Cloning of an acetylcholinesterase gene in Locusta migratoria manilensis related to organophosphate insecticide resistance

Pesticide Biochemistry and Physiology 93 (2009) 77–84

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Cloning of an acetylcholinesterase gene in Locusta migratoria manilensis related to organophosphate insecticide resistance Xiaoxia Zhou, Yuxian Xia * Genetic Engineering Research Center, Bioengineering College, Chongqing University, Chongqing 400030, PR China

a r t i c l e

i n f o

Article history: Received 12 February 2008 Accepted 19 November 2008 Available online 6 December 2008 Keywords: Acetylcholinesterase Insecticide resistance Locusta migratoria manilensis

a b s t r a c t An acetylcholinesterase (AChE, EC 3.1.1.7) cDNA was cloned and characterized from oriental migratory locust (Locusta migratoria manilensis). The complete cDNA contains a 1638-bp GC-rich (GC% = 66%) open reading frame encoding 546 amino acid residues. The key amino acid residues, including the three residues Ser151 (200 in Torpedo), Glu277 (327) and His391 (440) forming a catalytic triad, five out of six cysteines putatively forming intra-chain disulfide bonds, and 10 out of the 14 aromatic residues lining the active site of the Torpedo AChE, are conserved. Bioinformatics analysis shows that the cloned gene belongs to a member of ace1. RNA interfering (RNAi) with a specific double strand RNA (dsRNA) led to a significant decrease in AChE1 mRNA, enzyme synthesis and activity. Bioassay showed that the RNA interfered locust was more susceptible to malathion than the control. The results are discussed in the light of the hypothesis that the cloned AChE1 gene is related to the organophosphate insecticide resistance of L. migratoria manilensis. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction Acetylcholinesterase (AChE, EC 3.1.1.7) is an essential enzyme at cholinergic synapses in all animals. It terminates neurotransmission by catalyzing the hydrolysis of the neurotransmitter acetylcholine. The enzyme is the primary target of organophosphate (OP) and carbamate (CA) insecticides, which work as AChE inhibitors. Since the first insect AChE gene cloned from fruit fly [1], the cDNA encoding AChEs have been cloned from several species including Diptera [2,3], Coleoptera [4], Hemiptera [5–9], Lepitoptera [10,11], and Acari [12–14]. These insect AChEs are classified according to their homology to Drosophila AChE (ace). AChE homologous to ace is Ace-orthologous AChE (AO-AChE) or ace2. AChE with less similarity to ace is Ace-paralogous AChE (AP-AChE) or ace1 [15,10,11]. Insensitive AChE caused by ace2 mutation has been proven the mechanism of OP and CA resistance in fruit fly [16], Colorado potato beetle [17], house fly [18,19], Australian sheep blowfly [3], olive fruit fly [20] and cotton aphid [15]. Furthermore, the mutation of the ace1 gene as the mechanism of insecticide resistance has been reported in some insects having two ace genes [21–25]. Therefore, the biochemical or molecular biological information on the AChE gene is highly valuable for understanding the mechanism of insecticide resistance.

* Corresponding author. Fax: +86 23 65120490. E-mail address: [email protected] (Y. Xia). 0048-3575/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2008.11.007

The oriental migratory locust, Locusta migratoria manilensis (Meyen), a common subspecies of L. migratoria, is widely distributed in Southeast Asia [26]. It feeds on bulrush and gramineous plants, and is a major agricultural pest due to periodic massive outbreaks [27]. Extensive use of insecticides has caused less sensitive AChE and OP resistance in some locust populations [27]. Although the insensitive AChE is an important mechanism for insecticide resistance in many pest species, the molecular basis of the altered forms of AChE, is unknown for L. migratoria manilensis. Here we report a cDNA sequence encoding AChE cloned from L. migratoria manilensis and elucidate the relationship between the enzyme and the malathion resistance by RNA interfering (RNAi) and bioassay. To our knowledge, this is the first report on the ace gene and molecular properties of AChE in Orthoptera. The study furthers our understanding of the molecular properties of insect AChEs and should help elucidate molecular mechanisms of OP resistance in the locust in the future. 2. Experiments 2.1. Insects The colony of L. migratoria manilensis was routinely reared in the laboratory under conditions as described by He et al. [28]. Briefly, locusts were raised with corn sprout in 20  20  20 cm cages at 28 ± 2 °C, under a 12-h light/12-h dark photoperiod.

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2.2. Extraction of the genomic DNA and the total RNA Genomic DNA was isolated from the oriental migratory locusts by the method of Sambrook et al. [29] with slight modifications. Briefly, 3 g of adult oriental migratory locusts, without wings and alimentary canal, were pulverized in liquid nitrogen. After the nitrogen had dissipated, the sample was mixed with 10 ml of extraction buffer (100 mM Tris, 40 mM EDTA, 2% SDS) and incubated for 60 min at 65 °C, swirling every 10 min. The DNA was extracted with phenol/chloroform and precipitated with ethanol. The precipitated DNA was washed with 70% ethanol and dissolved in 500 lL of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) containing 200 lg/ml RNase A, and incubated at 37 °C overnight. The DNA was again extracted with phenol/chloroform, precipitated with ethanol and washed with 70% ethanol once. The DNA was redissolved in the TE buffer and stored at 80 °C. Total RNA was extracted from 30 mg of the brain of oriental migratory locusts with SV Total RNA Isolation System (Promega Co., USA) according to the manufacturer’s instructions. A mixture of juveniles and adults were used as a source of material for RNA extraction. First strand cDNAs were immediately synthesized after extraction. 2.3. Cloning and sequencing Degenerate primers were designed according to the conserved amino acid sequences reported in other insect species to amplify the partial fragment of AChE cDNA, in order to decrease the complexity of the primers, the bias of codon usage of L. migratoria manilensis was considered (http://www.kazusa.or.jp/codon). The sense primer is 50 -TGGATBTWSGGCGGBGG-30 and the antisense primer is 50 -CCNGCVGACTCSCCGAA-30 . The conserved regions targeted with the degenerate primers encoded the amino acid sequences WIFGGG and FGESAG respectively. The PCR was performed using the L. migratoria manilensis genome DNA as template. Conditions were 35 cycles, each with 95 °C for 30 s; 55 °C for 30 s; and 72 °C for 30 s. PCR products were cloned into pMD18-T vector (TaKaRa). The inserted fragment was send to Shanghai Songon Biologic Technology Co., Ltd. for sequencing. The first strand of cDNA for 30 -RACE was synthesized with oligo dT-3 sites adaptor primer provided by the 30 -Full RACE Core Set (TaKaRa) according to the manufacturer’s instructions, the 30 -end fragment of the AChE cDNA sequence was amplified with gene specific forward primers GSPF1, GSPF2 (Fig. 1), and the 3 sites adaptor primer 50 -CTGATCTAGAGGTACCGGATCC-30 . The following parameters were used. 95 °C for 1 min followed by 40 cycles at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 2 min and one additional cycle at 72 °C for 10 min. The 50 -end of the cDNA was amplified by a modified 50 -RACE method using CreatorTM SMARTTM cDNA Library Construction Kit (Clontech). The total RNA was annealed at 72 °C for 2 min with gene specific reverse primer GSPR1 (Fig. 1) and SMART IVTM Oligonucleotide provided in the kit, then the first strand cDNA was synthesized at 42 °C according to the direction. Two-step PCR protocol was employed to amplify the 50 -end of L. migratoria manilensis AChE gene with primers GSPR1, GSPR2, and SMART IVTM. 2.4. Alignment and phylogenetic analysis Identity was investigated using BLASTP (http://www.ncbi.nlm. nih.gov/blast/Blast.cgi) and CLUSTALW (http://align.genome.jp). Locations of signal peptide cleavage sites were predicted using SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP). Hydropathy profile was drawn using protein hydrophobicity plots server

