A nicotinic acetylcholine receptor mutation (Y151S) causes reduced agonist potency to a range of neonicotinoid insecticides

Authors


Address correspondence and reprint requests to Dr Neil S. Millar, Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT, UK. E-mail: n.millar@ucl.ac.uk

Abstract

Neonicotinoid insecticides are potent selective agonists of insect nicotinic acetylcholine receptors (nAChRs). Since their introduction in 1991, resistance to neonicotinoids has been slow to develop, but it is now established in some insect field populations such as the planthopper, Nilaparvata lugens, a major rice pest in many parts of Asia. We have reported recently the identification of a target-site mutation (Y151S) within two nAChR subunits (Nlα1 and Nlα3) from a laboratory-selected field population of N. lugens. In the present study, we have examined the influence of this mutation upon the functional properties of recombinant nAChRs expressed in Xenopus oocytes (as hybrid nAChRs, co-expressed with a rat β2 subunit). The agonist potency of several nicotinic agonists has been examined, including all of the neonicotinoid insecticides that are currently licensed for either crop protection or animal health applications (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam). The Y151S mutation was found to have no significant effect on the maximal current (Imax) observed with the endogenous agonist, acetylcholine. In contrast, a significant reduction in Imax was observed for all neonicotinoids (the Imax for mutant nAChRs ranged from 13 to 81% of that observed on wild-type receptors). In addition, nAChRs containing the Y151S mutation caused a significant rightward shift in agonist dose–response curves for all neonicotinoids, but of varying magnitude (shifts in EC50 values ranged from 1.3 to 3.6-fold). The relationship between neonicotinoid structure and their potency on nAChRs containing the Y151S target-site mutation is discussed.

Abbreviations used
AChBP

acetylcholine binding protein

nAChR

nicotinic acetylcholine receptor

Neonicotinoid insecticides are insect-selective nicotinic acetylcholine receptor (nAChR) agonists that are used extensively in areas of crop protection and animal health (Matsuda et al. 2001; Tomizawa and Casida 2005). Since the introduction of imidacloprid in the early 1990s, neonicotinoids have become one of the most widely used classes of pesticide. Although resistance to neonicotinoid insecticides has been relatively slow to develop, it has now been identified as an emerging problem in several pest species (Nauen and Denholm 2005). In most cases, resistance has been attributed to enhanced oxidative detoxification of neonicotinoids by overexpressed monooxygenase enzymes. Recently, however, studies with the brown planthopper, Nilaparvata lugens, a major rice pest in many parts of Asia, have identified a target-site mutation within nAChR α subunits, which is associated with neonicotinoid insecticide resistance (Liu et al. 2005).

Previous studies of wild-type and imidacloprid-resistant N. lugens (a laboratory strain which was selected from a field population collected in Jiangsu, China) have identified a single point mutation (Y151S) within the extracellular, agonist-binding domain of two nAChR α subunits (Liu et al. 2005). Radioligand binding studies, performed with both native nAChR preparations and with heterologously-expressed recombinant nAChRs, demonstrated that the Y151S resistance-associated point mutation is responsible for a dramatically reduced level of specific [3H]imidacloprid binding. In the present study, we have used two-electrode voltage-clamp techniques to examine the influence of the Y151S mutation upon the functional properties of recombinant nAChRs expressed in Xenopus oocytes. The potencies of a range of nAChR agonists have been compared on recombinant nAChRs containing either the wild-type or mutated N. lugens nAChR subunit Nlα1. All seven of the neonicotinoid insecticide compounds that have been released for commercial use (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam; Fig. 1) have been examined in the present study, and have been compared with other nicotinic agonists, such as acetylcholine, nicotine and epibatidine.

Figure 1.

 Chemical structure of nicotinic agonists examined in this study. The structure of three classical nAChR agonists (acetylcholine, nicotine and epibatidine) and seven commercial neonicotinoid insecticides are shown. The neonicotinoids include four chloropyridyl compounds (acetamiprid, imidacloprid, nitenpyram and thiacloprid), two chlorothiazolyl compounds (clothianidin and thiamethoxam) and a tetrahydrofuryl compound (dinotefuran).

