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Mutation in acetylcholinesterase1 associated with triazophos resistance in rice stem borer, Chilo suppressalis (Lepidoptera: Pyralidae)
Biochemical and Biophysical Research Communications 378 (2009) 269–272
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Mutation in acetylcholinesterase1 associated with triazophos resistance in rice stem borer, Chilo suppressalis (Lepidoptera: Pyralidae) Xiaojing Jiang a,1, Mingjing Qu b,1, Ian Denholm c, Jichao Fang d, Weihua Jiang a, Zhaojun Han a,* a
Key Laboratory of Monitoring and Management of Plant Diseases and Insects, Ministry of Agriculture, Nanjing Agricultural University, Weigang No. 1, Nanjing 210095, China Shandong Peanut Research Institute, Qingdao 266100, China c Plant and Invertebrate Ecology Division, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK d Institute of Plant Protection, Jiangsu Academy of Agricultural Science, Nanjing 210014, China b
a r t i c l e
i n f o
Article history: Received 5 November 2008 Available online 24 November 2008
Keywords: Chilo suppressalis Acetylcholinesterase Gene mutation Insecticide resistance Triazophos
a b s t r a c t Two full-length genes encoding different acetylcholinesterases (AChEs), designated as Ch-ace1 and Ch-ace2, were cloned from strains of the rice stem borer (Chilo suppressalis) susceptible and resistant to the organophosphate insecticide triazophos. Sequence analysis found an amino acid mutation A314S in Ch-ace1 (corresponding to A201 in Torpedo californica AChE) that was consistently associated with the occurrence of resistance. This mutation removed an MspA1 I restriction site from the wild type allele. An assay based on restriction fragment length polymorphism (RFLP) analysis was developed to diagnose A314S genotypes in field populations. Results showed a strong correlation between frequencies of the mutation and phenotypic levels of resistance to triazophos. The assay offers a prospect for rapid monitoring of resistance and assisting with the appropriate choice of insecticide for combating damage caused by C. suppressalis. Ó 2008 Elsevier Inc. All rights reserved.
Although one of the most important insect pests in Asia, the rice stem borer, Chilo suppressalis (Walker), is difficult to control because most of its life-cycle is spent inside rice stems. Only insecticides that combine good penetration or systemicity, low toxicity to fish, and affordability to farmers are appropriate for stem borer control. In the past, hexachlorocyolohexane (BHC), chlordimeform and monosultap were used extensively in China. However, the first two have been banned for ecotoxicological reasons, and monosultap has lost its effectiveness due to resistance. The organophosphate triazophos initially proved an excellent replacement for monosultap, but recently its effectiveness has declined. A previous publication confirmed resistance to triazophos in C. suppressalis from some locations in China, and suggested reduced sensitivity of acetylcholinesterase – the target site of organophosphates – to be the primary resistance mechanism [1]. Acetylcholinesterase (AChE, EC3.1.1.7) is a key enzyme in neurotransmission. It regulates nerve impulses at cholinergic synapses by rapidly catalyzing the hydrolysis of the neurotransmitter acetylcholine. Aside from some species of Diptera, insects have two different AChE genes [2], but it is believed that only one of these (ace1) plays a key role in insecticide toxicology [3–5]. Inhibition of AChE by insecticides causes excessive excitement in nerves, blockage of neurotransmission and subsequent death. Mutations * Corresponding author. Fax: +86 25 84395245. E-mail address: [email protected] (Z. Han). 1 These authors contributed equally. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.11.046
making AChE less sensitive to insecticides can confer potent resistance and have been described in a range of insect species including Drosophila melanogaster, Musca domestica (housefly), Bactrocera oleae (olive fruit fly), Leptinotarsa decemlineata (Colorado beetle), Anopheles gambiae and Culex pipiens (mosquitoes), Myzus persicae (peach-potato aphid), Cydia pomonella (codling moth) and Putella xylostella (diamondback moth) [6–13]. In the present work, AChE genes were cloned from triazophosresistant and -susceptible C. suppressalis to investigate possible mutations responsible for resistance in this species. The results demonstrated a mutation in Ch-AChE1 consistently associated with resistance. Furthermore, the frequency of the mutation was confirmed to indicate well the level of triazophos resistance in field populations. Materials and methods Insects. The susceptible strain (Gy-S) of C. suppressalis was collected from Ganyu County in Jiangsu Province in 2006, where little triazophos had been used for rice pest control. The standard resistant strain (Tp-R) with 1172-fold resistance to triazophos was produced by eight generations of laboratory selection from a field strain with 203-fold resistance collected from Cangnan County in Zhejiang Province, where triazophos had been extensively used for over 7 years. Additional field strains were collected from Cangnan County in Wenzhou in 2008 (WZCN2008), Ruian County in Wenzhou in 2007 and 2008 (WZRA2007 and WZRA2008,
