Association between the R81T mutation in the nicotinic acetylcholine receptor β1 subunit of Aphis gossypii and the differential resistance to acetamiprid and imidacloprid

Abstract

The Aphis gossypii clone, Kushima, which was first discovered in Miyazaki Prefecture, Japan, exhibits significant resistance to neonicotinoid insecticides. We investigated the resistance mechanism involved by sequencing the nicotinic acetylcholine receptor (nAChR) gene, by electrophysiological analysis, and by insecticidal tests in the presence or absence of the oxidase inhibitor, piperonyl butoxide. The Kushima clone showed higher resistance to nitro-substituted neonicotinoids, such as imidacloprid, than to cyano-substituted neonicotinoids, such as acetamiprid. Sequencing of the nAChR subunit genes of a susceptible clone and the Kushima clone revealed an R81T mutation in loop D of the β1 subunit in the resistant clone. This mutation led to a significant shift in the pEC₅₀ value of imidacloprid for the Drosophila melanogaster Dα2-chicken β2 subunit, while it barely affected the concentration-response curves of acetylcholine and acetamiprid.

Introduction

The cotton aphid, Aphis gossypii, is an important sucking pest that causes severe crop losses, both in the field and in greenhouses. Neonicotinoid insecticides (Fig. 1), which show excellent control of A. gossypii, target insect nicotinic acetylcholine (ACh) receptors (nAChRs).^1–4) nAChRs are members of the cys-loop superfamily of ligand-gated ion channels that conduct fast-moving excitatory cholinergic neurotransmission.⁵⁾ Targeting nAChRs has raised concerns about mammalian toxicity, yet neonicotinoids show highly selective toxicity to insects over vertebrates.^1,2,4)

Fig. 1. Chemical structures of neonicotinoid insecticides.

Insecticide resistance is a major hindrance to the effective control of insect pests worldwide. Neonicotinoids have been used for more than 20 years to protect economically important crops.^6–8) However, although resistance to neonicotinoids has developed relatively slowly, it is now recognized as an emerging issue.⁹⁾ In most cases, P450-mediated detoxification plays a primary role in insecticide resistance in a variety of insects, including the whitefly (Bemisia tabaci) and the Colorado potato beetle (Leptinotarsa decemlineata).^10,11) On the other hand, laboratory selection of Nilaparvata lugens with imidacloprid resulted in an α-subunit Y151S mutation that was responsible for the acquisition of neonicotinoid resistance.^12,13) However, this mutation has not been identified in any pest in the field. Instead, a mutation in the nAChR β1 subunit was found to be a major factor in imidacloprid resistance in a field population of Myzus persicae.¹⁴⁾

We previously reported the emergence of neonicotinoid resistance in the aphid and recently isolated an A. gossypii (Kushima) clone in Miyazaki Prefecture, Japan, that exhibited relatively high neonicotinoid resistance.¹⁵⁾ Interestingly, the level of the Kushima clone’s resistance to neonicotinoids with nitro substituents (e.g., imidacloprid) was higher than for those with cyano substituents (e.g., acetamiprid; see Fig. 1 for the structures). However, the mechanisms of neonicotinoid resistance remain unknown.

In this study, we attempted to identify the mechanisms of neonicotinoid resistance using an insecticidal assay with and without piperonyl butoxide (PBO) pretreatment, along with sequencing of the nAChR gene and voltage-clamp electrophysiology. Our results suggest that a mutation in the β1 subunit is the main mechanism of resistance and that differences in resistance levels are probably associated with differential interactions between nitro- and cyano-type neonicotinoids and nAChRs.

Materials and Methods

1. Insects

In this study, we used a field-isolated clone of A. gossypii (Kushima) and a susceptible clone that has been maintained since 1993 at the Odawara Research Center, Nippon soda Co., Ltd. (Odawara, Kanagawa, Japan). The Kushima clone was collected from a field in Miyazaki Prefecture, Japan, in 2012. The Kushima clone was reared for four months and used for insecticide bioassays without insecticide selection. These aphid clones were reared on cucumber seedlings at 25°C and a relative humidity (RH) of 60% under a 16 : 8-hr light : dark photoperiod.

