Corrigendum: Cloning and expression of cockroach α7 nicotinic acetylcholine receptor subunit.

[This corrects the article DOI: 10.3389/fphys.2020.00418.].

Effect of Acetamiprid, a Neonicotinoid Insecticide, on Locomotor Activity of the American Cockroach.

Toxicological studies have shown that the American cockroach Periplaneta americana (Linnaeus) is a classical model for studying the mode of action of commonly used insecticides. In a previous study, we demonstrated that thiamethoxam and clothianidin decreased locomotor activity in an open-field-like apparatus. Here, we tested the effect of the neonicotinoid acetamiprid when applied orally, topically, or injected into the haemolymph. We found that acetamiprid was also able to impair locomotor activity in the open-field-like apparatus. When treated with acetamiprid, a strong alteration in locomotor activity was observed 1 h, 24 h, and 48 h after haemolymph and topical applications. Oral application induced an impairment of locomotor activity at 24 h and 48 h. A comparison of the present data with our previously published results showed that neonicotinoids were more active when injected into the haemolymph compared to oral and topical applications. These findings increased our understanding of the effect of neonicotinoid insecticides on insect locomotor activity, and demonstrated that the cyano-substituted neonicotinoid, acetamiprid, was able to alter cockroach locomotor activity.

Isolation and electrophysiological recording of Ixodes ricinus synganglion neurons.

The central nervous system of hard ticks (Ixodidae) consists of a concentrated merged nerve mass known as the synganglion. Although knowledge of tick neurobiology has dramatically improved over the last two decades, this is the first time that isolation and electrophysiological recordings have been carried out on tick neurons from the synganglion. Method: We developed a simple protocol for synganglion neuron isolation and used a whole-cell patch clamp to measure ionic currents induced by acetylcholine, nicotine and muscarine. Relatively large neurons (∼ 25 µm and âˆ¼ 35 µm) were isolated and 1 mM acetylcholine was used to induce strong inward currents of -0.38 ± 0.1 nA and - 1.04 ± 0.1 nA, respectively, with the corresponding cell capacitances being at around 142 pF and 188 pF. In addition, successive application of 1 mM acetylcholine through ∼25 µm and âˆ¼ 35 µm cells for increasing amounts of time resulted in a rapid reduction in current amplitudes. We also found that acetylcholine-evoked currents were associated with a reversible increase in intracellular calcium levels for each neuronal type. In contrast, 1 mM muscarine and nicotine induced a strong and non-reversible increase in intracellular calcium levels. This study serves as a proof of concept for the mechanical isolation of tick synganglion neurons followed by their electrophysiological recording. This approach will aid investigations into the pharmacological properties of tick neurons and provides the tools needed for the identification of drug-targeted sites and effective tick control measures.

Identification of sulfonamide compounds active on the insect nervous system: Molecular modeling, synthesis and biological evaluation.

Insect nicotinic acetylcholine receptors (nAChRs) are a recognized target for insecticide design. In this work, we have identified, from a structure-based approach using molecular modeling tools, ligands with potential selective activity for pests versus pollinators. A high-throughput virtual screening with the Openeye software was performed using a library from the ZINC database, thiacloprid being used as the target structure. The top sixteen molecules were then docked in α6 cockroach and honeybee homomeric nAChRs to check from a theoretical point of view relevant descriptors in favor of pest selectivity. Among the selected molecules, one original sulfonamide compound has afterward been synthesized, together with various analogs. Two compounds of this family have been shown to behave as activators of the cockroach cholinergic synaptic transmission.

Effect of the combination of DEET and flupyradifurone on the tick Ixodes ricinus: Repellency bioassay and pharmacological characterization using microtransplantation of synganglion membranes.