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respectively), Gaochun County in Nanjing in 2008 (NJGC2008), Jiande County in Hangzhou in 2007 (HZJD2007), Guilin County in Guangxi in 2008 (GXGL2008), and Ganyu County in northern Jiangsu in 2008 (JSGY2008). All insects were reared in the laboratory on rice seedlings [14] at 28 ± 1 °C under a 16 h photoperiod and >80% relative humidity. Total RNA extraction and cDNA synthesis. Total RNAs were extracted with TRIzolÒ reagent (Invitrogen Co., USA) from individual fourth-instar larva according to the manufacturer’s specifications. First-strand cDNA was synthesized from the total RNA using ThermoScriptTM reverse transcriptase (Invitrogen). The reaction vessel contained 3 lg of total RNA, 1 lM of OligodT-Adaptor primer (50 -AAGCAGTGGTATCAACGCAGAGTAC(T30)VN), 1 ll of reverse transcriptase and the reaction mix in a final volume of 20 ll. The following cycling parameters were used: 65 °C for 50 min to synthesize cDNA and 37 °C for 20 min to remove RNA by adding RNaseH (Invitrogen). Cloning of cDNA fragments of AChE genes. Two sets of primers (Table S1) based on known differences between the two AChE genes found in insects (Ch-ace1 and Ch-ace2) were designed to clone cDNA fragments from C. suppressalis. PCR for cloning Ch-ace1 fragments was performed by Taq polymerase (TaKaRa Co., Japan) with following parameters: 94 °C for 2 min followed by 40 cycles at 94 °C for 30 s, 45 °C for 30 s, and 72 °C for 40 s, and one additional cycle at 72 °C for 6 min. For Ch-ace2 fragments, only the annealing temperature was adjusted to 43 °C. Rapid amplification of cDNA end (RACE) was used to obtain the complete gene. The downstream region to the 30 -end of Ch-ace1was amplified by LATaq polymerase and 10 LA PCR Buffer II (Takara) with specific and adaptor primers (Table S1). The PCR reaction was performed as follows: 94 °C for 2 min followed by 40 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 2 min, and a final extension at 72 °C for 6 min. 50 -RACE was performed with cDNA synthesized with GeneRacersTM kit (Invitrogen). LATaq polymerase and 2 GC Buffer II (Takara) were used to amplify the 50 region with the following parameters: 94 °C for 2 min followed by 40 cycles at 94 °C for 30 s, 61 °C for 30 s, and 72 °C for 1 min and a final exten-
sion at 72 °C for 6 min. For Ch-ace2, LATaq polymerase and 2 GC Buffer I (Takara) were used to amplify the 30 region. The 50 -RACE was performed with the BD SMARTTM RACE kit (Clontech, USA) and UPM primers in the kit were used as sense and antisense primers. RACE PCR was performed under manufacturer’s recommended conditions. Amplification of the full length of two AChE genes. All primers were designed with primer5.0 software (Table S1). All PCR reactions were performed with 2 GC Buffer and LATaq polymerase (Takara) in a MyCyclerTM Thermal Cycler (Bio-RAD Inc., USA) under the conditions: 25 ll reaction mixture containing 12.5 ll 2 GC buffer I or II, 0.4 mM dNTPs, 0.4 lM of each primer, 1 ll cDNA template, and 1.25 U LATaq polymerase. The following parameters were used: 94 °C for 2 min followed by 40 cycles at 94 °C for 30 s, 54 °C for 30 s, 72 °C for 2.5 min, and a final extension step at 72 °C for 6 min. DNA sequencing. PCR products were separated by agarose gel electrophoresis, purified with AxyPrepTM DNA Gel Extraction Kit (Axygen Biosciences, USA), and then cloned into pGEM-T easy vector (Promega, USA). The ligation reactions were used for transformations with the DH5a competent cells. Successful stains were screened with blue/white and standard ampicillin selection. Recombinant plasmids were fully sequenced on an Applied Biosystems 377 automated sequencer. RFLP-PCR diagnostic test for detection of the specific point mutation. A C/T nucleotide transition caused an alanine/serine amino acid substitution (A314S) in the insecticide-insensitive Ch-ace1. To detect this mutation at the genomic level, a method was developed based on the restriction fragment length polymorphism analysis of an appropriate PCR product (RFLP-PCR) encompassing this nucleotide position. As template, C. suppressalis genomic DNA was extracted from single fourth-instar larvae with Universal Genomic DNA Extraction Kit V.3.0 (Takara). PCR was performed by LATaq polymerase and 2 GC Buffer II (Takara) with the primers (Table S1) for thirty cycles (94 °C for 30 s, 59 °C for 30 s and 72 °C for 50 s). The PCR product fragments were digested with MspA1 I restriction enzyme (New England BioLabs Inc., USA) according to the manufacturer’s instructions and fractionated on a 2% agarose
Fig. 1. The deduced amino acid sequences of Ch-ace1 (GenBank: EF453724) and Ch-ace2 (GenBank: EF470245) from a susceptible strain of Chilo suppressalis. The putative signal peptide in the N-terminal region is underlined. The putative hydrophobic amino acid tail in the C-terminal region is double-underlined. The characteristic ‘FGESAG’ motif surrounding the active serine is shown in boxes. Acyl pocket residues are encircled. Anionic subsites are indicated by black triangles. The residues identified with black squares shape the oxianion holes. The double-underlined Cys residues indicate the interfaces for intra- and inter-molecular disulfide bonds. The shaded residues of the catalytic triads are boxed.
