- and 5'-positions of the phenoxybenzyl moiety are

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near the heme plane (less than 3.0 Å). Therefore, it is reasonable to conjecture that

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hydroxylation in the phenoxybenzyl moiety is one of the deltamethrin metabolism pathways 14 Page 14 of 24

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by CYP9AQ2. Deltamethrin don’t contain hydroxyl group; however, deltamethrin has a

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similar nucleophilie (i.e., cyanogroup). In our docking model, cyanogroup of deltamethrin is

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near by the Ser311 of the oxygen region. It is, therefore, speculated that

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the cyanogroup of the deltamethrin should participate in the catalyzing process of CYP9AQ2

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[36].

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We realized that the sequence homology of CYP9AQ2 with CYP3A4 is relatively low;

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therefore, our CYP9AQ2 model may not be sufficiently accurate to simulate the catalytic

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reaction of CYP9AQ2 for deltamethrin. Nevertheless, a detailed analysis of the catalytic

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mechanism of CYP9AQ2 was beyond the scope of our research. Further work is necessary to

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determine the products of deltamethrin metabolism catalyzed by CYP9AQ2.

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4. Conclusion

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We identified and sequenced CYP9AQ2 cDNA from L. migratoria. Analysis of

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characteristic motifs of its deduced protein sequence indicated its highly conserved structures

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as compared with those of known insect CYP9 family. Tissue-specific and developmental

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expression patterns of CYP9AQ2 suggested that CYP9AQ2 is likely to be involved in

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detoxification of xenobiotics. The expression of CYP9AQ2 can be up-regulated by

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deltamethrin. Furthermore, injection of dsRNA specific to CYP9AQ2 can effectively suppress

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the gene expression, and consequently result in a significantly increased susceptibility of the

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locusts to deltamethrin. Computational docking studies suggested that hydroxylation of the

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phenoxybenzyl moiety might be one of the deltamethrin metabolic pathways by CYP9AQ2.

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These results support our notion that CYP9AQ2 may play a significant role in deltamethrin

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detoxification in the locust.

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15 Page 15 of 24

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Acknowledgements

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This research was supported by National Natural Science Foundation of China (International

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Cooperation and Exchange Program Grant No. 31320103921 and Research Grant No.

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31172161, 31301723), research fund for the Doctoral Program of Higher Education of China

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No. 20111401110006. The authors give special thanks to Dr. D. Nelson for help in the

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nomenclature of CYP9AQ2 and to Prof. Yuanhuai Han (Shanxi Agricultural University) for

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helping with the manuscript preparation.

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16 Page 16 of 24

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Figure Legends:

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Fig. 1. Alignment of amino acid sequences of CYP9AQ2 with nine other members of CYP9A

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subfamily and CYP3A4 (51247719) from human. The nine CYP9A members include

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CYP9A1 from Heliothis virescens; CYP9A12 (No. AAC25787) from Helicoverpa zea;

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CYP9A14 (AAR37015), CYP9A17 (ACB30272) and CYP9A18 (ABB69055) from

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Helicoverpa armigera; and CYP9A19 (ABQ08709), CYP9A20 (NP_001077079), CYP9A21

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(NP_001103394) and CYP9A22 (ABQ08707) from Bombyx mori. The conserved motifs are

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boxed with red dashed solid lines. Boxed with blue solid lines represent six substrate

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recognition sites.

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Fig. 2. Expression patterns of L. migratoria CYP9AQ2 gene in different tissues of the fifth-

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instar nymphs and in the whole body of different developmental stages as evaluated by RT-

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qPCR. (A) Their relative expression levels were examined in seven different tissues including

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foregut (FG), midgut (MG), gastric caecum (GC), hindgut (HG), Malpighian tubules (MT),

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fat bodies (FB), muscles (MC), hemolymph (HL) and brain (BR). (B) Their relative

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expression levels were examined in seven different developmental stages including egg (EG);

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first-instar (N1), second-instar (N2), third-instar (N3), fourth-instar (N4) and fifth-instar (N5)

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nymphs; and adult (AD). β -actin was used as an internal reference gene.

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Fig. 3. Effect of deltamethrin on the expression of CYP9AQ2 in locust nymphs. Five different

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concentrations (0.01, 0.02, 0.04, 0.08 and 0.12 μg/mL) of deltamethrin and acetone as control

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(0) were used to expose third-instar nymphs for 12 h. The mRNA level in the control and

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each treatment was normalized using β-actin as a reference gene. The relative levels of gene

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expression shown on Y axis are the ratio of the gene expression in the treatment in 22 Page 22 of 24

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comparison with that of the control in which acetone alone was used to treat insects. The

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vertical bars indicate standard errors of the mean (n = 4). One and two asterisks on the

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standard error bars indicated significant difference of the means at P<0.05 and P<0.01,

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respectively, among the control and each of the five concentrations of deltamethrin.

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Fig. 4. Changes in the transcript levels of CYP9AQ2 after the locust nymphs were injected

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with its corresponding dsRNA. The second-instar nymphs (2-day old) were used for the

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injection. (A) Silencing efficiency of CYP9AQ2 after the locust nymphs were injected with 3

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μg dsRNA. Control nymphs were injected with equivalent volumes of dsGFP. The transcript

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of CYP9AQ2 was examined by RT-qPCR. RNA was extracted and quantified by RT-qPCR at

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12, 24 and 48 h after the injection. β-actin was used as an internal reference gene. Vertical

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bars indicated standard errors of the mean (n = 3). Different letters next to the standard

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deviation bars indicate statistically significant differences in gene transcript levels between

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dsRNA treated and control nymphs based on Fisher’s LSD multiple comparison test

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(P<0.05). (B) Changes in the susceptibility of the locusts to different insecticides after the

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injection of CYP9AQ2 dsRNA in second-instar nymphs. Insecticides bioassays were

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conducted 24 h after the injections by topical application. The mortalities of the locusts were

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assessed 24 h after the deltamethrin treatments at the dose of 1.5 ng per gram of body weight.

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Results were mean and standard errors with S.E. of three biological replications (n = 3). An

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asterisk next to the standard deviation bars indicated significant differences in the mortalities

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among the control and treatments (student’s t-test, **P<0.01).

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Fig. 5. Predicted three-dimensional structures of CYP9AQ2 based on homology modeling

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(A) and docking of deltamethrin and CYP9AQ2 (B). The black arrows indicated putative

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binding sites of heme, SRSs and serine instead of conserved threonine. Image colored by 23 Page 23 of 24

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rainbow N

C terminus.

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