Materials and methods

Materials

Neonicotinoid insecticides were purchased from Sigma/Riedel-deHaën (Seelze, Germany), or were generously provided by Bayer CropSciences (Monheim, Germany) or Mitsui Chemicals (Mobara, Japan).

Plasmids and subcloning

Cloning of the N. lugens nAChR subunits Nlα1, Nlα3 and Nlβ1, and of the mutant (Y151S) subunit Nlα1Y151S, has been described previously (Liu et al. 2005). The rat nAChR β2 subunit (Deneris et al. 1988) in plasmid pcDNAI/Neo was provided by Dr Jim Patrick (Baylor College of Medicine, Houston, TX, USA). The N. lugens nAChR subunit cDNAs were subcloned into EcoRI and XbaI sites of pGH19, a modified version of plasmid pGEMHE (Liman et al. 1992; Trudeau et al. 1995). The rat nAChR β2 subunit was subcloned into the BamHI and XbaI sites of pGEMHE(NotI), a modified version of pGEMHE (Liman et al. 1992), in which a NotI site was introduced by the ligation of synthetic oligonucleotides between the NheI and PstI sites. All plasmid constructs were verified by nucleotide sequencing.

In vitro transcription

Plasmid pGH19 constructs encoding N. lugens nAChR subunits were linearized with NotI. Plasmid pGEMHE(NotI)-rβ2 was linearized by digestion with NdeI. In vitro transcription, to generate cRNA, was performed using the mMESSAGE mMACHINE T7 transcription kit (Ambion, Huntington, UK). Reactions were carried out according to the manufacturer's protocol. Transcripts were recovered by precipitation with propan-2-ol, dissolved in nuclease-free water at a final concentration of 0.5 mg/mL and stored at − 80°C prior to use.

Oocyte preparation and electrophysiology

Ovarian lobes were isolated from female Xenopus laevis frogs using standard procedures, as described previously (Boorman et al. 2000). Clumps of stage V–VI oocytes were dissected in a sterile modified Barth's solution (NaCl 88 mm; KCl 1 mm; MgCl2 0.82 mm; CaCl2 0.77 mm; NaHCO3 2.4 mm; Tris-HCl 15 mm; with 50 U/mL penicillin and 50 µg/mL streptomycin; pH 7.4 adjusted with NaOH). The dissected oocytes were treated with collagenase (type IA, Sigma, Sunnyvale, CA, UK; 65 min at 18°C, 245 collagen digestion units/mL in Barth's solution, 10–12 oocytes/ml), rinsed, stored at 4°C overnight, and manually defolliculated the following day before injection with cRNA (46 nL/oocyte). To express heteromeric nAChRs, cRNAs were injected at a ratio of 1 : 1. The oocytes were incubated for approximately 60 h at 18°C in Barth's solution containing 5% heat-inactivated horse serum (Gibco/Invitrogen, Paisley, UK) and then stored at 4°C. Experiments were carried out at a room temperature of 18–20°C between 2 and 10 days after injection.

Oocytes, held in a 0.2 mL bath, were perfused at 4.5 mL/min with modified Ringer's solution (NaCl 150 mm, KCl 2.8 mm, HEPES 10 mm, MgCl2 2 mm, 0.5 µm atropine sulfate, Sigma; pH 7.2 ,adjusted with NaOH) and voltage clamped at − 70 mV using the two-electrode clamp mode of an Axoclamp-2B amplifier (Molecular Devices, Poole, UK). Electrodes were pulled from Clark borosilicate glass GC150TF (Harvard Apparatus, Edenbridge, UK) and filled with 3 m KCl. The electrode resistance was 0.5–1 MΩ on the current-passing side. Experiments were terminated if the total holding current exceeded 2 µA in order to reduce the effect of series resistance errors. A nominally calcium-free solution was used in order to minimize the contribution of the endogenous calcium-gated chloride conductance (Sands and Barish 1991).