X. Jiang et al. / Biochemical and Biophysical Research Communications 378 (2009) 269–272
271
gel. In order to check the identity of the amplified fragments, sequences were also done directly on PCR products. Results Identification and characterization of AChE genes from C. suppressalis Two full-length AChE genes were successfully cloned and designated as Ch-ace1 and Ch-ace2 (Fig. 1). Ch-ace1 (GenBank: EF453724) contained an open reading frame (ORF) of 2082 bp encoding 694 amino acids, 235 bp of 50 untranslated region (UTR) and 204 bp of 30 UTR containing a polyadenylation signal. Ch-ace2 (GenBank: EF470245) contained an ORF of 1914 bp encoding 638 amino acids, 321 bp of 50 UTR and 277 bp of 30 UTR containing a polyadenylation signal. Both were predicted to contain the sequence of a signal peptide by SignalP V.3.0 software (http://www.cbs.dtu.dk/services/SignalP/). The alignment of the deduced amino acid sequences did not show very high homology between Ch-AChE1 and Ch-AChE2. Nevertheless, both had the features shared by members of AChE family (Fig. 1). Compared with AChE in Torpedo californica [15], the functional motifs were highly conserved: the catalytic triads (S313, E439 and H553 in Ch-AChE1, and S266, E395 and H509 in Ch-AChE2), the characteristic ‘FGESAG’ motif surrounding the active serine, 3 intra-chain disulfide bridges (C181–C208, C367–380 and C515–C637 in Ch-AChE1, and C115–C143, C320–C335 and C471–C590 in Ch-AChE2), a anionic choline-binding site (W198 in Ch-AChE1 and W133 in Ch-AChE2), acyl pocket residues (W346, F402 and F443 in Ch-AChE1, and W299, F358 and F399 in Ch-AChE2) that accommodate the acyl moiety of the active site, and the residues that form the oxyanion hole (G232, G233 and A314 in Ch-AChE1, and G179, G180 and A267 in Ch-AChE2) that helps to stabilize the tetrahedral molecule during catalysis [16]. The C-terminal Cys residues that contributed to an intermolecular dimerization were Cys660 and Cys608, respectively. Following these Cys residues, both genes had amino acid tails enriched in hydrophobic residues as predicted using ProtScale (http://expasy.org/cgi-bin/protscale.pl). Sequence blast also showed that the two cloned genes were similar to previously reported insect AChE genes, especially those from Lepidoptera (Table 1). Thus, these two genes were concluded to be the relevant AChE genes in C. suppressalis. Difference in AChE sequence between susceptible and resistant strains When the cDNA sequences of AChE genes amplified from resistant and susceptible individuals were aligned, considerable non-silent single nucleotide polymorphisms (SNPs) and deduced amino acid replacements were found (Table S2). However, only one of these – GCG to TCG in Ch-ace1 – was exclusively associated with the resistant phenotype. This change accounted for an A314S
Table 1 Identity of cloned genes with some other insect AChE genes previously reported. Genes types
Host insects
Genbank accession no.