Ticks are vectors of many human and animal pathogens, and represent a major threat to public health. In recent years, an increase in tick-borne diseases has been observed, and new strategies are therefore needed in order to control tick numbers and reduce human tick bites. In the present study, we adapted the previous tick repellency bioassay based on the exploration behavior of the tick, using the ToxTrac software and video-tracking, to compare the repellent effect of two compounds on the tick Ixodes ricinus: N,N-diethyl-methyl-m-toluamide (DEET), and butenolide, flupyradifurone (FLU). We found that when applied alone, 10% DEET or FLU have no/or low repellency effect. But, the combination of both 10% DEET and FLU demonstrated a significant repellency effect against I. ricinus, similar to the repellency of 20% DEET. Using membrane microtransplantation, we evaluated the effect of DEET and FLU on native acetylcholine receptors expressed on the tick synganglion. We found that DEET has no effect on acetylcholine-evoked currents, but significantly reduced nicotine-induced current amplitudes. FLU induced an ionic current but was not able to reduce acetylcholine or nicotine evoked currents. The combination of both DEET and FLU strongly reduced nicotine-evoked currents. Finally, we demonstrated that our recording device for repellency, as well as the use of membrane microtransplantation, could be used as methods to study the mode of action of active compounds on ticks.

Impairments in learning and memory performances associated with nicotinic receptor expression in the honeybee Apis mellifera after exposure to a sublethal dose of sulfoxaflor.

Sulfoxaflor is a new insecticide which acts on the nicotinic acetylcholine receptor (nAChRs) in a similar way to neonicotinoids. However, sufloxaflor (SFX) is thought to act in a different manner and is thus proposed as an alternative in crop protection. The goal of this study is to evaluate the toxicity of SFX and its sublethal effect on the honeybee Apis mellifera after acute exposure. In toxicological assay studies, the LD50 value and sublethal dose (corresponding to the NOEL: no observed effect level) were 96 and 15 ng/bee, respectively. Using the proboscis extension response paradigm, we found that an SFX dose of 15 ng/bee significantly impairs learning and memory retrieval when applied 12 h before conditioning or 24 h after olfactory conditioning. SFX had no effect on honeybee olfactory performance when exposure happened after the conditioning. Relative quantitative PCR experiments performed on the six nicotinic acetylcholine receptor subunits demonstrated that they are differently expressed in the honeybee brain after SFX exposure, whether before or after conditioning. We found that intoxicated bees with learning defects showed a strong expression of the Amelß1 subunit. They displayed overexpression of Amelα9 and Amelß2, and down-regulation of Amelα1, Amelα3 and Amelα7 subunits. These results demonstrated for the first time that a sublethal dose of SFX could affect honeybee learning and memory performance and modulate the expression of specific nAChR subunits in the brain.

Transcriptome of the synganglion in the tick Ixodes ricinus and evolution of the cys-loop ligand-gated ion channel family in ticks.

BACKGROUND: Ticks represent a major health issue for humans and domesticated animals. Exploring the expression landscape of the tick's central nervous system (CNS), known as the synganglion, would be an important step in understanding tick physiology and in managing tick-borne diseases, but studies on that topic are still relatively scarce. Neuron-specific genes like the cys-loop ligand-gated ion channels (cys-loop LGICs, or cysLGICs) are important pharmacological targets of acaricides. To date their sequence have not been well catalogued for ticks, and their phylogeny has not been fully studied. RESULTS: We carried out the sequencing of transcriptomes of the I. ricinus synganglion, for adult ticks in different conditions (unfed males, unfed females, and partially-fed females). The de novo assembly of these transcriptomes allowed us to obtain a large collection of cys-loop LGICs sequences. A reference meta-transcriptome based on synganglion and whole body transcriptomes was then produced, showing high completeness and allowing differential expression analyses between synganglion and whole body. Many of the genes upregulated in the synganglion were associated with neurotransmission and/or localized in neurons or the synaptic membrane. As the first step of a functional study of cysLGICs, we cloned the predicted sequence of the resistance to dieldrin (RDL) subunit homolog, and functionally reconstituted the first GABA-gated receptor of Ixodes ricinus. A phylogenetic study was performed for the nicotinic acetylcholine receptors (nAChRs) and other cys-loop LGICs respectively, revealing tick-specific expansions of some types of receptors (especially for Histamine-like subunits and GluCls). CONCLUSIONS: We established a large catalogue of genes preferentially expressed in the tick CNS, including the cysLGICs. We discovered tick-specific gene family expansion of some types of cysLGIC receptors, and a case of intragenic duplication, suggesting a complex pattern of gene expression among different copies or different alternative transcripts of tick neuro-receptors.

Calmidazolium induces a decrease in nicotine-induced currents and intracellular calcium levels after pulse application of nicotine onto insect neurosecretory cells.

