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Keywords:

  • acetylcholinesterase;
  • RNA interference;
  • rice stem borer;
  • nontypical functions;
  • larvae growth;
  • motor ability

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Acetylcholinesterase (AChE, EC 3.1.1.7) is a key enzyme in terminating synaptic transmission. We knocked down the expression of Csace1 or Csace2 using chemically synthesized small interfering RNAs (siRNAs) designed from divergent regions. The mRNA abundance of the two ace genes was reduced to 50–70% of control levels. The enzyme activities were decreased to 40–70%. Silencing of Csace1 or Csace2 resulted in a ∼25% mortality rate. Knockdown of Csace1 had major effects on larval growth inhibition and resulted in reduced larval weight and length, malformation and motor disability, whereas silencing of Csace2 had only minor effects. These results suggested that both AChE-1 and AChE-2 have important roles in maintaining life in this insect and indicated that AChE-1 might have nontypical functions in regulating larval growth and motor ability.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Acetylcholinesterase (AChE, EC 3.1.1.7) is a key enzyme in the termination of synaptic transmission which it does by hydrolysing the neurotransmitter acetylcholine. It has been intensively studied in insects because it is the target of organophosphorous (OP) and carbamate pesticides (Dong et al., 2005). In 2002, two ace genes were found in Aphis gossypii (Li & Han, 2002) and Anopheles gambiae (Weill et al., 2002). Since then, two ace genes have been identified in many other insects, such as Culex pipiens (Huchard et al., 2006), Aedes aegypti (Mori et al., 2007), Culex tritaeniorhynchus (Nabeshima et al., 2004), Myzus persicae (Nabeshima et al., 2003), Rhopalosiphum padi and Sitobion avenae (Chen & Han, 2006), Helicoverpa assulta (Lee et al., 2006), Plutella xylostella (Baek et al., 2005; Lee et al., 2007) and Bombyx mori (Chen et al., 2009). It has been suggested that the two aces in insects originated from duplication before the diversification of arthropods (Labbe et al., 2007). However, only the ace-2 gene was found in the Cyclorrapha suborder of Diptera, suggesting that a secondary loss of the ace-1 gene may have happened in evolution (Huchard et al., 2006).

In those insects that have two ace genes, AChE-1 has been reported to be the main target of OP and carbamate insecticides and most resistance-associated mutations have been reported in the ace-1 gene (Baek et al., 2005; Lee et al., 2006, 2007). However, in Cyclorrapha insects that have only one ace gene, AChE-2 is the only target of OP and carbamate insecticides. Mutations in ace-2 were reported to confer insecticide resistance in Drosophila melanogaster (Weill et al., 2002), Lucilia cuprina (Chen et al., 2001) and Musca domestica (Walsh et al., 2001; Temeyer & Chen, 2007). AChE-1 therefore plays key roles in some insects, whereas AChE-2 has critical functions in other insects. This raises questions as to why two ace genes exist in most insects and what is the functional difference between these genes.

Although AChE has been best known for its roles in the termination of synaptic transmission, increasing evidence suggests that AChE has nontypical functions in mammals (Jiang & Zhang, 2008) and zebrafish (Seibt et al., 2009). These nontypical functions include regulating cell–matrix interactions in bone (Inkson et al., 2004), participating in neuroblastoma cell adhesion and neurite outgrowth (Johnson & Moore, 2000), playing an interactive major role in neocortical development by alternative splicing (Dori et al., 2005), regulating AChE-mediated axonal growth (Bigbee & Sharma, 2004), as well as having roles in synaptogenesis, memory formation and stress responses (Zimmerman & Soreq, 2006), cell proliferation and apoptosis (Jin et al., 2004). However, whether AChEs have nontypical functions in insects remains unclear.

RNA interference (RNAi) is an efficient technique for studying gene function by suppressing the expression of that gene (Fire et al., 1998). It has been widely used in insects to investigate the functions of important genes (Moriyama et al., 2009; Chen et al., 2010; Tang et al., 2010; Zhang et al., 2010). RNAi was also used to silence ace genes in insect pests. The suppression of ace genes in Helicoverpa armigera led to mortality, growth inhibition, malformation and drastically reduced fecundity (Kumar et al., 2009). In Blattella germanica, Bgace1 and Bgace2 were knocked down separately by using dsRNA synthesized from divergent regions of the two ace genes, proving that Bgace1 encodes a predominant AChE (Revuelta et al., 2009). There are two ace genes in the rice stem borer, Chilo suppressalis, an important pest worldwide that causes serious damage to rice production. Csace1 was known to encode the main AChE enzyme in this insect. An amino acid mutation A314S in Csace1 was found to confer resistance to triazophos (Jiang et al., 2009). At present, functional differences between the two ace genes has still not been well investigated. In the present study, we used small interfering RNAs (siRNAs) to study their functions and found that the ace genes in the rice stem borer might have nontypical functions in larval growth.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Knockdown of Csace1 and Csace2

We designed two single siRNAs for each ace gene and all siRNAs were fluorescence-labelled by FAM. Either 1 µl single siRNA at a concentration of 1 µg/µl (ace1-siRNA-1, ace1-siRNA-2, ace2-siRNA-1 and ace2-siRNA-2) or 1 µl mixes of two siRNAs (ace1-siRNA-mix, ace2-siRNA-mix) were injected into larvae, which were then observed under fluorescence microscopy. The ace1-siRNA mix contained equal amounts of ace1-siRNA-1 and ace1-siRNA-2, and the ace2-siRNA mix contained equal amounts of ace2-siRNA-1 and ace2-siRNA-2. We also used a mix of ace1-siRNA mix and ace2-siRNA mix to silence the two ace genes simultaneously. The injected siRNAs spread to the whole body, including the head where ace genes were highly expressed. This indicated that siRNAs could pass through the blood-brain barrier (BBB) and reach the central nervous system (CNS) (Fig. 1).

image

Figure 1. Injection of fluorescence-labelled small interfering RNAs (siRNAs)-mix (mix of equal amounts of two siRNAs for the same ace gene). (A) The siRNAs were spread to the whole body of the larvae. (B) siRNAs pass through the blood-brain barrier and reached the central nervous system.

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Two ace genes were successfully knocked down separately using sequence-specific siRNAs. The mRNA abundance of ace genes was monitored at 36, 48, 72 and 96 h after siRNA injection. The mRNA levels of both Csace1 and Csace2 reached the lowest values at 72 h (Fig. 2). Subsequently, the RNAi efficacy and potential off-target effects were assessed at 72 h after injection. When using ace1-siRNA mix or ace2-siRNA mix to silence ace genes, the mRNA level of Csace1 in ace1-siRNA mix-treated rice stem borers was <50% of the control level, whereas Csace2 was decreased to 70% of the control level (Fig. 3). When using a single siRNA to silence each ace gene separately, similar results were obtained. The silencing of either ace gene had negligible effects on the other ace gene, suggesting the absence of off-target effects (Fig. S1). This enabled us to study the functional differences between the two ace genes.

image

Figure 2. Relative mRNA abundance of Csace1 and Csace2 after small interfering RNA (siRNA)-mix injection. The mRNA abundance was monitored at 36, 48, 72 and 96 after siRNA injection for both ace genes. The mRNA levels reached the lowest values at 72 h.

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image

Figure 3. The RNA interference (RNAi) efficacy and potential off-target effects were examined at 72 h. Csace1 or Csace2 were separately knocked down with negligible effect on the other ace gene. The RNAi efficacies were about 40–70%.

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Acetylcholinesterase activities were measured at 72, 96 and 120 h after siRNA injection. The enzyme activities were apparently reduced and reached the lowest values at 96 h (Fig. 4). RNAi of Csace1, Csace2 or both ace genes using siRNA mixes led to a reduction of AChE activity to ≈40, 70 and 40% of control level, respectively (Fig. 5). Similar results were also obtained when using single siRNAs to silence the ace genes (Fig. S2).

image

Figure 4. Enzyme activities measured after small interfering RNA (siRNA) mix injection. The acetylcholinesterase (AChE) activities were measured at 72, 96 and 120 h after siRNA injections for each ace gene. The AChE activities reached their lowest levels at 96 h.

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image

Figure 5. Acetylcholinesterase (AChE) activities measured at 96 h from different treatments. The AChE activities were reduced to 40–70% of the control level.