Identity % Ch-AChE1
Ch-AChE2
AChE1 AChE1 AChE1 AChE1 AChE1 AChE2 AChE2 AChE2 AChE2 AChE2
Plutella xylostella Cydia pomonella Helicoverpa armigera Helicoverpa assulta Bombyx mori Plutella xylostella Cydia pomonella Helicoverpa armigera Helicoverpa assulta Bombyx mori
AAY34743 ABB76666 AAY59530 AAY42136 NP001037380 AAL33820 ABB76665 AAM90333 AAV65638 NP001037366
81 86 77 86 83 31 31 32 32 31
30 31 34 30 31 93 92 94 94 94
Fig. 2. RFLP-PCR assay to detect the presence of the A314S mutation in Chilo suppressalis. Genomic DNA amplification produces a 758 bp fragment, which is cut into two fragments (224 and 534 bp) by MspA1 I restriction enzyme for susceptible homozygotes (SS), but is undigested for resistant homozygotes (RR). Heterozygous individuals (RS) display all three bands.
mutation in the oxyanion hole of the enzyme and involved an amino acid residue homologous to A201 in T. californica AChE. Frequency of the mutation in field populations with different resistance The mutation GCG to TCG in Ch-ace1 removes an MspA1 I restriction site from the wild type allele. An RFLP-PCR assay was therefore designed using primers able to amplify specifically a 758-bp fragment encompassing the mutation site in Ch-ace1 from genomic DNA, and by checking the digestion of the resulting fragment with MspA1 I. As shown in Fig. 2, the PCR product amplified from the wild type allele was completely cut into two pieces (534 and 224 bp long). That amplified from the heterozygote was partially cut and presented as three bands (758, 534 and 224 bp long), whereas that amplified from the mutated allele remained intact. With this method, individual larvae of C. suppressalis collected from different field sites were analysed, and results showed a good correlation between mutation frequencies and levels of triazophos resistance (Table 2). Discussion Previous work suggested that target-site insensitivity was a major mechanism of triazophos resistance in C. suppressalis [1]. We have now cloned two full-length AChE genes in this species that show very high identity to insect AChE genes reported in other species [17,18]. We found a single amino acid substitution, A314S in Ch-AChE1, which associates consistently with the resistance phenotype. This alanine residue is located in the characteristic ‘FGESAG’ motif surrounding the active serine, and lies in the oxyanion hole that contributes to stabilizing the tetrahedral molecule during catalysis. The alanine to serine substitution changes the side group from –CH3 to –CH2OH, which is believed to alter the conformation of the adjacent serine of the catalytic triad and to affect the interaction between AChE and both substrates and inhibitors. This site corresponds to A201 in T. californica AChE, and the same amino acid substitution has been reported as a resistance mutation in cotton aphid, Aphis gossypii [19–21], and diamondback moth, P. xylostella [22]. The frequency of this mutation correlated well with levels of triazophos resistance, known to vary substantially between geographical regions [23]. Thus, A314S in Ch-AChE1 is proposed as a widespread and functional mutation contributing to triazaphos resistance in C. suppressalis.
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Table 2 Frequency of A314S genotypes in field strains of Chilo suppressalis with different levels of resistance to triazophos. Field population
Individuals Mutant homozygote
Mutant heterozygote
Wildtype homozygote
WZCN2008 WZRA2007 WZRA2008 NJGC2008 HZJD2007 GXGL2008 JSGY2008
20 19 9 8 4 2 0
0 1 11 12 13 9 0
0 0 0 0 3 9 20
*
Mutation frequency (%)
Resistance ratio to triazophos (monitoring date)*
100 97.5 72.5 70.0 52.5 32.5 0
895 (2006) 568 (2006) 58.1 (2006) – 52.6 (2005) 1.45 (2006)
Data were adopted from He et al. [23].
Ch-ace2 did not show any changes associated with resistance, which supports a view that in those insects with two different AChE genes, only ace1 plays a major role in insecticide toxicology. The discovery of two different AChE genes in most insects [17,24] prompted work to identify their relative importance as insecticide targets. In P. xylostella, the bollworm Helicoverpa assulta and the cockroach Blattella germanica, expression studies using quantitative real time-PCR showed that transcription levels of ace1 were significantly higher than those of ace2 in all tissues examined [4,13,25]. Furthermore, functional expression studies with Culex tritaeniorhynchus and A. gossypii showed susceptible ace1 to have much higher affinity to acetylcholine than ace2 [26,27]. These findings lead to an expectation that mutations conferring resistance to organophosphate and carbamate insecticides will reside in ace1 rather than the ace2 counterpart. The RFLP-PCR assay developed to diagnose the A314S mutation in field populations provides a potentially valuable way of monitoring resistance and investigating the relative frequencies of resistance genotypes. It overcomes many of the difficulties encountered with time-consuming bioassays against C. suppressalis, and offers the prospect of a rapid and high throughput kit to assist with choosing insecticides and combating resistance in this species.
[8]
[9]
[10]
[11]
[12]
[13]
[14] [15]
[16]
Acknowledgments [17]
This work was supported by the National Basic Research Programme (Grant No. 2006CB102003), Bumper Harvest Programme (Grant No. 2006BAD02a16), National Natural Science Foundation of China (Grant No. 30471145), National Science & Technology Pillar Programme (Grant No. 2006BAD08a03) and Jiangsu Key Research Project (Grant No. BE2006303).