Dorsal unpaired median (DUM) neurons, are a class of insect neurosecretory cells, which are involved in the control of several functions, such as excretion and reproduction, or the release of neurohormones. Previous studies demonstrated that they express different nicotinic acetylcholine receptor subtypes, in particular α-bungarotoxin-insensitive receptors, with nAChR1 and nAChR2 subtypes. Here, we demonstrated that pulse application of 1 mM nicotine (300 ms pulse duration) induced inward currents which were reduced under bath application of 15 µM calmidazolium, a calmodulin inhibitor. Bath application of 0.5 µM α-bungarotoxin had no effect on calmidazolium action, suggesting that it could have an indirect effect through α-bungarotoxin-insensitive receptors. Indeed, nicotine-evoked currents were reduced by 10 µM d-tubocurarine, and completely blocked by 5 µM mecamylamine, which affected nAChR1 and nAChR2 subtypes, respectively. Our results demonstrated that nAChR2 subtypes are involved in the indirect effect of calmidazolium. Moreover, we found that this calmidazolium effect was associated to a strong reduction in intracellular calcium levels after pulse application of 1 mM nicotine. Thus, compared to previous studies on mammalian cells, calmidazolium did not cause an increase in intracellular calcium levels in DUM neurons, suggesting that different calcium mechanisms are involved in the calmidazolium effect.

Mode of Action of Neonicotinoid Insecticides Imidacloprid and Thiacloprid to the Cockroach Pameα7 Nicotinic Acetylcholine Receptor.

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.

Nicotinic acetylcholine receptors in the synganglion of the tick Ixodes ricinus: Functional characterization using membrane microtransplantation.

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.

Synergic effect of a quinuclidine benzamide complexed with borane, the LMA10233, in combination with seven pesticides.

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.

An Overview on the Effect of Neonicotinoid Insecticides on Mammalian Cholinergic Functions through the Activation of Neuronal Nicotinic Acetylcholine Receptors.

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 α4ß2 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.

Cloning and Expression of Cockroach α7 Nicotinic Acetylcholine Receptor Subunit.

Understanding insect nicotinic acetylcholine receptor (nAChR) subtypes is of major interest because they are the main target of several insecticides. In this study, we have cloned a cockroach Pameα7 subunit that encodes a 518 amino acid protein with futures typical of nAChR subunit, and sequence homology to α7 subunit. Pameα7 is differently expressed in the cockroach nervous system, in particular in the antennal lobes, optical lobes and the mushroom bodies where specific expression was found in the non-compact Kenyon cells. In addition, we found that cockroach Pameα7 subunits expressed in Xenopus laevis oocytes can assemble to form homomeric receptors. Electrophysiological recordings using the two-electrode voltage clamp method demonstrated that nicotine induced an I max current of -92 ± 27 nA at 1 mM. Despite that currents are low with the endogenous ligand, ACh, this study provides information on the first expression of cockroach α7 homomeric receptor.

Mode of action of sulfoxaflor on α-bungarotoxin-insensitive nAChR1 and nAChR2 subtypes: Inhibitory effect of imidacloprid.

Cockroach neurosecretory cells, dorsal unpaired median (DUM) neurons, express two distinct α-bungarotoxin-insensitive nicotinic acetylcholine receptor subtypes, nAChR1 and nAChR2 which are differently sensitive to the neonicotinoid insecticides and intracellular calcium pathways. The aim of this study is to determine whether sulfoxaflor acts as an agonist of nAChR1 and nAChR2 subtypes. We demonstrated that 1 mM sulfoxaflor induced high current amplitudes, compared to acetylcholine, suggesting that it was a full agonist of DUM neuron nAChR subtypes. Sulfoxaflor evoked currents were not inhibited by the nicotinic acetylcholine receptor antagonist d-tubocurarine (dTC) which reduced nAChR1. But, sulfoxaflor evoked currents were reduced in the presence of 5 µM mecamylamine which is known to reduce nAChR2 subtype. Interestingly, when 1 µM imidacloprid was added in the extracellular solution, sulfoxaflor-induced currents were significantly suppressed. Moreover, when extracellular calcium concentration was increased, bath application of 1 µM imidacloprid partially reduced sulfoxaflor activated currents when nAChR1 was inhibited with 20 µM dTC and completely suppressed sulfoxaflor currents when nAChR2 was inhibited with 5 µM mecamylamine. Our data demonstrated therefore that sulfoxaflor activates both nAChR1 and nAChR2 subtypes.