(http://www.vivo.colostate.edu/molkit/hydropathy/index.html). Phylogenetic tree was constructed with neighbor-joining methods. The amino acid sequences of insect AChE precursors were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index. html). 2.5. RNAi Double-stranded RNA (dsRNA) was synthesized using MEGAscript high yield transcription set (Ambion) according to the producer’s instructions. The DNA template with T7 polymerase promoter sequence at both ends was prepared by PCR using the forward primer 50 -TAATACGACTCACTATAGGGGTTCGGCGAGTCGG30 , and the reverse primer 50 -TAATACGACTCACTATAGGG TACTCGCGCCCGTA-30 . The italic shows the minimum promoter sequence needed for efficient transcription, and the base in bold is the first base incorporated into RNA during transcription. Approximately 1 lg template was used for a 20 lL in vitro transcription reaction. The reaction mix was incubated for 10–16 h at 37 °C, followed by 15 min of DNase I treatment at 37 °C. The reaction mix was then purified and concentrated by phenol/chloroform extraction and isopropanol precipitation, and dissolved in TE buffer (1 mM Tris, 1 mM EDTA, pH 7.5). RNA was annealed in TE buffer by heating at 94 °C for 5 min, then cooling at room temperature for several hours. Annealing was confirmed by agarose gel electrophoresis. Fourth instar locusts of both sexes were anaesthetized on ice and injected with approximately 1.2 lg dsRNA in the abdomen between the second and third abdominal segments using a microsyringe (Shanghai Jiaan Analyze Apparatus Factory). Negative control was treated with the same amount of green fluorescence protein (gfp) dsRNA as the experimental group [30]. Quantitative real-time PCR (qrt-PCR) was performed to evaluate the efficiency of RNAi with SYBR-Green Realtime PCR Master Mix (TOYOBO) using the iCycler iQ real-time PCR detection system (Bio-Rad). A member of 70-kDa heat shock protein (hsp70, AY178988) was selected as reference gene [31]. Primers of 50 -C TGGTGTGCTCATTCAGGTAT-30 and 50 -TCGTGGGGCAGGTGGTAT T-30 were used for hsp70, and primers of 50 -GCCAACTTCGCCAAG ACCG-30 and 50 -TCAGGTACTCGCGCCCGTAC-30 were used for AChE gene. The amplification efficiency of each gene was estimated by using the equation, E = 101/slope, where the slope was derived from the plot of amplification critical time (Ct value) versus serially diluted template cDNA concentration. Following qrt-PCR, homogeneity of PCR product was confirmed by the melting curve analysis. Quantification of the transcript level or relative copy number of target gene against reference gene was conducted according to the 2DDCt method [32]. Indirect ELISA was performed to evaluate the impact of RNAi on AChE synthesis. Anti-Lm-AChE1 antibody (1:500, prepared in our lab) and horseradish peroxidase (HRP) conjugated goat-antimouse IgG (1:2000, Jackson) were used as primary and secondary antibody in tandem. The results of absorbance were detected using microplate reader (BIO-RAD, Model 550) at wavelength of 490 nm. 2.6. AChE activity assay Locusta migratoria manilensis brains were separated in autoclaved water and homogenized individually in 300 lL of 0.1 M phosphate buffer (pH 7.5) containing 0.3% Triton-X 100 at 4 °C. The homogenates were centrifuged at 5000g and 4 °C for 15 min and the resultant supernatant was used as the enzyme source for the estimation of enzyme activity. AChE activities were determined based on the method of Ellman et al. [33] using acetylthiocholine iodide (ATC) as substrate and 5, 50 -dithio-bis(2-nitrobenzoic acid) (DTNB) as chromogenic

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Fig. 1. cDNA and deduced amino acid sequences of L. migratoria manilensis AChE1 (Accession No. EU231603). Arrows with F and R indicate the position of degenerate primers, and GSPF1, GSPF2 and GSPR1, GSPR2 show the specific primers used in the 30 - and 50 -ends amplification. Asterisks denote the catalytic triad. Triangle represents the cholinebinding site. Rectangles indicate the residues lining in the active gorge. Circles mark the five out of the six cysteines form the three intro-chain disulfide bonds. Dot underlines show the five potential N-linked glycosylation sites. The italic shows the multiple inframe stop codons upstream of the start codon. The stop codon at the end of the ORF is highlighted in bold.

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Fig. 1 (continued)

reagent. Briefly, 20 lL of enzyme solution was mixed with 180 lL of solution containing 0.28 mM ATC and 0.44 mM DTNB. The rate of reaction was determined for 5 min at room temperature (approximately 24 °C) at 405 nm on a microplate reader (BIORAD, Model 550). Protein content was determined by the method of Bradford [34] using bovine serum albumin (BSA) as the standard. The assays were carried out on a microplate reader (BIO-RAD, Model 550) at 595 nm.

2.7. Bioassay Malathion (West Chester, PA) was dissolved in acetone to give concentrations of 100, 150, 200, 300, and 400 ppm. The susceptibility to malathion between AChE dsRNA and gfp dsRNA treated locusts was determined by topical application of a 5 lL drop of malathion solution underneath the pronotum of the locust with a pipette (Finnpipette). A sample of 30 dsRNA treated locusts was used for bioassay, each assay was carried out with five malathion

