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Nicotinic acetylcholine receptors are an important class of excitatory receptors in the central nervous system of arthropods. In the ticks Ixodes ricinus, the functional and pharmacological properties of nicotinic receptors located in their neurons are still unknown. The objective of this study was to characterize the pharmacological properties of tick nicotinic receptors using membrane microtransplantation in Xenopus laevis oocytes and two-electrodes voltage clamp method. The membranes microtransplanted were extracted from the tick synganglion. We found that oocytes microtransplanted with tick synganglion membranes expressed nicotinic acetylcholine receptor subtypes which were activated by acetylcholine (1 mM) and nicotine (1 mM). Currents induced by pressure application of acetylcholine and nicotine were diminished by 10 nM α-bungarotoxin and methyllycaconitine, suggesting that they expressed two subtypes of nicotinic receptors, α-bungarotoxin-sensitive and -insensitive, respectively. In addition, we found that nicotine receptors expressed in the synganglion membranes were poorly sensitive to the neonicotinoid insecticides clothianidin (CLT), imidacloprid (IMI), acetamiprid (ACE) and thiamethoxam (TMX), in agreement with their lack of activity as acaricides. Interestingly, current amplitudes were strongly potentialized in the presence of 1 μM PNU-120596. CLT was more active as an agonist than IMI, TMX and ACE. Finally, we demonstrated that microtransplantation of purified membrane from the tick synganglion can be a valuable tool for the development and screening of compounds targeting tick nicotinic acetylcholine receptor subtypes., Graphical abstract Image 1

The functional expression of the cockroach Pameα7 nicotinic acetylcholine receptor subunit has been previously studied, and was found to be able to form a homomeric receptor when expressed in Xenopus laevis oocytes. In this study, we found that the neonicotinoid insecticide imidacloprid is unable to activate the cockroach Pameα7 receptor, although thiacloprid induces low inward currents, suggesting that it is a partial agonist. In addition, the co-application or 5 min pretreatment with 10 µM imidacloprid increased nicotine current amplitudes, while the co-application or 5 min pretreatment with 10 µM thiacloprid decreased nicotine-evoked current amplitudes by 54% and 28%, respectively. This suggesting that these two representatives of neonicotinoid insecticides bind differently to the cockroach Pameα7 receptor. Interestingly, the docking models demonstrate that the orientation and interactions of the two insecticides in the cockroach Pameα7 nAChR binding pocket are very similar. Electrophysiological results have provided evidence to suggest that imidacloprid and thiacloprid could act as modulators of the cockroach Pameα7 receptors.

International audience; Neonicotinoid insecticides are used worldwide and have been demonstrated as toxic to beneficial insects such as honeybees. Their effectiveness is predominantly attributed to their high affinity for insect neuronal nicotinic acetylcholine receptors (nAChRs). Mammalian neuronal nAChRs are of major importance because cholinergic synaptic transmission plays a key role in rapid neurotransmission, learning and memory processes, and neurodegenerative diseases. Because of the low agonist effects of neonicotinoid insecticides on mammalian neuronal nAChRs, it has been suggested that they are relatively safe for mammals, including humans. However, several lines of evidence have demonstrated that neonicotinoid insecticides can modulate cholinergic functions through neuronal nAChRs. Major studies on the influence of neonicotinoid insecticides on cholinergic functions have been conducted using nicotine low-affinity homomeric α7 and high-affinity heteromeric α4β2 receptors, as they are the most abundant in the nervous system. It has been found that the neonicotinoids thiamethoxam and clothianidin can activate the release of dopamine in rat striatum. In some contexts, such as neurodegenerative diseases, they can disturb the neuronal distribution or induce oxidative stress, leading to neurotoxicity. This review highlights recent studies on the mode of action of neonicotinoid insecticides on mammalian neuronal nAChRs and cholinergic functions.

Some quinuclidine benzamide compounds have been found to modulate nicotinic acetylcholine receptors in both mammals and insects. In particular, the quaternarization of 3-amino quinuclidine benzamide derivatives with dichloromethane gave charged N-chloromethylated quinuclidine compounds, disclosing an antagonist profile on homomeric α7 nAChRs. Here, we synthesized and studied the toxicological effect of LMA10233, a quinuclidine-borane complex analogue, the LMA10233, on the pea aphid Acyrthosiphon pisum and found that LMA10233 only exhibit proper toxicity on A. pisum larvae when applied in concentrations of over 10 μg/ml. We assessed the ability of LMA10233 to enhance the toxicity of different insecticides. When a sublethal concentration of LMA10233 was combined with the LC10 of each compound, we found a strong increase in toxicity at 24 h and 48 h of exposure for clothianidin, fipronil and chlorpyrifos, and only at 24 h for imidacloprid, acetamiprid and deltamethrin. However, when the pesticide was used at the LC50, only acetamiprid showed a synergistic effect with LMA10233. When the concentration of LMA10233 was decreased, we found that up to 80–90% of mortality was obtained due to the synergism between acetamiprid and LMA10233. No similar effect was observed with other insecticides. We conclude that such quinuclidine-borane complex compounds could increase the toxic effect of insecticides at low concentrations.