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Mortality of siRNA-treated rice stem borers

Silencing Csace1, Csace2 or both ace genes using siRNA mixes resulted in the death of some siRNA-treated rice stem borers. The mortality rate increased from 24 h to 96 h after siRNA injection and was similar between different treatments. The highest mortality rate was 25–30% at 96 h. Silencing of either Csace1 or Csace2 using siRNA mixes resulted in a similar mortality rate. Knockdown of both ace genes resulted in higher mortality rates than that of a single ace gene (Fig. 6). When a single siRNA was used, knockdown of Csace1 with ace1-siRNA-1 or ace1-siRNA-2 resulted in higher mortality rates (Fig. S3). These results indicated that both ace genes have key roles in maintaining life in this insect.

image

Figure 6. The mortality rates of small interfering RNA (siRNA)-mix treated rice stem borers at 24, 48, 72 and 96 h. Both Csace1- silenced and Csace2-silenced rice stem borers showed similar mortalities.

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RNA interference effect on motor ability

We designed a movement test to investigate the motor ability of siRNA-treated rice stem borers. Germinated rice seeds were put at one end of the box, whereas the starved rice stem borers were put at the opposite end of the box. The time taken for rice stem borers to crawl from one end to the other end was monitored. We used siRNA mixes in this experiment to achieve the best results. The average time taken by untreated rice stem borers was 380 s. Silencing of Csace1 using ace1-siRNA-mix significantly increased the time to 734 s. When both Csace1 and Csace2 were silenced, the transit time was increased to 490 s, representing a significant difference. However, silencing of Csace2 using the ace2-siRNA mix resulted in a similar transit time to the control (Fig. 7). In these motor coordination experiments, the Csace1-silenced and Csace1&Csace2-silenced stem borers showed apparent motor disability. These results indicated that Csace1 was more important than Csace2 for the control of motor ability in the rice stem borer (Video S1, supplemental material).

image

Figure 7. Movement tests assay after treating with small interfering RNA (siRNA)-mix. (A) The design of movement test assay. (B) The average time taken to feed by rice stem borers in different treatments. The Csace1-silenced and Csace1& Csace2- silenced rice stem borers took longer to feed than the controls.

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RNA interference effect on larvae growth

We used siRNA mixes to silence each ace gene and observed the effects on larval growth. The weights and lengths of normal and siRNA-treated larvae were measured at 4, 7, 14 and 21 days after siRNA injection. There were no significant differences between siRNA-treated stem borers and controls at 4 days and 7 days (Figs 8, 9). At 14 days, both the weights and lengths of Csace1-silenced and Csace1&Csace2-silenced rice stem borers were significantly lower than those of the controls (Fig. 10). Larval weights and lengths in Csace2-silenced stem borers were not significantly different to the controls. Larval growth of Csace1-silenced and Csace1&Csace2-silenced rice stem borers returned to normal at 21 days. To confirm the reliability of RNAi experiments, we also used single siRNAs to silence each ace gene separately, producing similar results (Figs S4, S5). In summary, knockdown of Csace1 had the major effects on larval growth, whereas silencing of Csace2 had only minor effects.

image

Figure 8. Average larval weight of small interfering RNA (siRNA)-mix treated rice stem borers at 4, 7, 14 and 21 days. There was a significant difference between the weight of Csace1- silenced and Csace1& Csace2-silenced larvae compared with the controls at 14 days.

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image

Figure 9. Average larval body length of small interfering RNA (siRNA)-treated rice stem borers at 4, 7, 14 and 21 days. (A) There was a significant difference in larval length between the controls and Csace1- silenced or Csace1& Csace2-silenced rice stem borers at 14 days.

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image

Figure 10. The larvae growth inhibition of small interfering RNA (siRNA)-treated rice stem borers results in short body length compared with controls.

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In Csace1-silenced and Csace1&Csace2-silenced rice stem borers, 2–4% of tested insects had abnormal morphological phenotypes between the 9th and 10th larval segments (Fig. 11) and these malformed insects lost their ability to move for feeding. These abnormal insects starved to death during the following days (Video S2, supplemental material). No such malformations were observed in Csace2-silenced stem borers.