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Fig. 2. Phylogenetic analysis based on AChE1 and AChE2 amino acid sequences using phylip Ver. 3.67. Bootstrap values of 100 trials are indicated at the branches. The bar indicates phylogenetic distance value. Tc-AChE from Torpedo californica is defined as an outgroup. The tree was made with the neighbor-joining method using multiple alignments of amino acid sequences. Anopheles gambiae, Anog-AChE1 (AJ488492) and Anog-AChE2 (AAAB01008846); Aphis gossypii, Ag-AChE1 (AF502081) and Ag-AChE2 (AF502082); Bactrocera dorsalis, Bd-AChE2 (AY155500); Blattella germanica, Bg-AChE1(DQ288249); Bactrocera oleae, Bo-AChE2 (AF452052); Boophilus microplus, Bm-AChE1 (AJ223965), Bm-AChE2 (AF067771), Bm-AChE3 (AY267337); Bombyx mori, Bom-AChE2 (AB161180) and Bom-AChE1 (AB189740); Culex pipiens pallens, Cpp-AChE1 (AY762905); Culex tritaeniorhynchus, Ct-AChE1 (AB122152) and Ct-AChE2 (AB122151); Drosophila melanogaster, Dm-AChE (X05893); Helicoverpa armigera, Harm-AChE2 (AF369793); Helicoverpa assulta, Ha-AChE1 (DQ001323) and Ha-AChE2 (AY817736); Leptinotarsa decemlineata, Ld-AChE2 (L41180); Lucilia cuprina, Lc-AChE2 (U88631); Musca domestica, Md-AChE2 (AF281167); Myzus persicae, Mp-AChE1 (AF287291) and Mp-AChE2 (AY147797); Nephotettix cincticeps, Nc-AChE1 (AF145235) and Nc-AChE2 (AY256851); Plutella xylostella, Px-AChE1 (AAV65825) and Px-AChE2 (AY061975); Rhipicephalus appendiculatus, Ra-AChE2 (AJ006338); Rhopalosiphum padi, Rp-AChE1 (AY667435) and Rp-AChE2 (AY707318); Schizaphis graminum, Sg-AChE1 (AF321574); Sitobion avenae, Sa-AChE1 (AY819704) and Sa-AChE2 (AY707319); Tetranychus urticae, Tu-AChE (AY188448).

doses and a solvent control. Mortality was recorded 24 h after treatment. Larvae were considered dead if they were not able to move in a coordinated way when touched with a brush. Control mortality never exceeded 10%. LD50 value was estimated by probit analysis. All the bioassays were conducted with three replicates. 3. Results 3.1. cDNA squence and its deduced amino acid of Lm-AChE A 263-bp fragment of AChE gene was isolated from L. migratoria manilensis genome DNA using the degenerate primers. There is no intron in the short DNA fragment, and the deduced amino acid sequence is in the conserved region of AChEs containing the catalytic site. A contiguous cDNA sequence with a total length of 2.2 kb was obtained by 30 - and 50 -end GSP primer walking (Fig. 1). The open reading frame (ORF), which encoding for an AChE precursor of 546 amino acid residues, is a GC-rich sequence (GC% = 66%). There is a 286 bp and 317 bp of non-translated region at 50 - and 30 -end, respectively. No polyadenylation signal was found within the 30 UTR, although a 14-bp span of poly (A) nucleotides was detected at its end. The cDNA-deduced amino acid sequence from the L. migratoria manilensis AChE (Lm-AChE) gene was compared with other sequences in GenBank with BLASTP. The AChE sequence shares 77.84% identity with Blattella germanica AChE1, 75.46% identity

with Nephotettix cincticeps AChE2 (which has little similarity with Drosophila AChE than the N. cincticeps AChE1). Because the LmAChE shares as little as 36.26% identity with Drosophila AChE, the cloned Lm-AChE gene is named as Lm-ace1. Phylogenetic tree (Fig. 2) also demonstrates that the Lm-AChE is a member of APAChEs. Indeed, Lm-AChE1 exhibits all the common features for an AChE including: (1) conserved active site triad, S151 (S200 in Torpedo), E277 (327) and H391 (440); (2) a choline-binding site W45 (84 in Torpedo); (3) five out of the six cysteines putatively forming intra-chain disulfide bonds; (4) 10 conserved aromatic amino acid residues out of 14 aromatic residues lining the catalytic gorge of the Torpedo AChE (Fig. 1); and (5) the sequence FGESAG, flanking S151, conserved in all cholinesterase. Signal peptide cleavage sites prediction (data not shown) with SignalP 3.0 server was failed in finding any possibility of signal peptide and signal anchor, so the Lm-AChE1 is most likely a nonsecretory protein. Nevertheless, the start codon can be identified because of the multiple in frame stop codons upstream of it and the possible Kozak’s context [35]. It seems that the Lm-AChE1 has 50 amino acid residues absence at the N-terminal compared with other AChEs. But AChE is highly divergent at both terminals, so the effect of the missing residues has on the size of mature peptide is uncertain. Another explanation for this is that the existence of alternative splicing at the 50 -end of insect AChE [36]. Whether it is true or not remains to be elucidated.