2. Chemicals

Acetamiprid and other neonicotinoids were synthesized at the Odawara Research Center. ACh chloride was purchased from Sigma-Aldrich (Tokyo, Japan). The purity of imidacloprid and acetamiprid was >99.9%.

3. Insecticide bioassays

All compounds were dissolved in a 5% N,N,-dimethylformamide aqueous solution and then diluted with distilled water and 2×10⁻⁴% (v/v) of the surfactant RABIDEN 3S (which is a trade name: 1.4% sodium dioctylsulfosuccinate, 8% polyoxyethylene alkyl ether, and 3% polyoxyethylene fatty acid ester). Wingless female A. gossypii (n=5–10) were incubated on cucumber seedlings at the first- to second-leaf stage of development. One day later, they were removed, and the test solutions were sprayed on the offspring, while the leaves were maintained under the same conditions (25°C, 60% RH, and a 16 : 8-hr light : dark photoperiod). Seventy-two hr after application, the effects of the compounds were assessed using median lethal dose (LC₅₀) values calculated by probit analysis. Each compound was tested in duplicate.

To assess the effects of the synergist PBO, aphids were sprayed with PBO solution (250 ppm, 5% N,N,-dimethylformamide aqueous solution) 5 hr prior to application of the insecticide solution. Each compound was tested in duplicate.

4. Sequence analysis of the A. gossypii nAChR α1, α2, and β1 subunits

Total RNA was isolated from adult aphids using TRIzol Reagent (Invitrogen Corporation, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. First strand cDNA was synthesized from total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, K.K., Tokyo, Japan) with oligo-dT primers. PCR amplification was performed using KOD plus Neo polymerase (Toyobo Co., Ltd., Osaka, Japan) and the following gene-specific primers: Ago nAChR β1-F (5′-CAA CAA ACT AAT CAG ACC TGT CC-3′), Ago nAChR β1-R (5′-GGC AAG TAG AAC ACT AGC ACG C-3′), Ago nAChR α1-F (5′-CTA CAA TCG GTT GAT CAG GC-3′), Ago nAChR α1-R (5′-CTA ACA CCG TCA TGA ATG TC-3′), Ago nAChR α2-F (5′-CTA GTA AGG CCT GTG CTC AAC-3′), and Ago nAChR α2-R (5′-GGA AGG TAA AAT ACA AGT ATC G-3′). PCR amplification was performed under the following cycling conditions: 2 min at 94°C, followed by 35 cycles of 10 s at 98°C, 30 s at 56°C, and 1 min at 68°C. Amplicons of the expected size were purified using the QIAquick PCR Purification Kit (Qiagen GmbH, Hilden, Germany) and directly sequenced in both directions using the BigDye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The direct sequences were confirmed using the following primers: Ago nAChR β1S-F (5′-CAG ACC TGT CCA GAA CAT GAC C-3′) and Ago nAChR β1S-R (5′-ATG AGG ACC GTG GGC AGT ATC-3′) for subunit β1, Ago nAChR α1S-F (5′-CAG TCC AAA ACC ATT CAG AG-3′) and Ago nAChR α1S-R (5′-GTA GAA CAA AGT CTT GCG TCG-3′) for subunit α1, and Ago nAChR α2S-F (5′-CAG TAC GGA TTA AGC TCA AAC-3′) and Ago nAChR α2S-R (5′-GGT ATG ATC AGA TTT ACT GTG-3′) for subunit α2.

5. Isolation of cDNA clones encoding nAChRs

cDNA encoding Drosophila melanogaster nAChR α2 subunit (Dα2) was amplified from first strand cDNA synthesized from larval total RNA using the Transcriptor First Strand cDNA Synthesis Kit with oligo-dT primers. PCR amplification was performed using the following primers: Dme nAChR α2-F (5′-GGT ACC ATG GCT CCT GGC TGC TGC ACC AC-3′) and Dme nAChR α2-R (5′-GGG CCC TTA ATT CTT CTT CTC GGT TAG-3′). PCR amplification was performed under the following cycling conditions with KOD plus Neo: 2 min at 94°C, followed by 30 cycles of 10 sec at 98°C, 30 sec at 56°C, and 2 min at 68°C. Amplicons were purified using the QIAquick Gel Extraction Kit (Qiagen GmbH) and cloned into pTA2 vectors (Toyobo Co., Ltd.). Several clones containing the Dα2 gene were sequenced. One clone identical to a published sequence (GenBank accession number X53583) was excised from the pTA2 vector using the restriction endonucleases KpnI and ApaI and then ligated into the pcDNA3.1(+) vector (Life Technologies, Japan, Tokyo, Japan).