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Figure 11. Some small interfering RNA (siRNA) mix-treated rice stem borers have a malformation between the 9th and 10th larval segments.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The existence of two ace genes in most insects is an interesting discovery with respect to insect toxicology because AChE is the target of widely used OP and carbamate insecticides. However, although there are two ace genes in most insect pests, only the homologues of ace1 were confirmed to be the target of insecticides. The expression of ace1 is more abundant than that of ace2 in most insects that have two ace genes (Kim et al., 2006; Lee et al., 2006), although we reported a special case in the silkworm in which mRNA abundance of ace2 was higher than that of ace1 (Chen et al., 2009). Moreover, mutations of ace1 but not ace2 conferred insecticide resistance (Nabeshima et al., 2003; Li & Han, 2004; Alon et al., 2008; Jiang et al., 2009; Khajehali et al., 2010). This raises the question of why the ace2 gene exists in most insects. The functional differences between ace1 and ace2 have attracted increasing attention. Gene-specific knockdown of the two ace genes enabled us to investigate their functions separately. This was successfully realized by injecting sequence-specific siRNAs into the larvae of rice stem borers. In the present study, we knocked down Csace1 and Csace2 separately using chemically synthesized siRNAs designed from divergent regions of the two ace genes. As a result, Csace1 or Csace2 were silenced without affecting the other ace gene. Silencing of Csace1 resulted not only in high mortality but also in larval growth defects, motor disability and malformation. However, silencing of Csace2 only led to high mortality without apparent abnormal phenotypes. It was reported that the mRNA abundance of Csace1 was about 4.8-fold higher than that of Csace2. Csace1 encodes the major AChE enzyme and its mutations confer the insecticide resistance (Jiang et al., 2009). The present study showed that both Csace1 and Csace2 are important for maintaining life because silencing of either ace gene would lead to death.

It should be noticed that the maximum inhibition obtained is around 50%, i.e. 50% of AChE activity remains. Since this level of activity would be sufficient for viability, the reasons for the mortality we observed require consideration. In the present study, the tested insects used for quantitative real-time PCR (qRT-PCR) or enzyme analysis were still alive because dead insects were not suitable for the experiments. This means that the silencing efficiencies we obtained were from surviving insects. We argue that silencing efficiency would probably be >50% in the dead insects. It has also been understood that the AChE enzyme may last up to many days in vivo. Thus, it is unclear why RNAi of ace genes in the rice stem borer would result in a reduction of AChE activity in 4 days (96 h). We conducted similar experiments in other insects and failed to find such a change in enzyme activity (unpubl. data). In the rice stem borer, AChE activities were apparently reduced after 96 h and we argue that this may simply reflect differences between insect species.

It is reasonable to consider why only Ch-AChE-1 is the main insecticide target, as evidenced by mutations conferring insecticide resistance. The expression of Csace1 is abundant in most tissues, whereas the mRNA abundance of Csace2 is relatively enriched in the CNS (Jiang et al., 2009 and unpubl. data). When chemical pesticides enter the body of insect pests, they would first inhibit the abundant Ch-AChE1 in tissues such as the gut, malpighian tubules and fat body. This inhibition may be enough to kill the insect pests. We reasoned that a large part of chemical insecticides may be degraded by the metabolizing enzymes such as cytochrome P450 mono-oxygenases and esterase before they reach the CNS. Therefore, Csace1 becomes the major target of OP and carbamate insecticides. Csace2, which is mainly expressed in the CNS, faces less selection pressure from insecticides than Csace1. In the present study, fluorescence microscopy showed that siRNAs can pass through the BBB and knockdown ace gene in the CNS. It should be noted that we used high-dose siRNAs that were not subject to degradation of metabolizing enzymes. Thus, silencing of Csace2 by siRNAs led to mortality. It remains possible that OP and carbamate insecticides readily cross into the CNS and would inhibit both AChE1 and AChE2 in this location. Since inhibition of AChE-2 would lead to death, AChE-2 should be subject to selection pressure and thus mutations of only AChE-1 would not protect the rice stem borer from the attack of OP and carbamate insecticides. In this case, we argue that most resistant pests have high expression of metabolizing enzymes which may further increase the degradation of insecticides before reaching the CNS. Moreover, the mutation of AChE-1 might have some degree of functional compensation for AChE-2. If this is the case, inhibition of AChE-2 in resistant strains of the rice stem borer would not result in death. We believe that this issue is worthy of further investigation to uncover the resistance mechanism.