Agonists, freshly prepared from frozen stock aliquots, were applied via bath perfusion for a period sufficient to obtain a stable plateau response (at low concentrations) or the beginning of a fall after a peak (at the higher concentrations); the resulting inward current was recorded on a flat bed chart recorder or digitized at 10 Hz for later analysis. An interval of 5 min was allowed between agonist applications, as this was found to be sufficient to ensure reproducible responses. In order to compensate for possible decreases in agonist sensitivity throughout the experiment, a standard concentration of agonist (approximately EC20 for the particular combination used) was applied every third response. The experiment was started only after checking that this standard concentration gave reproducible responses. Typically, the response to this agonist standard concentration observed by the end of the experiment was 60–90% of the initial response. All the data shown in the paper are compensated for the response rundown. Dose–response curves were fitted with the Hill equation {I = Imax ([A]nH/[A]nH + inline image)}, where I is the response, measured at its peak, [A] is the agonist concentration, Imax is the maximum response, EC50 is the agonist concentration for 50% maximum response and nH is the Hill coefficient.

Results

Heterologous expression in Xenopus oocytes

Nicotinic receptor subunits cloned previously from the brown planthopper, N. lugens, (Liu et al. 2005) were expressed in Xenopus oocytes, and their ability to generate functional recombinant nAChRs examined by two-electrode voltage-clamp recording. In agreement with previous expression studies performed in Drosophila S2 cells (Liu et al. 2005), no evidence of functional nAChRs could be detected in Xenopus oocytes when either the Nlα1 or Nlα3 subunits were expressed alone, or when co-expressed with other previously cloned N. lugens nAChR subunits such as Nlβ1. This finding is consistent with previously reported difficulties associated with the heterologous expression of insect nAChRs (Bertrand et al. 1994; Lansdell et al. 1997; Hermsen et al. 1998; Huang et al. 1999; Lansdell and Millar 2000, 2002).

Previous studies performed with recombinant nAChRs expressed in Drosophila S2 cells demonstrated that the Nlα1 subunit (but not the Nlα3 subunit) is capable of generating a high-affinity binding site for nicotinic radioligands when co-expressed with the rat β2 subunit (Liu et al. 2005). To examine whether expression of functional hybrid (N. lugens/rat) nAChRs could be detected, Xenopus oocytes were injected with in vitro-transcribed mRNA, encoding N. lugens and rat nAChR subunits and examined by two-electrode voltage-clamp recording. In oocytes co-injected with mRNA encoding the Nlα1 and rat β2 nAChR subunits, large inward currents were detected in response to applications of acetylcholine (Fig. 2). In contrast, no evidence of functional nAChRs was detected when the Nlα3 subunit was co-expressed with rat β2 nAChR subunits. In experiments in which the two N. lugensα subunits (Nlα1 and Nlα3) were co-expressed with either Nlβ1 or rat β2, no evidence was obtained to suggest that either Nlα3 or Nlβ1 was able to contribute to the formation of functional recombinant nAChRs. Rather than indicating that these subunits do not contribute to functional nAChRs in the insect nervous system, it is possible that these findings may simply reflect the widely encountered problems associated with the heterologous expression of insect nAChRs (for review see Millar 1999).

Figure 2.

 Representative whole-cell responses to nAChRs expressed in Xenopus oocytes, measured by two-electrode voltage-clamp recording. (a) Representative responses elicited by acetylcholine and imidacloprid in oocytes expressing Nlα1 + Rβ2. (b) Representative responses elicited by acetylcholine and imidacloprid in oocytes expressing Nlα1Y151S + Rβ2.