The chicken nAChR β2 subunit was amplified from first strand cDNA synthesized from chicken total RNA obtained from BioChain Institute, Inc. (Newark, CA, U.S.A.). PCR was performed using KOD-plus-Neo polymerase with the gene-specific primers Gga nAChR β2-F (5′-GGG GTA CCG CCA CCA TGG CGC TGC TCC GCG TCC TC-3′) and Gga nAChR β2-R (5′-GGG AAT TCC CTA TTT GGA GGT GGG GGT GCC CTG GC-3′). PCR amplification was performed under the following cycling conditions: 2 min at 94°C, followed by 30 cycles of 10 sec at 98°C, 30 sec at 56°C, and 1.5 min at 68°C. Amplicons were cloned into pTA2 vectors (Toyobo Co., Ltd.) and sequenced, while the β2 gene was cloned into the pcDNA3.1(+) vector.

6. Preparation of Dα2 and chicken β2 cRNA

Templates for in vitro transcription were obtained by PCR amplification of each plasmid using KOD-plus-Neo polymerase and the gene-specific primers pcDNA3 cRNA-F (5′-CTC TCT GGC TAA CTA GAG AAC C-3′) and pcDNA3 cRNA-R (5′-CTA GAA GGC ACA GTC GAG GCT G-3′). The capped RNA transcripts were synthesized using the T7 polymerase included with the mMessage mMachine® T7 Ultra Kit (Life Technologies). The quality and quantity of cRNAs were verified by agarose gel electrophoresis and absorption spectroscopy. The cRNA was stored at −80°C for further use.

7. nAChR expression in Xenopus oocytes by cRNA injection and electrophysiology

All experiments were performed at 18–19°C. Female Xenopus laevis were purchased from Hamamatsu Seibutsukyozai (Shizuoka, Japan) and maintained in tap water. Oocytes were surgically obtained using 0.03% benzocaine (Sigma-Aldrich) and then enzymatically defolliculated by incubation for 1–1.5 hr in Ca²⁺-free Standard Oocyte Saline (SOS) solution (100 mM NaCl, 2.0 mM KCl, 1.0 mM MgCl₂, 5.0 mM HEPES, pH 7.6)¹⁶⁾ containing 1 mg/mL collagenase (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Healthy stage V–VI oocytes were injected with equivalent amounts (10 ng each) of cRNA encoding Dα2 and chicken β2 using the Nanoject II™ Auto-Nanoliter Injector (Drummond Scientific Company, Broomall, PA, U.S.A.) and then incubated in SOS solution supplemented with 50 µg/mL gentamycin, 100 U/mL penicillin, 100 µg/mL streptomycin, 2.5 mM sodium pyruvate, and 4% horse serum for 1–4 days. The medium was replaced daily and unhealthy oocytes were discarded.

Two-electrode voltage clamp electrophysiological analysis was performed as described by Ihara et al.¹⁶⁾ Currents were recorded using the TEV-200A amplifier (Dagan Corporation, Minneapolis, MN, U.S.A.) and digitized at 1 kHz using the PowerLab 8/30 data acquisition and analysis system (ADInstruments, Nagoya, Japan). Signals were filtered at 50 Hz using a 4-pole Bessel filter. Recording electrodes were prepared from borosilicate glass tubes (CG150TF-10; Warner Instruments, Hamden, CT, U.S.A.) using a P-1000 Puller (Sutter Instruments, Novato, CA, U.S.A.). Electrodes were filled with 3 M KCl and had resistance of 0.5–2.0 MΩ when measured in SOS solution. Oocytes were continuously perfused with SOS solution throughout the recording session at a rate of 5 mL/min using a gravity-fed system¹⁶⁾ and voltage-clamped at −100 mV, while those with excessive leak currents (>−1000 nA) were not used for further analysis.