Acetylcholinesterase is best known as the key enzyme in synaptic transmission. However, increasing evidence suggests that AChE also has some nonclassic functions. In humans, AChE is expressed in bone – a tissue that lacks expression of other neuronal proteins (Inkson et al., 2004). Generally, nontypical functions of AChE genes include axon growth, neurite outgrowth, cell proliferation and apoptosis (Johnson & Moore, 2000; Bigbee & Sharma, 2004; Jin et al., 2004; Dori et al., 2005). However, little has been understood about the atypical functions of AChE genes in insects. In the present study, the survivors of Csace1-silenced and Csace1&Csace2-silenced rice stem borers showed larval growth inhibition, motor disability and an abnormal phenotype, suggesting that Csace1 had similar nontypical functions to its counterparts in mammals. We reasoned that growth inhibition and malformation of larvae might be ascribed to cell proliferation and apoptosis during larval growth. The silencing of Csace2 did not induce an apparent abnormal phenotype, suggesting it has only typical functions in cholinergic signalling, and this is consistent with its expression pattern that is mainly enriched in the CNS.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Insects

Rice stem borers were collected from Cangnan County in Zhejiang province in 2003 and maintained in the laboratory for 7 years. This strain was sensitive to almost all pesticides. All insects were reared in the laboratory on rice seedlings at 28 ± 1°C under a 16-h photoperiod and >80% relative humidity. For RNAi experiments, we collected rice stem borers at the egg stage and the third instar larvae were used for siRNA injection.

SiRNA design and injection

We first aligned the nucleotide sequences of two ace genes and searched the divergent regions. Then, two siRNAs were designed from divergent regions for each ace gene. To avoid off-target effects, the designed siRNAs were used to align with the other ace gene followed by BLAST (http://www.ncbi.nlm.nih.gov/BLAST) with transcriptome data from the rice stem borer (unpubl. data). The siRNAs that contained more than 11 contiguous base pairs of homology to the other ace gene or 16–17 contiguous base pairs of homology to other protein coding sequences were eliminated from consideration.

SiRNAs were chemically synthesized by Shanghai GenePharma Co., Ltd (Shanghai, China) and fluorescently labelled by FAM at the 5'end of sense strand. The siRNAs were double-stranded and purified by high-performance liquid chromatography. SiRNAs were dissolved in the diethylpyrocarbonate-treated water (Milli-Q-grade). The final concentration was 1 µg siRNA/µl H2O. The 1 µl fluorescence-labelled siRNAs (either a single siRNA or a mix of equal amounts of two siRNAs) were injected between the third and fourth segments of 3rd instar larvae using the Eppendorf InjectMan NI 2 microinjection system (Eppendorf, Hamburg, Germany). The mixes of two siRNAs contained only 50% of the dose of each siRNA. The needles were pulled from glass capillaries (1.0 mm outer diameter and 0.50 mm inner diameter) using a micropipetter puller (Model P-87, Sutter Instruments Co., Novato, CA, USA). To avoid leakage of siRNA from the insect body, needles were kept still at the injection point for 30 s (Fig. 1). One set of siRNAs with a random sequence was used as the negative control. The sequences of siRNAs are given in Table 1. Thirty insects were used for each treatment and all experiments were conducted in triplicate.

Table 1.  The sequences of siRNA for RNA interference and primers for quantitative real-time PCR
 NameSequences
  • siRNA, small interfering RNA.

  • *

    The FAM-labelled nucleotides in siRNAs were underlined.

siRNAace1-siRNA-15′-*CAGAAGAUCCCGUGAGAAATT-3′
5′-UUUCUCACGGGAUCUUCUGTT-3′
ace1-siRNA-25′-GCUAAAUCCCGGAAAGAAUTT-3′
5′-AUUCUUUCCGGGAUUUAGCTT-3′
ace2-siRNA-15′-CAUUAAAGGUUACGCCAAATT-3′
5′-UUUGGCGUAACCUUUAAUGTT-3′
ace2-siRNA-25′-GCGAAAGAGCACAGGACAUTT-3′
5′-AUGUCCUGUGCUCUUUCGCTT-3′
Negative control5′-UUCUCCGAACGUGUCACGUTT-3′
5′-ACGUGACACGUUCGGAGAATT-3′
qPCR primersElongation factor-1 (EF-1) Forward5′-TGAACCCCCATACAGCGAATCC-3′
Elongation factor-1(EF-1) Reverse5′-TCTCCGTGCCAACCAGAAATAGG-3′
AChE1-F5′-GAGGAAAGCATTTTACGAGGCACA-3′
AChE1-R5′-GTTCGTCAGCACTCTTCTTCCTTA-3′
AChE2-F5′-TGTGGATATACGGCGGTGGTTACA-3′
AChE2-R5′-AAAAATCCGAACGCTCCTACCCTA-3′