Agonist potency of acetylcholine on wild-type and mutant nAChRs

To examine the influence of the recently identified nAChR Y151S point mutation (Liu et al. 2005) on the functional properties of nAChRs, wild-type (Nlα1) and mutant (Nlα1Y151S) subunits were co-expressed with rat β2 in Xenopus oocytes and examined by two-electrode voltage-clamp methods. The maximum current (Imax) observed in response to application of acetylcholine in oocytes expressing Nlα1 + β2 nAChRs (220 ± 7.1 nA; n = 4) was not significantly different to that observed with Nlα1Y151S + rat β2 receptors (221 ± 2.1 nA; n = 4; Figs 3 and 4). The Y151S mutation resulted in a small (1.4-fold) but significant rightward shift in the agonist dose–response curve for acetylcholine, reflecting an increase in EC50 (Figs 3 and 4). The mean EC50 value for acetylcholine was 33 ± 1.0 µm on Nlα1 + β2 nAChRs, and 47 ± 1.3 µm on Nlα1Y151S + rat β2 nAChRs (p = 0.0001; n = 4).

Figure 3.

 Agonist dose–response curves from nAChRs expressed in Xenopus oocytes. (a) Dose–response curves for acetylcholine determined from oocytes expressing Nlα1 + Rβ2 and Nlα1Y151S + Rβ2. (b) Dose–response curves for imidacloprid determined from oocytes expressing Nlα1 + Rβ2 and Nlα1Y151S + Rβ2. In all cases, representative dose–response curves are shown that are typical of four independent experiments obtained from different oocytes.

Figure 4.

 Influence of Y151S mutation on Imax and EC50 for nicotinic agonists. (a) Maximal current (Imax) and (b) EC50 values determined from three to four independent agonist dose–response curves performed in Xenopus oocytes. Data are shown for both Nlα1 + Rβ2 (open bars) and Nlα1Y151S + Rβ2 (closed bars). Note, EC50 values for epibatidine are presented as 10−8 m, whereas those for all other agonist are presented as 10−6 mm). (c) Data presented in (a) have been re-plotted to show the maximal current observed with Nlα1Y151S + Rβ2 as a percentage of that observed with the same agonist on Nlα1 + Rβ2. (d) Data presented in (b) have been re-plotted to show data as a fold effect (EC50 obtained with Nlα1Y151S + Rβ2 is compared with that observed with the same agonist on Nlα1 + Rβ2). All data are mean values (n = 3–4) ± SEM.

Agonist potency of neonicotinoids on wild-type and mutant nAChRs

Further dose–response experiments were performed to examine the influence of the Y151S mutation on the agonist potency of the neonicotinoid insecticide, imidacloprid (Fig. 2). Imidacloprid activated Nlα1 + rat β2 receptors with an Imax of 172 ± 2.3 nA (n = 4) and EC50 of 67 ± 2.6 µm (n = 4). Although the Y151S mutation had no significant effect on the maximal current detected with acetylcholine, significantly smaller maximal whole-cell currents were observed in response to imidacloprid in oocytes expressing Nlα1Y151S + β2 nAChRs (22 ± 0.5 nA; n = 4) than in oocytes expressing Nlα1 + β2 nAChRs (Figs 3 and 4). In addition, imidacloprid exhibited a large (2.7-fold) shift in EC50 on receptors containing the Nlα1Y151S subunit (EC50 = 177 ± 3.4 µm; n = 4) compared with receptors containing the wild-type subunit (Fig. 4). Data are summarized in Table 1.

Table 1.   Agonist potency of neonicotinoid and non-neonicotinoid compounds on recombinant nAChRs containing wild-type (Nlα1) or mutant (N1α1Y151S) subunits expressed in Xenopus oocytes
Agonist Nlα1 + β2 Nlα1Y151S + β2Percent/fold effect of Y151S mutation
Imax (nA)EC50m)Imax (nA)EC50m)Imax (%)EC50 (fold effect)
  1. Data are means of three to four independent experiments ± SEM.