Test compounds were first dissolved in dimethyl sulfoxide (DMSO) and later diluted to the appropriate test concentration in SOS solution (100 mM NaCl, 2.0 mM KCl, 1.0 mM MgCl₂, 1.8 mM CaCl₂, 5.0 mM HEPES, pH 7.6) containing 0.5 µM atropine. The concentration of DMSO was maintained at <0.1% (v/v) to avoid the effects of the channel or cell. A solution of ACh and SOS was prepared by adding ACh chloride directly to the atropine-SOS solution immediately before use.

The LabChart 7 (ADInstruments, Nagoya, Japan) and GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, U.S.A.) software packages were used for data analysis and the preparation of graphs. Normalized concentration-response data were analyzed by nonlinear regression using GraphPad Prism to obtain I_max (the maximum normalized response), EC₅₀ (the concentration inducing the half-maximal normalized response), and pEC₅₀ (−log EC₅₀). The peak amplitude of the current recorded in response to each challenge was normalized to the maximum amplitude of the response to ACh. Data from the Dα2β2, T77R, and T77R+E79V mutants were normalized to the response to 1 mM ACh.

Results

1. Neonicotinoid insecticide bioassays

The A. gossypii (Kushima) clone, which was discovered in Miyazaki Prefecture, Japan, exhibited resistance to neonicotinoid insecticides as compared with a susceptible clone (Table 1), which was associated with resistance factors (RFs) 23.8–394. Additionally, the Kushima clone had a high level of resistance (RFs 216–394) to nitro-substituted neonicotinoids (Fig. 1), such as imidacloprid. In contrast, the Kushima clone exhibited low to moderate levels of resistance (RFs 23.8 and 65.4) to cyano-substituted neonicotinoids (e.g., acetamiprid).

Table 1. Insecticidal activity of neonicotinoids to susceptible aphids and the Kushima clone with and without pretreatment with piperonyl butoxide (PBO)

Compound	Susceptible clone		Kushima clone		RFa)	SFb)
	LC50 (ppm)	95% confidence limit of LC50	LC50 (ppm)	95% confidence limit of LC50
Acetamiprid	0.10	0.080–0.12	6.54	4.62–9.73	65.4
Thiacloprid	0.30	0.20–0.61	7.15	4.40–11.8	23.8
Imidacloprid	0.35	0.20–0.85	75.7	44.7–193	216
Clothianidin	0.10	0.061–0.18	39.4	28.0–58.5	394
Thiamethoxam	0.19	0.12–0.31	56.0	40.6–73.7	295
Dinotefuran	0.66	0.40–1.04	167	114–285	253
Nitenpyram	0.30	0.19–0.54	71.0	27.5–103	237
Acetamiprid+PBO	0.028	0.016–0.048	1.91	1.03–3.08		3.4
Imidacloprid+PBO	0.050	0.032–0.079	7.01	3.46–12.0		11

Experiments were performed in duplicate.a) Resistance factor (LC₅₀ Kushima clone/LC₅₀ susceptible clone).b) Synergistic factor (LC₅₀ Kushima clone/LC₅₀ Kushima clone with PBO).

When PBO was used as a pretreatment synergist with the Kushima clone, the LC₅₀ value decreased (Table 1). The LC₅₀ values of acetamiprid and imidacloprid to the Kushima clone without PBO pretreatment were 6.54 and 75.7 ppm, but 1.91 and 7.01 ppm with PBO pretreatment, respectively. The synergistic factor (SF) of acetamiprid was lower than that for imidacloprid (3.4 vs. 11, respectively). These results indicate that the Kushima clone acquired a resistance mechanism other than an enhanced oxidative metabolism.