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Fig. 3. Hydropathy profiles of some insect AChEs. Bg-AChE1, Blattella germanica AChE1; Dm-AChE, Drosophila melanogaster AChE; Lm-AChE1, Locusta migratoria manilensis AChE1; Nc-AChE2, Nephotettix cincticeps AChE2.

3.2. Lm-AChE RNAi A 929 bp double stranded RNA (dsRNA) from Lm-AChE1 mRNA was prepared in vitro for RNA interfering (RNAi). The Lm-ace1 RNAi-treated locusts were compared with gfp dsRNA treated controls daily, but no distinct phenotype difference were observed among the RNAi groups and controls at a serial of doses and different instars. To evaluate the efficiency of RNAi, quantitative real-time PCR was performed to evaluate the efficiency of RNAi at the level of transcription, ELISA was employed to estimate the RNAi effect at the level of translation, the specific activity of AChE was measured to estimate the impact of RNAi on enzyme activity. As shown in Table 1, the transcription level of Lm-ace1 in Lm-AChE dsRNA in-

140 Relative activity (%)

Despite the absence of signal peptide and missing amino acid residues at the N-terminal, Lm-AChE1 has a quite similar hydropathy profile with that of B. germanica AChE1, N. cincticeps AChE2, and Drosophila AChE (Fig. 3). Like other AChEs, the hydrophobic region in the C-terminus suggested that Lm-AChE1 also had an anchor for glycosyl phosphatydil inositol replacement [37].

120 100 80 60 40 20 0 0

2

4

6 Time (Day)

8

10

12

Fig. 4. The effect of RNAi on AChE activity. Enzyme activity was measured every 2 days after 1.2 lg dsRNA injection. The results were expressed as relative activity, which was defined as the ratio of the specific activity between Lm-ace1 RNAi locusts and control locusts. The results are expressed as the mean ± STD (n = 3).

jected locusts only took up 53.86% against that of the gfp dsRNA injected locusts. The qrt-PCR result indicated that the RNAi was

Table 1 Comparison of the RNAi results and malathion susceptibility between ace 1 and gfp dsRNA treated locusts.a

Control group RNAi group

Transcription level (%)

Translation level (%)

LD50 (95% CIb)

Slope ± SE

v2

cc

98.08 ± 10.20 52.83 ± 16.25

109.70 ± 12.73 51.97 ± 13.32

4.34 (2.32–8.11) 1.73 (1.43–2.10)

1.49 ± 0.14 2.49 ± 0.043

1.33 3.01

0.97 0.98

A significant difference between the LD50 of the two groups was based on the non-overlapping 95% CI of LD50. a Locusts injected with 1.2 lg of ace 1 and gfp dsRNA were used. The insects sampled on the 5th day were used for qrt-PCR, and sampled on the sixth day were used for ELISA or bioassay. The transcription and translation level are given as the average ± STD compared with the non-treated ones at the same stage (n = 3). Thirty locusts were used in each group for malathion susceptibility detection. b Confidence interval, all values are lg/each. c Correlation coefficient.