Acetylcholine220 ± 7.133 ± 1.0221 ± 2.147 ± 1.3101 ± 1.01.40 ± 0.04
Epibatidine202 ± 3.10.42 ± 0.0127 ± 1.01.0 ± 0.0113 ± 0.52.42 ± 0.03
Nicotine58 ± 1.6115 ± 5.435 ± 1.7158 ± 2.161 ± 3.01.38 ± 0.02
Chloropyridyl neonicotinoids
 Acetamiprid128 ± 3.367 ± 2.820 ± 1.4160 ± 6.415 ± 1.12.39 ± 0.09
 Imidacloprid172 ± 2.367 ± 2.622 ± 0.5177 ± 3.413 ± 2.82.64 ± 0.05
 Nitenpyram57 ± 1.961 ± 2.712 ± 1.1172 ± 4.321 ± 1.92.80 ± 0.07
 Thiacloprid134 ± 4.247 ± 1.329 ± 1.4136 ± 2.422 ± 1.12.87 ± 0.05
Chlorothiazolyl neonicotinoids
 Clothianidin115 ± 3.462 ± 2.024 ± 1.3149 ± 6.321 ± 1.12.40 ± 0.11
 Thiamethoxam107 ± 1.248 ± 1.622 ± 1.3176 ± 5.120 ± 1.33.66 ± 0.11
Tetrahydrofuryl neonicotinoids
 Dinotefuran102 ± 5.0122 ± 6.982 ± 3.7163 ± 5.181 ± 3.81.33 ± 0.04

The results of our electrophysiological studies with imidacloprid are in agreement with previous radioligand binding data obtained from studies conducted both with native nAChRs from N. lugens and with recombinant nAChRs expressed in Drosophila S2 cells (Liu et al. 2005). To examine whether the Y151S mutation has an effect on the agonist potency of other neonicotinoid compounds, a further series of dose–response experiments was performed on receptors expressed in Xenopus oocytes. In addition to imidacloprid, six other neonicotinoid compounds have been released for use as insecticides (acetamiprid, clothianidin, dinotefuran, nitenpyram, thiacloprid and thiamethoxam). The potency of all six of these neonicotinoid insecticides was examined. The Y151S mutation resulted in significantly lower Imax values and a significant increase in EC50 for all neonicotinoid compounds examined (Fig. 4). However, the Y151S mutation had a less profound influence on the agonist potency of dinotefuran than was seen with the other neonicotinoid compounds examined. Whilst the Y151S mutation had no significant effect on Imax detected with acetylcholine, it resulted in Imax values that were 81% of the wild-type response with dinotefuran and 13–22% of the wild-type response for other neonicotinoids (Fig. 4). The Y151S mutation resulted in a 1.3-fold shift in EC50 for dinotefuran and a 2.4–3.7-fold shift for all other neonicotinoids examined (Fig. 4). Table 1 gives a summary of Imax and EC50 data obtained in wild-type and mutant nAChRs.

Agonist potency of epibatidine and nicotine on wild-type and mutant nAChRs

To examine further the influence of the Y151S mutation on agonist potency, dose–response experiments were performed with epibatidine and nicotine. Although epibatidine does not display selectivity for insect nAChRs, it shares a chlorinated heterocyclic group common to all neonicotinoid insecticides with the exception of dinotefuran (Fig. 1). As was seen with all of the chlorinated neonicotinoids, the Y151S mutation had a considerable effect on the Imax (13% of wild-type) and EC50 (2.5-fold shift) observed with epibatidine. The Y151S mutation had a less profound effect on nicotine, which shows little structural similarity to either acetylcholine or to neonicotinoids (Fig. 1). The Imax observed with nicotine on Nlα1Y151S + β2 nAChRs was 61% of that observed with the wild-type nAChR, and the EC50 showed a 1.4-fold change compared with wild-type nAChRs (Fig. 4).

Discussion

Previous studies using radioligand binding techniques have identified a point mutation (Y151S), located within the extracellular domain of N. lugens nAChR α subunits, that is associated with resistance to imidacloprid (Liu et al. 2005). The mutation was identified in a laboratory-selected strain, isolated from a field population of N. lugens after several generations of selection with imidacloprid. This amino acid is located within loop B, one of three regions within nAChR α subunits that have been proposed to contribute to the agonist binding site (Corringer et al. 2000; Grutter and Changeux 2001). Whereas previous studies implicating the Y151S mutation in reduced sensitivity of nAChRs to imidacloprid have employed radioligand binding methods, the aim of the present study was to examine the functional consequence of the Y151S mutation by means of electrophysiological characterization of recombinant nAChRs.