2. Sequence analysis of A. gossypii nAChR subunit genes

The N-terminal regions of the A. gossypii (Kushima clone) nAChR β1, α1, and α2 subunits, which encompass the conserved domains (loop A–F) that comprise the ACh and neonicotinoid binding sites, were PCR-amplified and then sequenced. Although a limited number of silent single-nucleotide polymorphisms were detected, no amino acid changes were observed in the α1 and α2 subunits. In contrast, a single point mutation, Arg81 (AGA) to Thr (ACA), was detected in the β1 subunit (Fig. 2). Additionally, the mutation was detected heterozygously (data not shown).

Fig. 2. Alignment of amino acid sequences in loop D of the agonist binding region of vertebrate and insect nicotinic ACh receptor subunits (nAChRs). Homo sapiens (human), Gallus gallus (chicken), Rattus norvegicus (rat), Drosophila melanogaster (fruit fly), Bemisia tabaci (sweet potato whitefly), Myzus persicae (peach-potato aphid), Aphis gossypii (cotton aphid).

3. Agonist activity of ACh

A concentration–response curve of ACh to Drosophila melanogaster Dα2-chicken β2 nAChRs was used as a control in this study. We used the Dα2-vertebrate β2 hybrid receptor for the following reasons: Insect nAChR functional expression is difficult, but the Dα2-chicken β2 hybrid receptor has been well established for assaying neonicotinoids. The I_max and pEC₅₀ values of ACh for Dα2β2 nAChR were 1.08±0.03 and 4.60±0.04, respectively. The ACh values of the Dα2β2 nAChR were similar to those in previous reports^16–18) (Fig. 3, Table 2).

Fig. 3. Response to acetamiprid of Dα2Gβ2 nicotinic ACh receptors (nAChRs). A–C; Responses of oocytes expressing WT Dα2β2 nAChRs (A), the T77R single mutation (B), and the T77R+E79V double mutation (C) to 100 µM ACh and 100 µM acetamiprid. D–F; Responses of oocytes expressing the WT (D), the T77R single mutation (E), and the T77R+E79V double mutation Dα2β2 nAChRs (F) to 100 µM ACh and 30 µM imidacloprid. Concentration-response curves of ACh, acetamiprid, and imidacloprid of the WT (G), the T77R single mutation (H), and the T77R+E79V double mutation (I) Dα2Gβ2 nAChRs. Each point represents the mean±S.E.M. of four experiments (n=4). The peak amplitude of the current recorded in response to each challenge was normalized to the maximum amplitude of the response to ACh. Data from the Dα2β2, T77R, and T77R+E79V mutants were normalized to the response to 1 mM ACh.

Table 2. pEC₅₀ and I_max values of ACh, acetamiprid, and imidacloprid for WT, T77R, and T77R+E79V Dα2β2 nAChRs

Compounds	Dα2β2 WT		Dα2β2 T77R		Dα2β2 T77R+E79V
	pEC50	Imax	pEC50	Imax	pEC50	Imax
ACh	4.60±0.04	1.08±0.03	4.64±0.07	1.03±0.01	4.47±0.03	1.04±0.02
Acetamiprid	5.26±0.09	0.49±0.03	5.42±0.06	0.99±0.04*	5.38±0.04	0.97±0.03*
Imidacloprid	5.61±0.06	0.77±0.03	6.35±0.06*	0.79±0.03	6.18±0.02*	0.96±0.02*

Values shown are the results of a fit of the concentration-response data (mean±S.E.M, n=4) illustrated in Fig. 3. Statistical test (one-way ANOVA, Dunnett’s multiple-comparison test) is for significant differences from the wild-type data. *p<0.05. The peak amplitude of the current recorded in the response to each challenge was normalized to the maximum amplitude of the response to ACh. Data from the Dα2β2, T77R, and T77R+E79V mutants were normalized to the response to 1 mM ACh.