[18]
[19]
[20]
Appendix A. Supplementary data [21]
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2008.11.046.
[22]
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Mutation in acetylcholinesterase1 associated with triazophos resistance in rice stem borer, Chilo suppressalis (Lepidoptera: Pyralidae) Xiaojing Jiang a,1, Mingjing Qu b,1, Ian Denholm c, Jichao Fang d, Weihua Jiang a, Zhaojun Han a,* a
Key Laboratory of Monitoring and Management of Plant Diseases and Insects, Ministry of Agriculture, Nanjing Agricultural University, Weigang No. 1, Nanjing 210095, China Shandong Peanut Research Institute, Qingdao 266100, China c Plant and Invertebrate Ecology Division, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK d Institute of Plant Protection, Jiangsu Academy of Agricultural Science, Nanjing 210014, China b
a r t i c l e
i n f o
Article history: Received 5 November 2008 Available online 24 November 2008
Keywords: Chilo suppressalis Acetylcholinesterase Gene mutation Insecticide resistance Triazophos
a b s t r a c t Two full-length genes encoding different acetylcholinesterases (AChEs), designated as Ch-ace1 and Ch-ace2, were cloned from strains of the rice stem borer (Chilo suppressalis) susceptible and resistant to the organophosphate insecticide triazophos. Sequence analysis found an amino acid mutation A314S in Ch-ace1 (corresponding to A201 in Torpedo californica AChE) that was consistently associated with the occurrence of resistance. This mutation removed an MspA1 I restriction site from the wild type allele. An assay based on restriction fragment length polymorphism (RFLP) analysis was developed to diagnose A314S genotypes in field populations. Results showed a strong correlation between frequencies of the mutation and phenotypic levels of resistance to triazophos. The assay offers a prospect for rapid monitoring of resistance and assisting with the appropriate choice of insecticide for combating damage caused by C. suppressalis. Ó 2008 Elsevier Inc. All rights reserved.
Although one of the most important insect pests in Asia, the rice stem borer, Chilo suppressalis (Walker), is difficult to control because most of its life-cycle is spent inside rice stems. Only insecticides that combine good penetration or systemicity, low toxicity to fish, and affordability to farmers are appropriate for stem borer control. In the past, hexachlorocyolohexane (BHC), chlordimeform and monosultap were used extensively in China. However, the first two have been banned for ecotoxicological reasons, and monosultap has lost its effectiveness due to resistance. The organophosphate triazophos initially proved an excellent replacement for monosultap, but recently its effectiveness has declined. A previous publication confirmed resistance to triazophos in C. suppressalis from some locations in China, and suggested reduced sensitivity of acetylcholinesterase – the target site of organophosphates – to be the primary resistance mechanism [1]. Acetylcholinesterase (AChE, EC3.1.1.7) is a key enzyme in neurotransmission. It regulates nerve impulses at cholinergic synapses by rapidly catalyzing the hydrolysis of the neurotransmitter acetylcholine. Aside from some species of Diptera, insects have two different AChE genes [2], but it is believed that only one of these (ace1) plays a key role in insecticide toxicology [3–5]. Inhibition of AChE by insecticides causes excessive excitement in nerves, blockage of neurotransmission and subsequent death. Mutations * Corresponding author. Fax: +86 25 84395245. E-mail address: [email protected] (Z. Han). 1 These authors contributed equally. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.11.046
making AChE less sensitive to insecticides can confer potent resistance and have been described in a range of insect species including Drosophila melanogaster, Musca domestica (housefly), Bactrocera oleae (olive fruit fly), Leptinotarsa decemlineata (Colorado beetle), Anopheles gambiae and Culex pipiens (mosquitoes), Myzus persicae (peach-potato aphid), Cydia pomonella (codling moth) and Putella xylostella (diamondback moth) [6–13]. In the present work, AChE genes were cloned from triazophosresistant and -susceptible C. suppressalis to investigate possible mutations responsible for resistance in this species. The results demonstrated a mutation in Ch-AChE1 consistently associated with resistance. Furthermore, the frequency of the mutation was confirmed to indicate well the level of triazophos resistance in field populations. Materials and methods Insects. The susceptible strain (Gy-S) of C. suppressalis was collected from Ganyu County in Jiangsu Province in 2006, where little triazophos had been used for rice pest control. The standard resistant strain (Tp-R) with 1172-fold resistance to triazophos was produced by eight generations of laboratory selection from a field strain with 203-fold resistance collected from Cangnan County in Zhejiang Province, where triazophos had been extensively used for over 7 years. Additional field strains were collected from Cangnan County in Wenzhou in 2008 (WZCN2008), Ruian County in Wenzhou in 2007 and 2008 (WZRA2007 and WZRA2008,
