Neonicotinoid insecticides: historical evolution and resistance mechanisms

The use ofneonicotinoid insecticides has grown considerably since their introduction in 1990s. They are used extensively for the control of agriculturally important crop pests and also in the control of cat and dog fleas. Imidacloprid exploited through an elaborated structural and substituent optimization of nithiazine was launched to market in 1990. The selectivity ofneonicotinoid compounds for insect species has been attributed to their binding on nicotinic acetylcholine receptors in which the negatively charged nitro- or cyano-groups of neonicotinoid compounds interact with a cationic subsite within insect nicotinic acetylcholine receptors. The first example of a pest evolving resistance to field use of neonicotinoids was Bemisia tabaci. Resistance to neonicotinoids can arise either through nAChR subtypes expression, detoxification mechanisms and/or structural alterations of target-site proteins. Consequently, a number of derivatives and analogues ofimidacloprid have been generated to date. In 1992, a new neonicotinoid using acetylcholine as the lead compound has been found. This was dinotefuran, which has a characteristic tetrahydro-3-furylmethyl group instead of the pyridine-like rings of others neonicotinoids

Characterization of nicotine acetylcholine receptor subunits in the cockroach Periplaneta americana mushroom bodies reveals a strong expression of β1 subunit: involvement in nicotine-induced currents

Nicotinic acetylcholine receptors are ligand-gated ion channels expressed in many insect structures, such as mushroom bodies, in which they play a central role. We have recently demonstrated using electrophysiological recordings that different native nicotinic receptors are expressed in cockroach mushroom bodies Kenyon cells. In the present study, we demonstrated that eight genes coding for cockroach nicotinic acetylcholine receptor subunits are expressed in the mushroom bodies. Quantitative real-time polymerase chain reaction (PCR) experiments demonstrated that β1 subunit was the most expressed in the mushroom bodies. Moreover, antisense oligonucleotides performed against β1 subunit revealed that inhibition of β1 expression strongly decreases nicotine-induced currents amplitudes. Moreover, co-application with 0.5 μM α-bungarotoxin completely inhibited nicotine currents whereas 10 μM d-tubocurarine had a partial effect demonstrating that β1-containing neuronal nicotinic acetylcholine receptor subtypes could be sensitive to the nicotinic acetylcholine receptor antagonist α-bungarotoxin

Agonist actions of clothianidin on synaptic and extrasynaptic nicotinic acetylcholine receptors expressed on cockroach sixth abdominal ganglion

Clothianidin is new neonicotinoid insecticide acting selectively on insect nicotinic acetylcholine receptors (nAChRs). Its effects on nAChRs expressed on cercal afferent/giant interneuron synapses and DUM neurons have been studied using mannitol-gap and whole-cell patch-clamp techniques, respectively. Bath-application of clothianidin-induced dose-dependent depolarizations of cockroach cercal afferent/giant interneuron synapses which were not reversed after wash-out suggesting a strong desensitization of postsynaptic interneurons at the 6th abdominal ganglion (A6). Clothinidin activity on the nerve preparation was characterized by an increased firing rate of action potentials which then ceased when the depolarization reached a peak. Clothianidin responses were insensitive to all muscarinic antagonists tested but were blocked by co-application of specific nicotinic antagonists methyllicaconitine, alpha-bungarotoxin and d-tubocurarine. In a second round of experiment, clothianidin actions were tested on DUM neurons isolated from the A6. There was a strong desensitization of nAChRs which was not affected by muscarinic antagonists, pirenzepine and atropine, but was reduced with nicotinic antagonist alpha-bungarotoxin. In addition, clothianidin-induced currents were completely blocked by methyllicaconitine suggesting that (1) clothianidin acted as a specific agonist of nAChR subtypes and (2) a small proportion of receptors blocked by MLA was insensitive to alpha-bungarotoxin. Moreover, because clothianidin currents were blocked by d-tubocurarine and mecamylamine, we provided that clothianidin was an agonist of both nAChRs: imidacloprid-sensitive nAChR1 and -insensitive nAChR2 subtypes

Thiamethoxam, a poor agonist of nicotinic acetylcholine receptors expressed on isolated cell bodies, acts as a full agonist at cockroach cercal afferent/giant interneuron synapses

Thiamethoxam (TMX) is a second-generation neonicotinoid which is known to induce toxic effects on insects and mammalians. Recently, it has been proposed that TMX is a poor agonist of insect nicotinic acetylcholine receptors (nAChRs) on isolated cell bodies. Here, we have studied its effect on synaptic transmission. Our results demonstrate that TMX acts as an agonist of nAChRs expressed on cockroach cercal afferent giant/interneuron synapses as bath applications of TMX induce a strong reversible depolarization of the sixth abdominal ganglion. This response was reduced by the nicotinic antagonists mecamylamine and methyllicaconitine, but was insensitive to d-tubocurarine. Interestingly, TMX-induced depolarization was partially reduced by the muscarinic antagonist atropine, suggesting that TMX could bind to a \u27mixed nicotinic/muscarinic\u27 receptor. Compared to previous studies, we proposed that TMX is able to act as agonist of insect nAChRs expressed at cercal afferent/giant interneuron synapses. Moreover, our results suggest that nAChRs expressed on synaptic ganglion are distinct to nAChRs expressed on isolated cell bodies and that synaptic receptors have higher affinity to TMX resulting to a depolarization of postsynaptic nicotinic receptors

Electrophysiological studies and pharmacological properties of insect native nicotinic acetylcholine receptors

The existence of several nicotinic acetylcholine receptor genes in insects suggests that many nicotinic receptor subtypes are present, but the identification and characterization of these subtypes in native neurons has been limited. Their pharmacological properties came from electrophysiological studies in which variations in the sensitivity of insect neurons were correlated with time course, current amplitudes, desensitization rates occurring in varying proportions in different cells. Thus pressure application of agonists on cultured cells induced inward currents showing that acetylcholine and nicotine were partial agonists of some cells with a lower efficacy while they were full agonists in other neurons. The variation in kinetics appeared to be due to differential expression of distinct nicotinic receptor subtypes as corroborated by the blocking activity induced by antagonists. In fact, the alpha-bungarotoxin-sensitive nicotinic receptor subtypes described as homomeric could be also heteromeric receptors. Interestingly, some receptors mediating nicotinic responses have been termed ‘mixed’ receptors because they were blocked by a range of nicotinic and muscarinic antagonists. Following electrophysiological studies, it has been also demonstrated that insect nicotinic receptors were modulated by Ca2+ pathways. Ca2+ permeability through insect nicotinic receptors, voltage-gated Ca2+ channels or released from intracellular stores represents an important indication of insect native nicotinic acetylcholine receptor modulation. The Ca2+ flow may trigger a variety of cytosolic Ca2+ pathways underlying many cellular processes such Calmodulin kinase, PKA and PKC. Most of the studies suggested that the effect of phosphorylation mechanism was dependent on the receptor subtype

Effect of calcium on nicotine-induced current expressed by an atypical alpha-bungarotoxin-insensitive nAChR2

Two distinct native alpha-bungarotoxin (alpha-Bgt)-insensitive nicotinic acetylcholine receptors (nAChRs), named nAChR1 and nAChR2, were identified in the cockroach Periplaneta americana dorsal unpaired median (DUM) neurons. They differed in their electrophysiological, pharmacological properties and intracellular regulation pathways. nAChR2 being an atypical nicotinic receptor closed upon agonist application and its current-voltage relationship resulted from a reduction in potassium conductance. In this study, using whole-cell patch-clamp technique, we demonstrated that calcium modulated nAChR2-mediated nicotine response. Under 0.5 microM alpha-Bgt and 20 mM d-tubocurarine, the nicotine-induced inward current amplitude was strongly reduced in the presence of intracellularly applied BAPTA or bath application of calcium-free solution. In addition, using cadmium chloride, we showed that nicotine response was modulated by extracellular calcium through plasma membrane calcium channels. Moreover, extracellular application of caffeine and thapsigargin reduced nAChR2-mediated response. Together these experiments revealed a complex calcium-dependent regulation of nAChR2

Neonicotinoid insecticides imidacloprid and clothianidin affect differently neural Kenyon cell death in the cockroach Periplaneta americana

The intracellular toxicity of the neonicotinoid insecticides imidacloprid and clothianidin was studied on cockroach Periplaneta americana Kenyon cells using the trypan blue exclusion test and the adenylate kinase (AK) detection reagent. To evaluate cytotoxicity. Kenyon cells were exposed to different concentrations (1, 5, 10, 50 and 100 mu M) of both imidacloprid and clothianidin at different delays (1, 3, 5, 8 and 24 h). Our data show that both imidacloprid and clothianidin decreased cell viability, with a more pronounced effect following imidacloprid exposure. Indeed, a significant decrease of cell viability was observed for 50 and 100 mu M imidacloprid at 8 and 24 h, with trypan blue exclusion test. Study of the AK activity revealed that 50 and 100 mu M imidacloprid induced an increase of AK activity, except for 50 mu M at 24 h whereas at the same concentrations, clothianidin induced a transient effect at 5 and 8 h. According to previous studies showing that imidacloprid was a partial agonist and clothianidin a full agonist of insect nicotinic acetylcholine receptors, we demonstrated that both imidacloprid and clothianidin were also able to induce distinct intracellular toxic effects