International audience; Structural features and selected physicochemical properties of four common pesticides: acetamiprid (neonicotinoid), chlorpyriphos (organophosphate insecticide), deltamethrin (pyrethroid) and fipronil (phenylpyrazole) have been investigated by Density Functional Theory quantum chemical calculations. The high flexible character of these insecticides is revealed by the numerous conformers obtained, located within a 20 kJ mol(-1) range in the gas phase. In line with this trend, a redistribution of the energetic minima is observed in water medium. Molecular electrostatic potential calculations provide a ranking of the potential interaction sites of the four insecticides. The theoretical studies reported in the present work are completed by comparative toxicological assays against three aphid strains. Thus, the same toxicity order for the two susceptible strains Myzus persicae 4106A and Acyrthosiphon pisum LSR1: acetamiprid > fipronil > deltamethrin > chlorpyriphos is revealed. In the resistant strain M. persicae 1300145, the toxicity order is modified: acetamiprid > fipronil > chlorpyriphos > deltamethrin. Interestingly, the strain 1300145 which is known to be resistant to neonicotinoids, is also less sensitive to deltamethrin, chlorpyriphos and fipronil. (C) 2015 Elsevier Ltd. All rights reserved.

International audience; The modes of action of most insecticides are known, but little information exists regarding the toxicological interactions involving insecticide mixtures at low doses. The effects of mixtures of four insecticides were investigated using LC10 values (concentration leading to 10% mortality), acetamiprid (ACE, 0.235 mu g/mL), chlorpyriphos (CHL, 107.0 mu g/mL), deltamethrin (DEL, 5.831 mu g/mL), and fipronil (FIP, 3.775 mu g/mL) on the larvae of the pea aphid, Acyrthosiphon pisum. After 24 h exposure, 6 of the 11 tested combinations, DEL/FIP, ACE/DEL, CHL/FIP, ACE/DEL/FIP, ACE/CHL/FIP, and ACE/DEL/CHL/FIP, were toxic through an additive effect. Four combinations, ACE/FIP, DEL/CHL, ACE/CHL, and ACE/DEL/CHL had a synergistic effect, whereas only one DEL/CHL/FIP showed an antagonistic effect. The toxic effect of these mixtures was confirmed after 48 h of exposure, revealing an enhanced toxicity of CHL, DEL, and FIP in combination with ACE. We suggest that an insect pest management strategy should be evaluated in the future using different combinations of insecticides.

International audience; Here, we describe the synthesis of two new fluorescent derivatives of thiamethoxam and compared their toxicity on aphid Acyrthosiphon pisum and their mode of action on insect nicotinic acetylcholine receptors expressed on the sixth abdominal ganglion. The compound 3 with two 2-chlorothiazole moieties was found to be more toxic using toxicological bioassays 24 h and 48 h after exposure while compound 4 appeared more active using cockroach ganglionic depolarization. Interestingly, thiamethoxam appeared more effective than component 3 and 4, respectively. Our results demonstrated that component 3 and 4 act as agonists of insect nicotinic acetylcholine receptors

Cellular responses to Ca2+ require intermediary proteins such as calcium/calmodulin-dependent protein kinase II (CaMKII), which transduces the signal into downstream effects. We recently demonstrated that the cockroach genome encodes five different CaMKII isoforms, and only PaCaMKII-E isoform is specifically expressed in the dorsal unpaired median neurosecretory cells. In the present study, using antisense oligonucleotides, we demonstrated that PaCaMKII-E isoform inhibition reduced nicotine-induced currents through α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptor subtypes. Specifically, PaCaMKII-E isoform is sufficient to repress nicotinic current amplitudes as a result of its depression by antisense oligonucleotides. Similar results were found using the neonicotinoid insecticide clothianidin, which acted as a full agonist of dorsal unpaired median neuron nicotinic acetylcholine receptors. Clothianidin current amplitudes are strongly reduced under bath application of PaCaMKII-E antisense oligonucleotides but no significant results are found with α-bungarotoxin co-applied, demonstrating that CaMKII-E isoform affects nicotine currents through α-bungarotoxin-sensitive and -insensitive receptor subtypes whereas clothianidin currents are reduced via α-bungarotoxin-insensitive receptors. In addition, we found that intracellular calcium increase induced by nicotine and clothianidin were reduced by PaCaMKII-E antisense oligonucleotides, demonstrating that intracellular calcium increase induced by nicotine and clothianidin are affected by PaCaMKII-E inhibition. Cellular responses to Ca2+ require intermediary proteins such as calcium/calmodulin-dependent protein kinase II (CaMKII). We recently demonstrated that the cockroach genome encodes five different CaMKII isoforms and only PaCaMKII-E isoform was specifically expressed in the dorsal unpaired median neurosecretory cells. Here we show that specific inhibition of PaCaMKII-E isoform is associated with a decrease in nicotine- and clothianidin-induced currents. In addition, analysis of calcium changes demonstrates that PaCaMKII-E inhibition induces a decrease in intracellular calcium concentration.

International audience; Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [H-3]-IMI (Kd of 0.16 +/- 0.04 nM and 41.7 +/- 5.9 nM) and for the nicotinic antagonist [I-125]-alpha- bungarotoxin (Kd of 0.008 +/- 0.002 nM and 1.135 +/- 0.213 nM). Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [I-125]-alpha-bungarotoxin binding sites while CLT affinity was similar for both [I-125]-alpha-bungarotoxin and [H-3]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 mu g/ml) and TMX (LC50 = 0.034 mu g/ml) were more toxic than CLT (LC50 = 0.118 mu g/ml). The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisum alpha 10 and Apisum beta 1 expressions levels, whereas Apisum beta 2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies