Comparative analysis of neonicotinoid binding to insect membranes: I. A structure-activity study of the mode of [3H]imidacloprid displacement in Myzus persicae and Aphis craccivora

Neonicotinoids bind selectively to insect nicotinic acetylcholine receptors with nanomolar affinity to act as potent insecticides. While the members of the neonicotinoid class have many structural features in common, it is not known whether they also share the same mode of binding to the target receptor. Previous competition studies with [3H]imidacloprid, the first commercialised neonicotinoid, indicated that thiamethoxam, representing a novel structural sub-class, may bind in a different way from that of other neonicotinoids. In the present work we analysed the mode of [3H]imidacloprid displacement by established neonicotinoids and newly synthesized analogues in the aphids Myzus persicae Sulzer and Aphis craccivora Koch. We found two classes of neonicotinoids with distinct modes of interference with [3H]imidacloprid, described as direct competitive inhibition and non-competitive inhibition, respectively. Competitive neonicotinoids were acetamiprid, nitenpyram, thiacloprid, clothianidin and nithiazine, whereas thiamethoxam and the N-methyl analogues of imidacloprid and clothianidin showed non-competitive inhibition. The chloropyridine or chlorothiazole heterocycles, the polar pharmacophore parts, such as nitroimino, cyanoimino and nitromethylene, and the cyclic or acyclic structure of the pharmacophore were not relevant for the mode of inhibition. Consensus structural features of the neonicotinoids were defined for the two mechanisms of interaction with [3H]imidacloprid binding. Furthermore, two sub-classes of non-competitive inhibitors can be discriminated on the basis of their Hill coefficients for imidacloprid displacement. We conclude from the present data that the direct competitors share the binding site with imidacloprid, whereas non-competitive compounds, like thiamethoxam, bind to a different site or in a different mode.     

Efficacy of plant metabolites of imidacloprid against Myzus persicae and Aphis gossypii (Homoptera: Aphididae)

The metabolism of the chloronicotinyl insecticide imidacloprid is strongly influenced by the method of application. Whilst in foliar application most of the residues on the leaf surface display unchanged parent compound, most of the imidacloprid administered to plants by soil application or seed treatment is metabolized more or less completely, depending on plant species and time. The present study revealed that certain metabolites of imidacloprid which have been described in crop plants are highly active against aphid pests in different types of bioassays. Some of these metabolites showed a high oral activity against the green peach aphid (Myzus persicae), and the cotton aphid (Aphis gossypii). The aphicidal potency of the metabolites investigated was weaker in aphid dip tests than in oral ingestion bioassays using artificial double membranes. The most active plant metabolite was the imidazoline derivative of imidacloprid. The LC₅₀ values of this metabolite for M. persicae and A. gossypii in oral ingestion bioassays were in the lower ppb-range, i.e. 0·0044 and 0·0068 mg litre^-1, respectively. Most of the other reported metabolites showed much weaker activity. Compared to imidacloprid, the imidazoline derivative showed superior affinity to housefly (Musca domestica) head nicotinic acetylcholine receptors, while all other metabolites were less specific than imidacloprid. It seems possible that, after seed treatment or soil application, a few of the biologically active metabolites arising are acting in concert with remaining levels of the parent compound imidacloprid, thus providing good control and long-lasting residual activity against plant-sucking pests in certain crops. © 1998 SCI.     

Insecticidal and binding activities of N3-substituted imidacloprid derivatives against the housefly Musca domestica and the alpha-bungarotoxin binding sites of nicotinic acetylcholine receptors

N3-substituted imidacloprid congeners containing C1-C6 alkyl groups or various analogous groups, and their corresponding nitromethylene analogues, were used in this study. Their insecticidal activity against the housefly, Musca domestica, and their binding activity toward the nicotinic acetylcholine receptor were determined. The insecticidal test was conducted using the synergists piperonyl butoxide and propargyl propyl phenylphosphonate. The binding assay was performed with housefly head membrane preparations using radio-labelled alpha-bungarotoxin. Both insecticidal and binding activities were drastically lowered by the introduction of alkyl/allyl groups at the imidazolidine NH sites of both nitroimino and nitromethylene compounds. The binding activity of N3-substituted nitromethylene analogues was much higher than that of the corresponding nitroimino analogues. However, the insecticidal activity of both series of compounds with a given substituent was nearly identical. The insecticidal activity correlated positively with the binding activity after taking into account the structural difference of the nitroimino and nitromethylene moieties and a structural feature of the N3-substituents.     