The present study provides evidence that the Y151S mutation has a significant effect upon agonist potency of imidacloprid, a neonicotinoid insecticide used extensively in Asia to protect rice plants from attack by pests such as N. lugens. This is in agreement with previous evidence indicating that the Y151S mutation results in reduced binding of imidacloprid. Since the introduction of imidacloprid as a commercial insecticide in 1990, six other neonicotinoid compounds have been approved for use as insecticides. These include nitenpyram (in 1995), acetamiprid (in 1996), thiamethoxam (in 1998), thiacloprid (in 2000), clothianidin (in 2002) and dinotefuran (in 2002). Our study examines, for the first time, the influence of the Y151S mutation upon neonicotinoids other than imidacloprid. By means of two-electrode voltage-clamp recording, we have demonstrated that the Y151S mutation confers reduced potency to all of the neonicotinoid compounds examined. Even for imidacloprid, the Y151S mutation caused a relatively small rightward shift in the dose–response curve (an approximately 3.7-fold change in EC50; see Table 1). This is substantially less than the loss in sensitivity to the cyclodiene insecticide, dieldrin, which is caused by the target-site Rdl mutation in insect GABA receptors (ffrench-Constant et al. 1993; Buckingham et al. 1996). However, the Y151S mutation is responsible for a dramatic reduction in the maximal observed whole-cell current. For imidacloprid, the maximum response on receptors containing the Y151S mutation was 13% of that observed with the wild-type receptor (see Table 1).

Despite evidence that the potency of all neonicotinoid compounds is reduced by the mutation, an interesting finding is that this effect is less pronounced for the tertrahydrofuryl compound, dinotefuran. In contrast to dinotefuran, all of the other neonicotinoid insecticides examined contain a chlorinated heterocyclic (chloropyridyl or chlorothiazolyl) group (Fig. 1). Significantly, the potency of epibatidine, which also contains a chloropyridyl group (Fig. 1), is affected profoundly by the Y151S mutation, a finding that is consistent with our previous radioligand binding studies with [3H]epibatidine (Liu et al. 2005). Although epibatidine shares structural similarity with many neonicotinoid insecticides, it does not exhibit the marked selectivity for insect nAChRs that is a feature of neonicotinoid insecticides (Tomizawa et al. 2000; Tomizawa and Casida 2003). The importance of the heterocyclic group in determining the extent to which agonist potency is influenced by the Y151S mutation is illustrated clearly when comparing two neonicotinoids, clothianidin and dinotefuran, that are otherwise chemically identical (Fig. 1). As shown in Fig. 4, the Y151S mutation causes a significantly greater reduction in the agonist potency of clothianidin (which contains a chlorothiazolyl group) than of dinotefuran (which contains a tetrahydrofuryl group).

As has been discussed previously (Liu et al. 2005), the Y151 residue is highly conserved in both insect and mammalian nAChR subunits. Consequently, despite the profound effect of the Y151S mutation upon the binding and agonist potency of neonicotinoids, it cannot be responsible for the remarkable selectivity of neonicotinoid compounds for insect nAChRs. It has been proposed that the selectivity of neonicotinoids for insect nAChRs is due to interactions between an electronegative pharmacophore in these compounds and a cationic subsite in insect nAChRs (Zhang et al. 2002; Tomizawa et al. 2003). Now, use of techniques such as site-directed mutagenesis is beginning to identify a variety of amino acids that influence either the selectivity or agonist potency of neonicotinoids (Matsuda et al. 2000; Shimomura et al. 2002, 2003, 2004, 2005).