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respectively), Gaochun County in Nanjing in 2008 (NJGC2008), Jiande County in Hangzhou in 2007 (HZJD2007), Guilin County in Guangxi in 2008 (GXGL2008), and Ganyu County in northern Jiangsu in 2008 (JSGY2008). All insects were reared in the laboratory on rice seedlings [14] at 28 ± 1 °C under a 16 h photoperiod and >80% relative humidity. Total RNA extraction and cDNA synthesis. Total RNAs were extracted with TRIzolÒ reagent (Invitrogen Co., USA) from individual fourth-instar larva according to the manufacturer’s specifications. First-strand cDNA was synthesized from the total RNA using ThermoScriptTM reverse transcriptase (Invitrogen). The reaction vessel contained 3 lg of total RNA, 1 lM of OligodT-Adaptor primer (50 -AAGCAGTGGTATCAACGCAGAGTAC(T30)VN), 1 ll of reverse transcriptase and the reaction mix in a final volume of 20 ll. The following cycling parameters were used: 65 °C for 50 min to synthesize cDNA and 37 °C for 20 min to remove RNA by adding RNaseH (Invitrogen). Cloning of cDNA fragments of AChE genes. Two sets of primers (Table S1) based on known differences between the two AChE genes found in insects (Ch-ace1 and Ch-ace2) were designed to clone cDNA fragments from C. suppressalis. PCR for cloning Ch-ace1 fragments was performed by Taq polymerase (TaKaRa Co., Japan) with following parameters: 94 °C for 2 min followed by 40 cycles at 94 °C for 30 s, 45 °C for 30 s, and 72 °C for 40 s, and one additional cycle at 72 °C for 6 min. For Ch-ace2 fragments, only the annealing temperature was adjusted to 43 °C. Rapid amplification of cDNA end (RACE) was used to obtain the complete gene. The downstream region to the 30 -end of Ch-ace1was amplified by LATaq polymerase and 10 LA PCR Buffer II (Takara) with specific and adaptor primers (Table S1). The PCR reaction was performed as follows: 94 °C for 2 min followed by 40 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 2 min, and a final extension at 72 °C for 6 min. 50 -RACE was performed with cDNA synthesized with GeneRacersTM kit (Invitrogen). LATaq polymerase and 2 GC Buffer II (Takara) were used to amplify the 50 region with the following parameters: 94 °C for 2 min followed by 40 cycles at 94 °C for 30 s, 61 °C for 30 s, and 72 °C for 1 min and a final exten-
sion at 72 °C for 6 min. For Ch-ace2, LATaq polymerase and 2 GC Buffer I (Takara) were used to amplify the 30 region. The 50 -RACE was performed with the BD SMARTTM RACE kit (Clontech, USA) and UPM primers in the kit were used as sense and antisense primers. RACE PCR was performed under manufacturer’s recommended conditions. Amplification of the full length of two AChE genes. All primers were designed with primer5.0 software (Table S1). All PCR reactions were performed with 2 GC Buffer and LATaq polymerase (Takara) in a MyCyclerTM Thermal Cycler (Bio-RAD Inc., USA) under the conditions: 25 ll reaction mixture containing 12.5 ll 2 GC buffer I or II, 0.4 mM dNTPs, 0.4 lM of each primer, 1 ll cDNA template, and 1.25 U LATaq polymerase. The following parameters were used: 94 °C for 2 min followed by 40 cycles at 94 °C for 30 s, 54 °C for 30 s, 72 °C for 2.5 min, and a final extension step at 72 °C for 6 min. DNA sequencing. PCR products were separated by agarose gel electrophoresis, purified with AxyPrepTM DNA Gel Extraction Kit (Axygen Biosciences, USA), and then cloned into pGEM-T easy vector (Promega, USA). The ligation reactions were used for transformations with the DH5a competent cells. Successful stains were screened with blue/white and standard ampicillin selection. Recombinant plasmids were fully sequenced on an Applied Biosystems 377 automated sequencer. RFLP-PCR diagnostic test for detection of the specific point mutation. A C/T nucleotide transition caused an alanine/serine amino acid substitution (A314S) in the insecticide-insensitive Ch-ace1. To detect this mutation at the genomic level, a method was developed based on the restriction fragment length polymorphism analysis of an appropriate PCR product (RFLP-PCR) encompassing this nucleotide position. As template, C. suppressalis genomic DNA was extracted from single fourth-instar larvae with Universal Genomic DNA Extraction Kit V.3.0 (Takara). PCR was performed by LATaq polymerase and 2 GC Buffer II (Takara) with the primers (Table S1) for thirty cycles (94 °C for 30 s, 59 °C for 30 s and 72 °C for 50 s). The PCR product fragments were digested with MspA1 I restriction enzyme (New England BioLabs Inc., USA) according to the manufacturer’s instructions and fractionated on a 2% agarose
Fig. 1. The deduced amino acid sequences of Ch-ace1 (GenBank: EF453724) and Ch-ace2 (GenBank: EF470245) from a susceptible strain of Chilo suppressalis. The putative signal peptide in the N-terminal region is underlined. The putative hydrophobic amino acid tail in the C-terminal region is double-underlined. The characteristic ‘FGESAG’ motif surrounding the active serine is shown in boxes. Acyl pocket residues are encircled. Anionic subsites are indicated by black triangles. The residues identified with black squares shape the oxianion holes. The double-underlined Cys residues indicate the interfaces for intra- and inter-molecular disulfide bonds. The shaded residues of the catalytic triads are boxed.