4. Effect of mutation in the nAChR β subunit on neonicotinoid sensitivity

To examine the effects of the R81T mutation in loop D of the β1 subunit on neonicotinoid agonist activity, we constructed an equivalent single mutant (T77R) of the chicken β2 subunit (Fig. 2). We also evaluated the T77R+E79V double mutant because the cotton aphid nAChR β1 subunit has Val (Val83) at the equivalent amino acid residue of the chicken β2 subunit (E79). The E79 residue of the chicken β2 subunit may suppress interactions between the nitro group of neonicotinoids and Arg 77 through the negative electrostatic forces of Glu79.¹⁸⁾ The I_max and pEC₅₀ values of ACh for Dα2β2 nAChR T77R were 1.03±0.01 and 4.64±0.07, respectively. The I_max and pEC₅₀ values of ACh for Dα2β2 nAChR with the double mutation T77R+E79V were 1.04±0.02 and 4.47±0.03, respectively. The T77R and T77R+E79V mutations did not significantly affect the pEC₅₀ and I_max values for the natural agonist ACh (Table 2, Fig. 3), as compared with the wild type (WT) Dα2β2 nAChR, in accordance with the findings of previous report.¹⁸⁾ The pEC₅₀ values of acetamiprid and imidacloprid for WT Dα2β2 nAChR were 5.26±0.09 and 5.61±0.06, respectively. On the other hand, the pEC₅₀ values of acetamiprid and imidacloprid for the Dα2β2 nAChR single mutant (T77R) were 5.42±0.06 and 6.35±0.06, and 5.38±0.04 and 6.18±0.02 for the Dα2β2 nAChR double mutant (T77R+E79V), respectively. The pEC₅₀ value of imidacloprid was significantly shifted relative to that of the WT, but that for acetamiprid exhibited no significant change. The I_max values of acetamiprid and imidacloprid for the WT, T77R, and T77R+E79V Dα2β2 nAChRs were, respectively, 0.49±0.03 and 0.77±0.03, 0.99±0.04 and 0.79±0.03, and 0.97±0.03 and 0.96±0.02. Unexpectedly, and importantly, neither the T77R nor the T77R+E79V mutations induced significant changes in the pEC₅₀ values of acetamiprid, whereas significant changes were observed in the I_max values (Fig. 3, Table 2). On the other hand, the T77R+E79V double mutant induced significant changes in both the pEC₅₀ and the I_max values of imidacloprid. In addition, the T77R and T77R+E79V mutations did not significantly affect the maximum amplitudes of the ACh-induced current as compared with the WT (data not shown).

Discussion

In this study, we characterized a field isolate of A. gossypii (Kushima clone) that exhibited levels of neonicotinoid resistance (23.8–394-fold) high enough to impair field performance. Sequencing of the nAChR gene of the Kushima clone showed that it had at least one point mutation (R81T) in the loop D region of the nAChR β1 subunit. The ACh binding site contains loop D,¹⁹⁾ and many experimental studies have indicated that the amino acid residue at position 81 within this loop is a key determinant of neonicotinoid binding to nAChRs.^18,20,21) In addition, an R81T mutation in Myzus persicae was previously reported to be responsible for reduced sensitivity to neonicotinoids and affinity of nAChR to imidacloprid.¹⁴⁾

P450-mediated detoxification has been implicated in resistance mechanisms through the use of PBO. The LC₅₀ values of acetamiprid and imidacloprid to the Kushima strain pretreated with PBO were 1.91 and 7.01 ppm, respectively, which were reduced by 3.4–11-fold. PBO pretreatment also reduced the LC₅₀ values of the susceptible clone to acetamiprid and imidacloprid (0.028 and 0.050 ppm, respectively). The synergistic factors of acetamiprid and imidacloprid were 3.6 and 6.0, respectively. PBO reduced the LC₅₀ values of acetamiprid and imidacloprid to the Kushima clone as well as to the susceptible clone. These findings suggest that enhanced oxidative metabolism is not the main mechanism of neonicotinoid resistance.²²⁾

The Kushima clone exhibited high levels of neonicotinoid resistance and different RFs between the nitro- and cyano-substituted neonicotinoids. Specifically, the cyano-substituted neonicotinoid (Fig. 1) had smaller RFs than did the nitro-substituted ones (Table 1). We assessed the effect of the R81T mutation (WT Dα2β2, T77R single mutant, and T77R+E79V double mutant nAChRs) on the activity of a neonicotinoid agonist using electrophysiological methods. The Thr 77 residue of the chicken β2 subunit corresponds to the Arg 81 residue of the cotton aphid β1 subunit (Fig. 2). Interestingly, the T77R and T77R+E79V mutations did not significantly affect the pEC₅₀ value of the natural agonist ACh (Table 2, Fig. 3), as previously reported.¹⁸⁾ The R81T mutation, which had no effect on the pEC₅₀ values of ACh, may enable both a high level of resistance to neonicotinoids and a low fitness cost.