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effective at transcription level. Compared to gfp dsRNA treated ones, Lm-ace1 RNAi-treated locusts had a 47.36% decrease in enzyme synthesis (Table 1). Trends of enzyme activity fluctuation vs. time (Fig. 4) also showed that the dsRNA influenced the expression of Lm-AChE in a specific way. As shown in Fig. 4, AChE activity decreased 40.8% on the 4th day, 60% on the 6th day, and 64% on the 8th day. Because persistent enzyme suppression lasted from the 4th day to the 8th day, the dsRNA treated locusts on the 6th day were subjected to bioassay. 3.3. Malathion sensitivity In order to detect the effect of AChE activity depression on OP sensitivity, bioassay was done on the 6th day after 1.2 lg dsRNA injection. The results (Table 1) showed that the LD50 of the ace1 RNA interfered group was 1.73 lg, and that of the control group was 4.34 lg. The difference between treatments is significant (p < 0.05). As was expected, the ace1 dsRNA interfered locusts with decreased AChE activity, were more sensitive to malathion than the gfp dsRNA treated control. 4. Discussions Insect AChEs have been of interest because of their critical function in neurotransmission and as a target of OP and CA insecticides. In order to clone Lm-AChE gene, the databases of L. migratoria manilensis constructed by Kang et al. [38] were searched for the sake of finding a fragment of Lm-ace, but no ace sequence was found. Unlike other authors, we amplify the AChE conserved fragment from genome DNA, because our attempt to clone the Lm-AChE gene from mRNA resulted in nonspecific amplification. Two reasons were proposed for this failure. First, the abundance of the target template is extremely low. For example, in Leptinotarsa decemlineata, AChE accounts for as little as 0.0026% of the total protein in the head and prothorax which implies that the mRNA for AChE is expressed at extremely low level [4]. Second, the degenerate primer set used was designed from the esteratic subsite of AChE, a region that is highly conserved in many serine hydrolases. There were two fragments when genome DNA was used as template, both of them were cloned and sequenced, but only the 263 bp fragment was from the target gene. In addition to high GC content, the deduced amino acid sequence is shorter than other AChEs. There are two potential translation initiation sequences (ATGs) upstream of the largest ORF; the second is internal to the reading frame of the first. Both represent possible initiation codons, but the first seems to provide a superior match with the consensus for efficient initiation of translation [35]. Some insects have two AChE genes. We only cloned the ace1 from L. migratoria, which is the major form in some insects with two AChE genes, such as Plutella xylostella [39], helicovepa assulta [10], Aedes albopictus [40], and B. germanica [41]. The results’ accordance between qrt-PCR, ELISA (Table 1), and activity assay (Fig. 4), also implies that AChE1 is the predominant form in vivo. The reasons of the RNAi failed to show any phenotype and to reduce all AChE activity should be explained as following. Firstly, the efficiency of RNAi is limited, it can touch down the expression of a gene, but is incapable of delete a gene. Secondly, the half-life of AChE is long. In mammalian, the half-life of AChE is 7 days. Thirdly, insects can live normally with little proportion of AChE. For example, Drosophila can live with 25% AChE activity, and 10% is the minimal fraction of AChE compatible with life in spider mites [42]. The results of RNAi and bioassay indicated that the decrease in AChE activity increased the sensitivity of malathion. This indicates that the enzyme is the target of OP insecticide and the quantity of

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it is related to malathion sensitivity. These results agree well with the conclusion drawn by Yang et al. [43]. Many agricultural and medical pests have developed resistance to insecticides by decreased sensitivity of AChE [44]. Less sensitive AChE has been purified from resistant population of L. migratoria manilensis and proven to be related to malathion resistance [27,43]. However, insufficient information at the molecular level in this insect has greatly retarded the understanding of the resistance mechanism. Successful cloning and sequencing of the AChE1 cDNA from L. migratoria manilensis will allow us to design specific primers to directly amplify cDNA from different insecticide resistant strains by PCR. Once deduced amino acid sequences are available, mutational analyzes should reveal specific mutations that have resulted in altered AChEs conferring insecticide resistance. Acknowledgments The authors thank Anthony Keith Charnley, University of Bath, for critical reading of this manuscript. This work was supported by a grant from the Hi-Tech Research and Development Program (No. 2006AA10A212) of the Ministry of Science and Technology, China.