An atomic resolution three-dimensional structure has been determined for a soluble pentameric acetylcholine binding protein (AChBP) from the mollusc, Lymnaea stagnalis (Brejc et al. 2001), that displays significant sequence similarity to the nAChR ligand-binding domain (Smit et al. 2001). Comparison of nAChR and AChBP amino acid sequences reveals that Y151 in nAChR α subunits is at a position analogous to a histidine residue (H145) in the AChBP (Brejc et al. 2001; Liu et al. 2005). X-ray diffraction studies on the Lymnaea AChBP reveals that H145 lies in close proximity to the agonist binding site of the AChBP (Brejc et al. 2001; Celie et al. 2004), but the side chain of H145 points away from the agonist binding pocket (Fig. 5). Therefore, despite the likely close proximity of Y151 to the nAChR agonist binding site, it may not be involved directly in the binding of neonicotinoids. Rather, it is possible that the Y151S mutation might cause an induced conformational change within the nAChR binding site that may result in a substantial affect on neonicotinoid binding but only a relatively minor affect on the binding of acetylcholine.

Figure 5.

 Location of a histidine residue, analogous to Y151 of Nlα1, within the atomic resolution model of the acetylcholine binding protein (AChBP) from Lymnaea. The image (generated by deepview Swiss-Pdb Viewer; http://us.expasy.org/spdbv/mainpage.htm) shows selected AChBP amino acids (the loop B region from I140 to R148) and their proximity to bound nicotine. The figure was generated from an atomic resolution (2.2 Å) crystal structure of the Lymnaea AChBP co-crystallized with nicotine (Celie et al. 2004). The histidine residue (H145), which is at a position within the AChBP primary amino acid sequence analogous to tyrosine 151 in nAChRs α subunits, is approximately 8 Å from the bound nicotine.

Previous work has documented a ‘fitness cost’ associated with the presence of the mutation conferring imidacloprid resistance in N. lugens (Liu & Han 2006). An important conclusion from the present study is that the Y151S point mutation has relatively little effect on the potency of the endogenous agonist, acetylcholine, on recombinant nAChRs containing the Nlα1 subunit. Although the precise subunit composition of native N. lugens nAChRs is not known (and our data were obtained from hybrid receptors containing insect α subunits co-expressed with a vertebrate β subunit), our findings would suggest that insects containing the Y151S mutation may retain functional nAChRs, despite this mutation having a significant effect on neonicotinoid binding. Two issues which have not been addressed in the present study, but have been discussed previously (Liu et al. 2005), are (i) the importance of Y151S mutations within nAChR subunits other than Nlα1, and (ii) the importance of heterozygous and homozygous Y151S mutations. Both of these issues may need to be considered when attempting to assess the potential significance of these findings upon insecticide use in the field. In addition, further work is required to determine whether the agonist concentrations examined in the present study reflect concentrations responsible for activation of native receptors after foliar or systemic application of insecticides.

As yet there has been no work to establish the prevalence of the Y151S mutation in field populations of N. lugens. However, this is being investigated in conjunction with ongoing surveys of neonicotinoid resistance in several countries. An important next step in understanding the practical significance of the mutation is to relate data reported here with the phenotypic expression of resistance in laboratory bioassays and under field treatment regimes. The broad-spectrum reduction in potency disclosed by our work supports consideration of neonicotinoids as a single group of compounds for resistance management, as advocated by the Mode of Action Classification published by the Insecticide Resistance Action Committee (http://www.irac-online.org/documents/moa/MoAv5.1.doc) and as discussed elsewhere (Nauen and Denholm 2005).

Acknowledgements

We thank Ralf Nauen (Bayer CropSciences, Germany) and Masahiko Nakamura (Mitsui Chemicals, Japan) for generously providing neonicotinoid compounds used in this study. We are very grateful to Skevi Krahia and Lucia Sivilotti for their help and advice with oocyte electrophysiology. Research funding to Rothamsted Research and to UCL was provided by the Biotechnology and Biological Sciences Research Council. Zewen Lin was supported by a China Research Fellowship from The Royal Society.

Ancillary