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gel. In order to check the identity of the amplified fragments, sequences were also done directly on PCR products. Results Identification and characterization of AChE genes from C. suppressalis Two full-length AChE genes were successfully cloned and designated as Ch-ace1 and Ch-ace2 (Fig. 1). Ch-ace1 (GenBank: EF453724) contained an open reading frame (ORF) of 2082 bp encoding 694 amino acids, 235 bp of 50 untranslated region (UTR) and 204 bp of 30 UTR containing a polyadenylation signal. Ch-ace2 (GenBank: EF470245) contained an ORF of 1914 bp encoding 638 amino acids, 321 bp of 50 UTR and 277 bp of 30 UTR containing a polyadenylation signal. Both were predicted to contain the sequence of a signal peptide by SignalP V.3.0 software (http://www.cbs.dtu.dk/services/SignalP/). The alignment of the deduced amino acid sequences did not show very high homology between Ch-AChE1 and Ch-AChE2. Nevertheless, both had the features shared by members of AChE family (Fig. 1). Compared with AChE in Torpedo californica [15], the functional motifs were highly conserved: the catalytic triads (S313, E439 and H553 in Ch-AChE1, and S266, E395 and H509 in Ch-AChE2), the characteristic ‘FGESAG’ motif surrounding the active serine, 3 intra-chain disulfide bridges (C181–C208, C367–380 and C515–C637 in Ch-AChE1, and C115–C143, C320–C335 and C471–C590 in Ch-AChE2), a anionic choline-binding site (W198 in Ch-AChE1 and W133 in Ch-AChE2), acyl pocket residues (W346, F402 and F443 in Ch-AChE1, and W299, F358 and F399 in Ch-AChE2) that accommodate the acyl moiety of the active site, and the residues that form the oxyanion hole (G232, G233 and A314 in Ch-AChE1, and G179, G180 and A267 in Ch-AChE2) that helps to stabilize the tetrahedral molecule during catalysis [16]. The C-terminal Cys residues that contributed to an intermolecular dimerization were Cys660 and Cys608, respectively. Following these Cys residues, both genes had amino acid tails enriched in hydrophobic residues as predicted using ProtScale (http://expasy.org/cgi-bin/protscale.pl). Sequence blast also showed that the two cloned genes were similar to previously reported insect AChE genes, especially those from Lepidoptera (Table 1). Thus, these two genes were concluded to be the relevant AChE genes in C. suppressalis. Difference in AChE sequence between susceptible and resistant strains When the cDNA sequences of AChE genes amplified from resistant and susceptible individuals were aligned, considerable non-silent single nucleotide polymorphisms (SNPs) and deduced amino acid replacements were found (Table S2). However, only one of these – GCG to TCG in Ch-ace1 – was exclusively associated with the resistant phenotype. This change accounted for an A314S
Table 1 Identity of cloned genes with some other insect AChE genes previously reported. Genes types
Host insects
Genbank accession no.
Identity % Ch-AChE1
Ch-AChE2
AChE1 AChE1 AChE1 AChE1 AChE1 AChE2 AChE2 AChE2 AChE2 AChE2
Plutella xylostella Cydia pomonella Helicoverpa armigera Helicoverpa assulta Bombyx mori Plutella xylostella Cydia pomonella Helicoverpa armigera Helicoverpa assulta Bombyx mori
AAY34743 ABB76666 AAY59530 AAY42136 NP001037380 AAL33820 ABB76665 AAM90333 AAV65638 NP001037366
81 86 77 86 83 31 31 32 32 31
30 31 34 30 31 93 92 94 94 94
Fig. 2. RFLP-PCR assay to detect the presence of the A314S mutation in Chilo suppressalis. Genomic DNA amplification produces a 758 bp fragment, which is cut into two fragments (224 and 534 bp) by MspA1 I restriction enzyme for susceptible homozygotes (SS), but is undigested for resistant homozygotes (RR). Heterozygous individuals (RS) display all three bands.