References [1] L.M.C. Hall, P. Spierer, The ace locus of Drosophila melanogaster: structural gene for acetylcholinesterase with an unusual 50 leader, EMBO J. 5 (1986) 2949– 2954. [2] Y. Huang, C. Qiao, M.S. Williamson, A.L. Devonshire, Characterization of the acetylcholinesterase gene from insecticide resistant houseflies (Musca domestica), Chin. J. Biotechnol. 13 (1997) 177–183. [3] Z. Chen, R. Newcomb, E. Forbes, J. McKenzie, P. Batterham, The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina, Insect Biochem. Mol. Biol. 31 (2001) 805–816. [4] K.Y. Zhu, J.M. Clark, Cloning and sequencing of a cDNA encoding acetylcholinesterase in Colorado potato beetle, Leptinotarsa decemlineata (Say), Insect Biochem. Mol. Biol. 25 (1995) 1129–1138. [5] L.M.C. Hall, C.A. Malcolm, The acetylcholinesterase gene of Anopheles stephensi, Cell. Mol. Neurobiol. 11 (1991) 131–141. [6] N. Anthony, T. Rocheleau, G. Mocelin, H.J. Lee, R.ffrench-Constant, Cloning, sequencing and functional expression of an acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti, FEBS Lett. 368 (1995) 461–465. [7] T. Tomita, O. Hidoh, Y. Kono, Absence of protein polymorphism attributable to insecticide-insensitivity of acetylcholinesterase in the green rice leafhopper, Nephotettix cincticeps, Insect Biochem. Mol. Biol. 30 (2000) 325–333. [8] J.R. Gao, S. Kambhampati, K.Y. Zhu, Molecular cloning and characterization of a greenbug (Schizaphis graminum) cDNA encoding acetylcholinesterase possibly evolved from a duplicate gene lineage, Insect Biochem. Mol. Biol. 32 (2002) 765–775. [9] F. Li, Z.J. Han, Two different genes encoding acetylcholinesterase existing in cotton aphid (Aphis gossypii), Genome 45 (2002) 1134–1141. [10] D.W. Lee, S.S. Kim, S.W. Shin, W.T. Kim, K.S. Boo, Molecular characterization of two acetylcholinesterase genes from the oriental tobacco budworm, Helicoverpa assulta (Guenée), Biochim. Biophys. Acta 1760 (2006) 125–133. [11] A. Seino, T. Kazuma, A.J. Tan, H. Tanaka, Y. Kono, K. Mita, T. Shiotsuki, Analysis of two acetylcholinesterase genes in Bombyx mori, Pestic. Biochem. Physiol. 88 (2007) 92–101. [12] R. Hernandez, H. He, A.C. Chen, G.W. Ivie, J.E. George, G.G. Wagner, Cloning and sequencing of a putative acetylcholinesterase cDNA from Boophilus microplus (Acari: Ixodidae), J. Med. Entomol. 36 (1999) 764–770. [13] G.D. Baxter, S.C. Barker, Comparison of acetylcholinesterase genes from cattle ticks, Int. J. Parasitol. 29 (1999) 1765–1774. [14] Y. Anazawa, T. Tomita, Y. Aiki, T. Kozaki, Y. Kono, Sequence of a cDNA encoding acetylcholinesterase from susceptible and resistant two-spotted spider mite, Tetranychus urticae, Insect Biochem. Mol. Biol. 33 (2003) 509–514. [15] F. Li, Z.J. Han, Mutations in acetylcholinesterase associated with insecticide resistance in the cotton aphid, Aphis gossypii Glover, Insect Biochem. Mol. Biol. 34 (2004) 397–405. [16] A. Mutero, M. Pralavorio, J.M. Bride, D. Fournier, Resistance-associated point mutations in insecticide-insensitive acetylcholinesterase, Proc. Natl. Acad. Sci. USA 9 (1994) 5922–5926. [17] K.Y. Zhu, S.H. Lee, J.M. Clark, A point mutation of acetylcholinesterase associated with Azinphosmethyl resistance and reduced fitness in Colorado potato beetle, Pestic. Biochem. Physiol. 55 (1996) 100–108.

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[18] T. Kozaki, T. Shono, T. Tomita, Y. Kono, Fenitroxon insensitive acetylcholinesterases of the housefly, Musca domestica associated with point mutations, Insect Biochem. Mol. Biol. 31 (2001) 991–997. [19] S.B. Walsh, T.A. Dolden, G.D. Moores, M. Kristensen, T. Lewis, A.L. Devonshire, M.S. Williamson, Identification and characterization of mutation in the housefly (Musca domestica) acetylcholinesterase involved in insecticide resistance, Biochem. J. 359 (2001) 175–181. [20] J.G. Vontas, M.J. Hejazi, N.J. Hawkes, N. Cosmidis, M. Loukas, R.W. Janes, J. Hemingway, Resistance-associated point mutations of organophosphate insensitive acetylcholinesterase, in the olive fruit fly Bactrocera olea, Insect Mol. Biol. 11 (2002) 329–336. [21] M. Weill, P. Fort, A. Berthomieu, M.P. Dubois, N. Pasteur, M. Raymond, A novel acetylcholinesterase gene in mosquitoes codes for the insecticide target and is nonhomologous to the ace gene in Drosophila, Proc. R. Soc. Lond. B 269 (2002) 2007–2016. [22] M. Weill, G. Lutfalla, K. Mogensen, F. Chandre, A. Berthomieu, C. Berticat, N. Pasteur, A. Philips, P. Fort, M. Raymond, Comparative genomics: insecticide resistance in mosquito vectors, Nature 423 (2003) 136–137. [23] T. Nabeshima, T. Kozaki, T. Tomita, Y. Kono, An amino acid substitution on the second acetylcholinesterase in the pirimicarb-resistant strains of the peach potato aphid, Myzus persicae, Biochem. Biophys. Res. Commun. 307 (2003) 15–22. [24] S. Toda, S. Komazaki, T. Tomita, Y. Kono, Two amino acid substitutions in acetylcholinesterase associated with pirimicarb and organophosphorous insecticide resistance in the cotton aphid, Aphis gossypii Glover (Homoptera: Aphididae), Insect Mol. Biol. 13 (2004) 549–553. [25] M.C. Andrew, A. Callaghan, L.M. Field, M.S. Williamson, G.D. Moores, Identification of mutations conferring insecticide-insensitive AChE in the cotton-melon aphid, Aphis gossypii, Insect Mol. Biol. 13 (2004) 555–561. [26] Y.P. He, E.B. Ma, K.Y. Zhu, Characterizations of general esterases in relation to malathion susceptibility in two field populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen), Pestic. Biochem. Physiol. 78 (2004) 103–113. [27] E.B. Ma, Y.P. He, K.Y. Zhu, Comparative studies of acetylcholinesterase purified from two field populations of the oriental migratory locust (Locusta migratoria manilensis): implications of insecticide resistance, Pestic. Biochem. Physiol. 78 (2004) 67–77. [28] Z. He, Y. Cao, Z. Wang, Y. Yin, G. Peng, Y. Xia, Role of hunchback in segment patterning of Locusta migratoria manilensis revealed by parental RNAi, Dev. Growth Differ. 48 (2006) 439–445. [29] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, second ed., Cold Spring Harbor Laboratory Press, New York, 1989. [30] W. Zhang, Y. Yin, B. Zhang, Z. Wang, G. Peng, Y. Cao, Y. Xia, Cloning of a novel protease required for the molting of Locusta migratoria manilensis, Dev. Growth Differ. 49 (2007) 611–621.