The pEC₅₀ value of imidacloprid to the Dα2β2 nAChR T77R single mutant significantly shifted compared with that of the WT Dα2β2 nAChR. Furthermore, the T77R+E79V double mutant induced significant changes in the pEC₅₀ and I_max values of imidacloprid (Fig. 3, Table 2). The pEC₅₀ value of imidacloprid for the Dα2β2 nAChR T77R mutant was 443 nM, which is close to the value determined for native nAChRs on the native neurons of the cockroach (Periplaneta americana).²³⁾ On the other hand, the T77R and T77R+E79V mutants did not significantly change the pEC₅₀ value of acetamiprid, whereas a significant change was observed in the I_max value. Although the effect of the T77R+E79V double mutation on imidacloprid agonist activity has been previously reported,^18,24) the present study is the first to show its effect on acetamiprid activity. Although it remains unclear whether insecticidal activity is dependent on either the pEC₅₀ or I_max value, differences in the RFs between nitro- and cyano-substituted neonicotinoids were likely due to the effect of the R81T mutation on agonist affinity. The crystal structures of imidacloprid and thiacloprid complexed with AChBP^20,25) showed that the nitro- and cyano-substituted neonicotinoids had almost identical interactions. Homology modeling of Myzus persicae nAChR with imidacloprid showed its similarity to the crystal structure of AChBP with imidacloprid.²⁶⁾ However, the cyano-substituted neonicotinoid (thiacloprid) had an additional water bridge with the cyano group, which presumably enhanced interactions with loop C.²⁰⁾ Recent studies have indicated that the binding characteristics of thiacloprid are distinct from those of nitro-substituted neonicotinoids,²⁵⁾ and the binding characteristic of acetamiprid are probably the same as those of thiacloprid. In addition, electrophysiological studies, photoaffinity labeling experiments, and homology modeling have indicated that the cyano group of neonicotinoids may interact with loop C in the agonist binding domain of the α subunit.^26,27) Furthermore, recent electrophysiological experiments have indicated that the cyano group of thiacloprid may have unique interactions as compared with the nitro group of imidacloprid.²⁹⁾ Accordingly, the different effects of acetamiprid and imidacloprid, as indicated by the pEC₅₀ values, are probably due to their slightly different interactions with aphid nAChRs.

In conclusion, we have shown that the Kushima clone harbors an R81T mutation that conveys a high level of resistance to neonicotinoids. In addition, we investigated the effect of the R81T mutation on neonicotinoid affinity. An important finding of this study was the significant changes in the pEC₅₀ values of imidacloprid for the Dα2β2 nAChR T77R single mutant and the T77R+E79V double mutant, whereas those of acetamiprid showed no change. In this study, we used Dα2 and chicken β2 hybrid nAChRs. To elucidate further the effect of mutations on neonicotinoid agonist activities, further studies with aphid nAChR or complete insect nAChRs are required. Here, we have analyzed the sequences of the aphid nAChR α1, α2, and β1 subunits. However, aphids may carry additional mutations in other nAChR subunit genes or different genes altogether. Therefore, whole-genome analyses of both susceptible aphid strains and the Kushima clone are necessary to investigate other possible resistance mechanisms. Additionally, the mutation of Arg81 (AGA) to Thr (ACA) was detected heterozygously. However, the inheritance pattern remains unclear. Elucidation of this is necessary for understanding the mechanism of resistance. We trust that the findings obtained in this study will contribute to a better understanding of the different modes of action of nitro- and cyano-substituted neonicotinoids to improve resistance management.

Acknowledgment

We wish to thank Motoaki Sato for valuable comments that significantly improved the quality of our manuscript. This work was supported by a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Genomics-based Technology for Agricultural Improvement, PRM-2102).

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