Antifeedant effect,biological efficacy and high affinity binding of imidacloprid to acetylcholine receptors in Myzus persicae and Myzus nicotianae

It is known from laboratory studies that tobacco-associated forms of Myzus persicae (Sulzer) and the closely related tobacco aphid Myzus nicotianae (Blackman) are often somewhat less susceptible to imidacloprid than non-tobacco strains of M. persicae. Choice tests (floating leaf technique) showed that tobacco aphids were also less susceptible to the antifeedant potential of imidacloprid in contact bioassays. Synergists like piperonyl butoxide or DEF did not enhance the susceptibility of tobacco-associated morphs of Myzus ssp. to imidacloprid, thus providing evidence that neither oxidative detoxication nor hydrolytic metabolization took place. However, in an attempt to study the influence of endosymbiotic bacteria on the efficacy of imidacloprid, we allowed small populations of tobacco aphids to feed on diets containing the antibiotic chlortetracycline prior to imidacloprid treatment. While the effectiveness of imidacloprid, i.e. lower LC₅₀ values, could be improved in all strains, including the susceptible reference strain, there was no change in overall tolerance factors. In order to investigate any possible alteration of the target site, the affinity of imidacloprid and nicotine to nicotinic acetylcholine receptors in whole-aphid homogenates was measured. All strains (and clones) showed the same high-affinity binding sites and no detectable difference. Studies using the FAO dip method revealed that the lower susceptibility of M. nicotianae is not restricted to chloronicotinyls like imidacloprid or acetamiprid, because other insecticides with different modes of action such as pymetrozine and fipronil were also affected in laboratory studies. It is considered that the observed tolerance to chloronicotinyls in certain strains of Myzus ssp. is a natural variation in response, probably not coupled with any known mechanism of resistance in this species complex. © 1998 SCI     

Comparative analysis of neonicotinoid binding to insect membranes: II. An unusual high affinity site for [3H]thiamethoxam in Myzus persicae and Aphis craccivora

Neonicotinoids represent a class of insect-selective ligands of nicotinic acetylcholine receptors. Imidacloprid, the first commercially used neonicotinoid insecticide, has been studied on neuronal preparations from many insects to date. Here we report first intrinsic binding data of thiamethoxam, using membranes from Myzus persicae Sulzer and Aphis craccivora Koch. In both aphids, specific binding of [3H]thiamethoxam was sensitive to temperature, while the absolute level of non-specific binding was not affected. In M persicae, binding capacity (Bmax) for [3H]thiamethoxam was ca 450 fmol mg(-1) of protein at 22 degrees C and ca 700 fmol mg(-1) of protein at 2 degrees C. The negative effect of increased temperature was reversible and hence not due to some destructive process. The affinity for [3H]thiamethoxam was less affected by temperature: Kd was ca 11 nM at 2 degrees C and ca 15 nM at 22 degrees C. The membranes also lost binding sites for [3H]thiamethoxam during prolonged storage at room temperature, and upon freezing and thawing. In A craccivora, [3H]thiamethoxam was bound with a capacity of ca 1000 fmol mg(-1) protein and an affinity of ca 90 nM, as measured at 2 degrees C. Overall, the in vitro temperature sensitivity of [3H]thiamethoxam binding was in obvious contrast to the behaviour of [3H]imidacloprid studied in parallel. Moreover, the binding of [3H]thiamethoxam was inhibited by imidacloprid in a non-competitive mode, as shown with M persicae. In our view, these differences demonstrate that thiamethoxam and imidacloprid, which represent different structural sub-classes of neonicotinoids, do not share the same binding site or mode. This holds also for other neonicotinoids, as we report in a companion article.     

吡虫啉亚致死剂量处理对桃蚜乙酰胆碱酯酶的时间与剂量效应

采用微量滴度酶标板法测定了吡虫啉亚致死剂量对桃蚜乙酰胆碱酯酶(AChE)的影响。结果表明,用不同亚致死剂量吡虫啉处理桃蚜后AChE的活力明显降低,表现为抑制作用,且不同剂量间AChE的活力差异显著;酶动力学分析表明,AChE的K_m值显著降低,而V_max显著升高,表明吡虫啉亚致死剂量处理桃蚜后AChE与底物的亲和力明显增强,且最大反应速度显著加快。用LC₁₀的吡虫啉处理桃蚜后AChE的活力随时间的延长而变化,且表现为明显的抑制作用;AChE的K_m随时间的变化表现为先升高后降低的趋势,但V_max均升高。此外研究了LC₁₀的吡虫啉处理桃蚜后AChE在不同亚细胞层的分布特征,结果表明,AChE比活力的高低次序依次为:微粒体>细胞核与细胞碎片>线粒体>细胞质液;吡虫啉亚致死剂量处理后虽然没有影响AChE在亚细胞中的分布,但是却改变了对底物的亲和力和最大反应速度。     

Neural actions of imidacloprid and their involvement in resistance in the Colorado potato beetle, Leptinotarsa decemlineata (Say)