Total RNA isolation and complementary DNA synthesis

Larvae were frozen with liquid nitrogen and homogenized in a tissue grinder. Then, 1 ml Trizol reagent (Takara, Kyoto, Japan) was added to homogenized insects. Total RNA was isolated following the recommended procedures. Genomic DNA was removed from total RNAs by treating with DNAse I following the protocol of the DNA-free kit (Ambion, Austin, TX, USA). The integrity of RNA was checked on a 1.5% agarose gel. The first strand of the complementary DNA template was synthesized from 1 µg of total RNA using Oligo (dT18) as the anchor primer and M-MLV reverse transcriptase (Promega, Madison, WI, USA).

Quantitative real-time PCR

The qRT-PCR reactions were carried out with SYBR Premix Ex TaqTM (Takara) following the manufacturer's protocol using an ABI Prism 7000. The qRT-PCR primers were designed using the online tool PrimerQuest (http://www.idtdna.com/Scitools/Applications/Primerquest/). The housekeeping gene Elongation factor-1 (EF-1) was used as the internal control. The qRT-PCRs were performed using SYBR Green Premix kit (Takara). Standard qRT-PCR procedures consisted of one cycle of 95°C for 10 s, 40 cycles of 95°C for 5 s and then annealed at 60°C for 31 s, followed by one cycle of 95°C for 15 s, 60°C for 60 s, 95°C for 15 s and 60°C for 15 s. The specificity of the PCR reactions was monitored with melting curve analysis and gel electrophoresis. Amplification efficiencies were determined by template dilution. The relative abundance of ace genes was calculated according to the 2-ΔΔCt method (Pfaffl, 2001). The data were analysed using SAS software and a significance level of P < 0.05 was used in all cases.

Enzyme activity

The AChE activities were measured using the kinetic method with minor modifications (Han et al., 1998). Two rice stem borers were homogenized in a glass tissue grinder on ice with 1 ml phosphate buffer solution (0.02 M, pH 7.5) containing 0.1% Triton X-100. The homogenate was centrifuged at 10 000 ×g for 20 min at 4°C and the supernatant was used as the AChE solution. 50 µl of enzyme preparation was placed in a well of a microplate and then 50 µl phosphate buffer (pH 7.5, 0.02 M) and 50 µl DTNB (45 µM) were added and mixed. The reactions were initiated by the addition of 50 µl ATChI (1.56 mM) and monitored at 405 nm for 20 min using a Bio-Rad microplate reader. The reaction temperature was maintained at 25°C and the interval time between two reads was 8 s. The linearity of reactions during the incubation was >0.9. Protein content was estimated by the Bradford method using bovine serum albumin as the standard (Bradford, 1976).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We are very grateful to the anonymous reviewers and the editor for their valuable comments and English-writing assistance to improve the quality of this paper. This work was supported by the transgenic programme (2009ZX08001-002B), National Basic Research Programme of China (2009CB125902, 2010CB126200) and the National Science Foundation of China (30771417, 30871636).

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. The RNA interference efficacy and potential off-target were examined at 72 h. Chace1 or Chace2 was knocked down specifically with a negligible effect on another ace gene.

Figure S2. Remained enzyme activities measured at 96 h after small interfering RNA (siRNA) injection.

Figure S3. The mortalities of siRNA-treated rice stem borers at 24, 48, 72 and 96 h.

Figure S4. Average larvae weight of siRNA-treated rice stem borer at 7 days, 14 days, and 21 days.

Figure S5. Average larvae body lengths of siRNA-treated rice stem borer at 7 days, 14 days, and 21 days.

Video S1. Movement of siRNA-treated rice stem borers. The Csace1-silenced and Csace1& Csace2- silenced rice stem borers moved more slowly than the controls.

Video S2. Feeding test of siRNA-treated rice stem borers. The Csace1- silenced and Csace1& Csace2- silenced rice stem borers failed to reach the germinated rice seed.

FilenameFormatSizeDescription
IMB_1081_sm_FS1-5.doc218KSupporting info item
IMB_1081_sm_VideoS1.wmv3552KSupporting info item
IMB_1081_sm_VideoS2.wmv5092KSupporting info item

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