Permethrin enhances the agonist activity of dinotefuran on insect cholinergic synaptic transmission and isolated neurons.

Insect resistance mechanisms against pesticides lead to the development and the search of new pesticide combinations in order to delay the resistance. The combination of neonicotinoids with pyrethroids was currently proposed but the mode of action of these compounds at synaptic and extrasynaptic levels needs to be further explored. In the present study, we evaluated the effect of the combination of two insecticides, permethrin and dinotefuran, on cockroach cholinergic synaptic transmission and on isolated cell bodies. We first found that combination of 5 µM permethrin and dinotefuran enhances depolarization of the sixth abdominal ganglion compared to dinotefuran alone, without an inhibition of the spontaneous activity. However, a pretreatment with 1 µM dinotefuran or permethrin before bath application of the mixture inhibits the ganglionic depolarization. Compared to permethrin, 1 µM dinotefuran induces a persistent enhancement of spontaneous activity. Interestingly, at extrasynaptic level, using dorsal unpaired median neurons and Kenyon cells, we found that combination of both 1 µM dinotefuran and permethrin resulted in an increase of the mixture-induced current amplitudes. Pretreatment with 1 µM dinotefuran strongly decreases the currents whereas permethrin induces a time-dependent inhibition. These data demonstrate that the combination of dinotefuran and permethrin enhances the effect of dinotefuran.

Neonicotinoid insecticides differently modulate acetycholine-induced currents on mammalian α7 nicotinic acetylcholine receptors.

BACKGROUND AND PURPOSE: Neonicotinoid insecticides are described as poor agonists of mammalian nicotinic ACh receptors. In this paper, we show that their effects on mammalian nicotinic receptors differ between compounds. EXPERIMENTAL APPROACH: Two-electrode voltage-clamp electrophysiology was used to characterize the pharmacology of three neonicotinoid insecticides on nicotinic α7 receptors expressed in Xenopus oocytes. Single and combined application of clothianidin, acetamiprid and thiamethoxam were tested. RESULTS: Two neonicotinoid insecticides, clothianidin and acetamiprid, were partial agonists of mammalian neuronal α7 nicotinic receptors, whereas another neonicotinoid insecticide, thiamethoxam, which is converted to clothianidin in insect and plant tissues, had no effect. Pretreatment with clothianidin and acetamiprid (10 µM) ACh significantly enhanced the subsequent currents evoked by ACh (100 µM ) whereas pretreatment with thiamethoxam (10 µM) reduced ACh-induced current amplitudes.A combination of the three neonicotinoids decreased the ACh-evoked currents. CONCLUSIONS AND IMPLICATIONS: The present findings suggest that neonicotinoid insecticides differ markedly in their direct effects on mammalian α7 nicotinic ACh receptors and can also modulate ACh-induced currents. Furthermore, our data indicate a previously unknown modulation of mammalian α7 nicotinic receptors by a combination of clothianidin, acetamiprid and thiamethoxam. LINKED ARTICLES: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.

Neonicotinoid insecticides mode of action on insect nicotinic acetylcholine receptors using binding studies.

Nicotinic acetylcholine receptors (nAChRs) are the main target of neonicotinoid insecticides, which are widely used in crop protection against insect pests. Electrophysiological and molecular approaches have demonstrated the presence of several nAChR subtypes with different affinities for neonicotinoid insecticides. However, the precise mode of action of neonicotinoids on insect nAChRs remains to be elucidated. Radioligand binding studies with [3H]-α-bungarotoxin and [3H]-imidacloprid have proved instructive in understanding ligand binding interactions between insect nAChRs and neonicotinoid insecticides. The precise binding site interactions have been established using membranes from whole body and specific tissues. In this review, we discuss findings concerning the number of nAChR binding sites against neonicotinoid insecticides from radioligand binding studies on native tissues. We summarize the data available in the literature and compare the binding properties of the most commonly used neonicotinoid insecticides in several insect species. Finally, we demonstrate that neonicotinoid-nAChR binding sites are also linked to biological samples used and insect species.