BACKGROUND: The development of resistance to imidacloprid in eastern US populations of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), threatens this critical use for neonicotinoid insecticides. Previous pharmacokinetic studies with resistant adult CPBs provided no explanation for the high resistance level (over 200-fold) to topically applied imidacloprid. The authors assessed the neural activity of imidacloprid by recording spontaneous activity from a motor nerve leaving the isolated central nervous system to compare the sensitivity of the latter to imidacloprid between susceptible and resistant CPBs. RESULTS: On the isolated central nervous system, imidacloprid was initially neuroexcitatory, and neuroinhibitory at higher concentrations. The neuroexcitatory action of imidacloprid was blocked by coapplication of a specific nAChR antagonist, methyllycaconitine, indicating that it is a result of action on nAChRs. The sensitivity to the neuroexcitatory and inhibitory activities of imidacloprid varied independently among individuals in each population. The sensitivity of the central nervous system of resistant CPBs to excitation by imidacloprid did not differ from that of susceptible insects, but the sensitivity to inhibition by imidacloprid was reduced 52- to 58-fold, indicating a possible change in the sensitivity of at least one subgroup of nAChRs. CONCLUSION: This study provides evidence that reduced nerve sensitivity to the blocking action of imidacloprid is associated with imidacloprid resistance in the CPB.     

Nicotinic acetylcholine receptor chimeras of rat α7 and Drosophila SAD reveal species-specific agonist binding regions

Species-specific agonist binding regions of nicotinic acetylcholine receptors (nAChR) were examined. Imidacloprid and physostigmine (Phy) selectively activated insect nAChR composed of Drosophila second alpha-like subunit (SAD) and chick β2, in contrast to rat α7 nAChR. The Phy-activated currents were α-bungarotoxin (α-BGT) sensitive, suggesting activation at the agonist binding loop C. Several SAD-α7 chimeras were constructed, by switching agonist binding regions, and expressed in oocytes. Though none of the chimeras was activated by a range of nicotinic agonists, [¹²⁵I]α-BGT binding revealed homomeric assembly of all chimeric cDNAs. Phy differentially displaced [¹²⁵I]α-BGT from the nAChR chimeras, suggesting that the β subunit is not involved in Phy binding, and that Phy targets the insect agonist binding loop C.     

Insecticidal activity and nicotinic acetylcholine receptor binding of dinotefuran and its analogues in the housefly, Musca domestica

The insecticidal activity of dinotefuran and 23 related compounds against the housefly, Musca domestica (L) was measured by injection with metabolic inhibitors. Dinotefuran was less active than imidacloprid and clothianidin by a factor of 10 in molar concentrations. Their binding activities to the fly-head membrane preparation were measured by using [125I]alpha-bungarotoxin ([125I]alpha-BGTX) and [3H]imidacloprid ([3H]IMI) as radioligands. The activity of some selected compounds measured with [3H]IMI was 10(4)-fold higher than that measured with [125I]alpha-BGTX. With [3H]IMI as a radioligand, dinotefuran was 13-fold less active than imidacloprid. The inhibitory effect of dinotefuran on the binding of [3H]IMI to the membrane preparation was in a competitive manner. Quantitative analysis of the insecticidal activity of the test compounds with the binding activity measured with [3H]IMI showed that the higher the binding activity, the higher was the insecticidal activity.     

Toxicity and nicotinic acetylcholine receptor interaction of imidacloprid and its metabolites in Apis mellifera (Hymenoptera: Apidae)

Acute oral and contact toxicity tests of imidacloprid, an insecticide acting agonistically on nicotinic acetylcholine receptors (nAChR), to adult honeybees, Apis mellifera L var carnica, were carried out by seven different European research facilities. Results indicated that the 48-h oral LD50 of imidacloprid is between 41 and > 81 ng per bee, and the contact LD50 between 49 and 102 ng per bee. The ingested amount of imidacloprid-containing sucrose solution decreased with increasing imidacloprid concentrations and may be attributed to dose-related sub-lethal intoxication symptoms or to antifeedant responses. Some previously reported imidacloprid metabolites occurring at low levels in planta after seed dressing, i.e. olefine-, 5-OH- and 4,5-OH-imidacloprid, showed lower oral LD50 values (> 36, > 49 and 159 ng per bee, respectively) compared with the concurrently tested parent molecule (41 ng per bee). The urea metabolite and 6-chloronicotinic acid (6-CNA) exhibited LD50 values of > 99,500 and > 121,500 ng per bee, respectively. The pharmacological profile of the [3H]imidacloprid binding site in honeybee head membrane preparations is consistent with that anticipated for a nAChR. IC50 values for the displacement of [3H]imidacloprid by several metabolites such as olefine, 5-OH-, 4,5-OH-imidacloprid, urea and 6-CNA were 0.45, 24, 6600, > 100,000, and > 100,000 nM, respectively. Displacement of [3H]imidacloprid by imidacloprid revealed an IC50 value of 2.9 nM, thus correlating well with the observed acute oral toxicity of the compounds in honeybees. Neurons isolated from the antennal lobe of A mellifera and subjected to whole-cell voltage clamp electrophysiology responded to the application of 100 microM acetylcholine with a fast inward current of between 30 and 1600 pA at -70 mV clamp potential. Imidacloprid and two of the metabolites (olefine- and 5-OH-imidacloprid) acted agonistically on these neurons, whereas the others did not induce currents at test concentrations up to 3 mM. The electrophysiological data revealed Hill coefficients of approximately 1, indicating a single binding site responsible for an activation of the receptor and no direct cooperativity or allosteric interaction with a second binding site.     