mutation in the oxyanion hole of the enzyme and involved an amino acid residue homologous to A201 in T. californica AChE. Frequency of the mutation in field populations with different resistance The mutation GCG to TCG in Ch-ace1 removes an MspA1 I restriction site from the wild type allele. An RFLP-PCR assay was therefore designed using primers able to amplify specifically a 758-bp fragment encompassing the mutation site in Ch-ace1 from genomic DNA, and by checking the digestion of the resulting fragment with MspA1 I. As shown in Fig. 2, the PCR product amplified from the wild type allele was completely cut into two pieces (534 and 224 bp long). That amplified from the heterozygote was partially cut and presented as three bands (758, 534 and 224 bp long), whereas that amplified from the mutated allele remained intact. With this method, individual larvae of C. suppressalis collected from different field sites were analysed, and results showed a good correlation between mutation frequencies and levels of triazophos resistance (Table 2). Discussion Previous work suggested that target-site insensitivity was a major mechanism of triazophos resistance in C. suppressalis [1]. We have now cloned two full-length AChE genes in this species that show very high identity to insect AChE genes reported in other species [17,18]. We found a single amino acid substitution, A314S in Ch-AChE1, which associates consistently with the resistance phenotype. This alanine residue is located in the characteristic ‘FGESAG’ motif surrounding the active serine, and lies in the oxyanion hole that contributes to stabilizing the tetrahedral molecule during catalysis. The alanine to serine substitution changes the side group from –CH3 to –CH2OH, which is believed to alter the conformation of the adjacent serine of the catalytic triad and to affect the interaction between AChE and both substrates and inhibitors. This site corresponds to A201 in T. californica AChE, and the same amino acid substitution has been reported as a resistance mutation in cotton aphid, Aphis gossypii [19–21], and diamondback moth, P. xylostella [22]. The frequency of this mutation correlated well with levels of triazophos resistance, known to vary substantially between geographical regions [23]. Thus, A314S in Ch-AChE1 is proposed as a widespread and functional mutation contributing to triazaphos resistance in C. suppressalis.
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Table 2 Frequency of A314S genotypes in field strains of Chilo suppressalis with different levels of resistance to triazophos. Field population
Individuals Mutant homozygote
Mutant heterozygote
Wildtype homozygote
WZCN2008 WZRA2007 WZRA2008 NJGC2008 HZJD2007 GXGL2008 JSGY2008
20 19 9 8 4 2 0
0 1 11 12 13 9 0
0 0 0 0 3 9 20
*
Mutation frequency (%)
Resistance ratio to triazophos (monitoring date)*
100 97.5 72.5 70.0 52.5 32.5 0
895 (2006) 568 (2006) 58.1 (2006) – 52.6 (2005) 1.45 (2006)
Data were adopted from He et al. [23].
Ch-ace2 did not show any changes associated with resistance, which supports a view that in those insects with two different AChE genes, only ace1 plays a major role in insecticide toxicology. The discovery of two different AChE genes in most insects [17,24] prompted work to identify their relative importance as insecticide targets. In P. xylostella, the bollworm Helicoverpa assulta and the cockroach Blattella germanica, expression studies using quantitative real time-PCR showed that transcription levels of ace1 were significantly higher than those of ace2 in all tissues examined [4,13,25]. Furthermore, functional expression studies with Culex tritaeniorhynchus and A. gossypii showed susceptible ace1 to have much higher affinity to acetylcholine than ace2 [26,27]. These findings lead to an expectation that mutations conferring resistance to organophosphate and carbamate insecticides will reside in ace1 rather than the ace2 counterpart. The RFLP-PCR assay developed to diagnose the A314S mutation in field populations provides a potentially valuable way of monitoring resistance and investigating the relative frequencies of resistance genotypes. It overcomes many of the difficulties encountered with time-consuming bioassays against C. suppressalis, and offers the prospect of a rapid and high throughput kit to assist with choosing insecticides and combating resistance in this species.
[8]
[9]
[10]
[11]
[12]
[13]
[14] [15]
[16]
Acknowledgments [17]
This work was supported by the National Basic Research Programme (Grant No. 2006CB102003), Bumper Harvest Programme (Grant No. 2006BAD02a16), National Natural Science Foundation of China (Grant No. 30471145), National Science & Technology Pillar Programme (Grant No. 2006BAD08a03) and Jiangsu Key Research Project (Grant No. BE2006303).
[18]
[19]
[20]
Appendix A. Supplementary data [21]
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2008.11.046.
[22]
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