Characterization of nicotinic acetylcholine receptors from the insectsAphis craccivora,Myzus persicae, andLocusta migratoria by radioligand binding assays: Relation to thiamethoxam action

Abstract

Thiamethoxam, a new neonicotinoid insecticide acting at nicotinic acetylcholine receptors, was characterized in competition binding assays with [3‐H]‐imidacloprid, a specific nicotinic ligand, using membranes from the aphids Aphis craccivora and Myzus persicae, and from the locust Locusta migratoria. In all insects, Scatchard analysis suggested two binding sites for imidacloprid with Kd values in the range of 1 nM and 10 nM, respectively. The Hill values were significantly below 1 (range of 0.63 to 0.85). In contrast to imidacloprid and nicotine, the potency of thiamethoxam to displace [3‐H]‐imidacloprid varied considerably among these insects. Thiamethoxam was more active than nicotine on Aphis receptors but 100‐fold less in Locusta, a nontarget insect. Comparable relations were found to nithiazine. In Myzus, the inhibition curve for thiamethoxam was shallow. This suggested a heterogeneous receptor population displaying a range of binding affinities to thiamethoxam in this aphid. In all three insects, the other neonicotinoid insecticides studied competed with [3‐H]‐imidacloprid in the same order: thiacloprid > imidacloprid ≥ acetamiprid > nitenpyram. N‐Methylation of imidacloprid strongly reduced the affinity to the imidacloprid site, whereas N‐demethylation of thiamethoxam resulted in a comparable increase of affinity. Supplementary assays were performed with (−)‐[3‐H]‐nicotine and [3‐H]‐α‐bungarotoxin on locust membranes. Overall, the data suggested that the outstanding insecticidal properties of thiamethoxam may be due to either a different binding site on nicotinic receptors, or receptor isoforms, or specific pharmakokinetic behavior, rather than to exceptional affinity to one of the examined binding sites. © 2000 John Wiley & Sons, Inc. J Biochem Toxicol 14:221–230, 2000

The invertebrate pharmacology of insecticides acting at nicotinic acetylcholine receptors

“…A separate detailed study in M. persicae and Aphis craccivora (cow pea aphid) confirmed this observation. 67) However, in this study, the presence of a very high affinity binding site in neuronal tissue from Locusta migratoria (migratory locust) was also found. More recently, the presence of dual affinity binding sites for [ 3 H]-IMI has also been observed in another hemipteran species, Nilaparvarta lugens (brown plant hopper) further solidifying the argument that hemipteran pests have two binding sites.…”

“…This clearly implies a different mode of binding and/or channel modulation for DNF in comparison to the other neonicotinoids. 21,67) As demonstrated in Xenopus oocytes with wildtype Nlα1, DNF clearly has the potential to interact at the same nAChRs as the other neonicotinoids, although (as observed with TMX) the influence of native insect β receptors is unknown. This data strongly suggests that the specific amino acids of the nAChR that are involved in DNF recognition and/or channel modulation have differences to that of the other neonicotinoids.…”

“…68,69) Certainly, not all studies on hemipteran pests have identified dual binding sites and this is likely to be down to the difficult nature of the experiments to tease out the very high affinity but low abundance site, as well as differences in interpretation of the data. 67,70) However, a general trend appears to be that when low % Triton X-100 is included in incubations the duality of binding disappears and only a single binding site (low nM K d ) is observed in aphid and fly species. 66,71) The effects of including detergent are complex and not easily rationalised but low μM concentrations of Triton may interfere with [ 3 H]-IMI binding.…”

“…62,65) It has been suggested that two sites for IMI binding maybe the norm, but this certainly requires further evidence for insects outside of the hemipteran order. 67,71,72) Strengthening the idea that hemipteran neonicotinoidresponsive nAChRs display differences compared to other insect orders, Casida applied a photoaffinity-based approach using an azido non-cyclic N-methyl derivative of IMI and demonstrated that the nAChR subunits isolated differed significantly in molecular weight between hemipteran and dipteran insects. 73,74) The subunits required to form the two distinct IMI binding sites in a hemipteran pest have been reported.…”

Insect Nicotinic Acetylcholine Receptor: Conserved Neonicotinoid Specificity of [3H]Imidacloprid Binding Site

“…Four buffers were based on earlier studies Wiesner and Kayser, 2000): aphid membrane isolation buffer, consisting of 20 mM Na 2 HPO 4 (adjusted to pH 7.0) containing 150 mM NaCl, 1 mM EDTA, and protease inhibitors (1 M chymostatin, 1 M leupeptin, 1 M pepstatin, and 100 M phenylmethanesulfonyl fluoride); fly membrane isolation buffer, consisting of 100 mM Na 2 HPO 4 (adjusted to pH 7.4) containing 0.32 M sucrose, 0.1 mM EDTA, 1 M leupeptin, 1 M pepstatin, and 100 M phenylmethanesulfonyl fluoride used with and without Triton X-100 (0.1% wt/wt); binding buffer, consisting of 10 mM Na 2 HPO 4 (adjusted to pH 7.4) containing 50 mM NaCl used with and without Triton X-100 (0.1% wt/wt); and washing buffer, consisting of 10 mM Na 2 HPO 4 (adjusted to pH 7.4) containing 50 mM NaCl and no Triton X-100. Solutions and serial dilutions of test compounds were made in binding buffer (with no Triton X-100) and ethanol (final concentration, Ͻ0.05% vol/vol).…”