Monitoring for imidacloprid resistance in the tobacco‐adapted form of the green peach aphid,Myzus persicae (Sulzer) (Hemiptera: Aphididae), in the eastern United States

BACKGROUND: Imidacloprid is the primary insecticide for controlling the tobacco‐adapted form of the green peach aphid (TGPA), Myzus persicae (Sulzer), a major pest of tobacco worldwide. This study used leaf‐dip bioassays to assess TGPA resistance to imidacloprid in the eastern United States from 2004 through 2007. RESULTS: When combined over the 4 year study, 18, 14 and 3% of the TGPA had imidacloprid resistance ratios (RRs) of 10–20‐fold, 20–30‐fold and 30–90‐fold, respectively, compared with the most susceptible colony tested. This indicates that some colonies have developed moderate levels of resistance to imidacloprid. A colony collected near Clayton, North Carolina, had the highest RR of 91 (LC₅₀ value = 31 mg L^?1). This resistance declined for six tests over a 3 year period in the laboratory culture from >130‐fold RR (LC₅₀ = 48 mg L^?1) to 40‐fold RR (LC₅₀ = 15 mg L^?1). Over the same period, the most susceptible colony and a standard colony not exposed to imidacloprid for over 7 years had consistently low LC₅₀ values. CONCLUSION: Moderate levels of resistance to imidacloprid are noticed among TGPA colonies from the eastern United States. The variation in resistance indicates that the factors responsible are present in the populations at low frequencies and are just not enough to cause field failures yet. Copyright © 2010 Society of Chemical Industry     

Prediction of the binding mode of imidacloprid and related compounds to house-fly head acetylcholine receptors using three-dimensional QSAR analysis

The binding activity of imidacloprid and related compounds to nicotinic acetylcholine receptors (nAChR) of house flies was measured by use of radioactive α-bungarotoxin as a ligand. Variations in the activity were examined three-dimensionally using comparative molecular field analysis (CoMFA). The CoMFA results suggest that one conformer among the four stable ones is active and provide support for one of the proposed binding models for this class of compound, in which the nitrogen atom of the pyridine ring and the nitrogen atom at the 1-position of the imidazolidine ring interact with the hydrogen-donating and electron-rich sites of nAChR, respectively. The CoMFA field map showed that the nitroimino moiety and a portion of the imidazolidine ring were mainly surrounded by a sterically and electrostatically sensitive region of nAChR. © 1998 Society of Chemical Industry     

Ligands of the nicotinic acetylcholine receptor as insecticides

Insect nicotinic acetyl receptors (nAChR) are targets of growing importance and, since the early 1990s, the number of such highly effective insecticides as imidacloprid and spinosyn has grown. Several natural compounds, eg dihydro-β-erythroidine, methyl caconitine and paraherquamide, showing high affinity to the same receptor, were considerably less active as insecticides, most likely because of their antagonistic action. Our observations on aphids after ingestion of the antagonistic compound dihydro-β-erythroidine revealed anti-feedant-like properties. As a consequence, the symptomology of poisoning was totally different between agonists and antagonists of the nAChR. Electrophysiological (whole-cell voltage clamp) measurements in isolated housefly neurones revealed that agonism seems to be a prerequisite for insecticidal activity. Furthermore, we were able to demonstrate the existence of two different subtypes of the nAChR in isolated locust neurones with different pharmacology and ion-channel properties.     

Role of the α subunit of nicotonic acetylcholine receptor in the selective action of imidacloprid

Examination of agonist interactions of imidacloprid on recombinant chicken α4β2 and Drosophila SAD/Chicken β2 hybrid receptors, expressed in Xenopus oocytes by nuclear injection of the cDNAs, indicates that imidacloprid is a partial agonist. Replacement of the α4 subunit for the Drosophila SAD subunit lowered the imidacloprid EC₅₀ 37-fold, whereas EC₅₀s for other agonists increased 4-50 fold, suggesting that the α subunit contributes to the high affinity of insect nicotonic receptors for imidacloprid. ©1999 Society of Chemical Industry