[31] W. Qin, M.G. Tyshenko, B.S. Wu, V.K. Walker, R.M. Robertson, Cloning and characterization of a member of the hsp70 gene family from Locusta migratoria, a highly thermotolerant insect, Cell Stress Chaperones 8 (2003) 144–152. [32] M.W. Pfaffl, A new mathematical model for relative quantification in real-time RT-PCR, Nucleic Acids Res. 29 (2001) 2002–2007. [33] G.L. Ellman, K.D. Courtny, U. Andres, K.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88–95. [34] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254. [35] M. Kozak, Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes, Cell 44 (1986) 283–292. [36] G.D. Baxter, S.C. Barker, Acetylcholinesterase cDNA of the cattle tick, Boophilus microplus: characterisation and role in organophosphate resistance, Insect Biochem. Mol. Biol. 28 (1998) 581–589. [37] R. Haas, T.L. Marshall, T.L. Rosenberry, Drosophila acetylcholinesterase: Demonstration of a glycoinositol phospholipid anchor and an endogenous proteolytic cleavage, Biochemistry 27 (1988) 6453–6457. [38] L. Kang, X.Y. Chen, Y. Zhou, B.W. Liu, W. Zheng, R.Q. Li, J. Wang, J. Yu, The analysis of large-scale gene expression correlated to the phase of the migratory locust, PANS 51 (2004) 17611–17615. [39] J.H. Baek, J.I. Kim, D.W. Lee, B.K. Chung, T. Miyata, S.H. Lee, Identification and characterization of ace1-type acetylcholinesterase likely associated with organophosphate resistance in Plutella xylostella, Pestic. Biochem. Physiol. 81 (2005) 164–175. [40] H. Mizuno, T. Tomita, S. Kasai, O. Komagata, S. Imanishi, Y. Kono, Identification and expression of two types of acetylcholinesterases in a cultured cell line of Aedes albopictus, compared to mosquito whole body extracts, Appl. Entomol. Zool. 41 (2006) 445–453. [41] H. Mizuno, S. Oh, O. Komagata, S. Kasai, H. Honda, Y. Kono, T. Tomita, Differential tissue distribution of two acetylcholinesterase transcripts in the German cockroach, Blattella germanica, Appl. Entomol. Zool. 42 (2007) 643–650. [42] H.R. Smissaert, A. El Hamid, W.P.J. Overmeer, The minimum acetycholinesterase (AChE) fraction compatible with life derived by aid of a simple model explaining the degree of dominance of resistance to inhibitors in AChE ‘‘mutants”, Biochem. Pharmacol. 24 (1975) 1043–1047. [43] M. Yang, J. Zhang, K.Y. Zhu, T. Xuan, X. Liu, Y. Guo, E. Ma, Increased activity and reduced sensitivity of acetylcholinesterase associated with malathion resistance in a field population of the oriental migratory locust, Locusta migratoria manilensis (Meyen), Pestic. Biochem. Physiol. 91 (2008) 31–38. [44] D. Fournier, A. Mutero, Modification of acetylcholinesterase as a mechanism of resistance to insecticides, Comp. Biochem. Physiol. 108C (1994) 19–31.