Nanoencapsulated deltamethrin as synergistic agent potentiates insecticide effect of indoxacarb through an unusual neuronal calcium-dependent mechanism.

The use of neurotoxic chemical insecticides has led to consequences against the environment, insect resistances and side-effects on non-target organisms. In this context, we developed a novel strategy to optimize insecticide efficacy while reducing doses. It is based on nanoencapsulation of a pyrethroid insecticide, deltamethrin, used as synergistic agent, combined with a non-encapsulated oxadiazine (indoxacarb). In this case, the synergistic agent is used to increase insecticide efficacy by activation of calcium-dependant intracellular signaling pathways involved in the regulation of the membrane target of insecticides. In contrast to permethrin (pyrethroid type I), we report that deltamethrin (pyrethroid type II) produces an increase in intracellular calcium concentration in insect neurons through the reverse Na/Ca exchanger. The resulting intracellular calcium rise rendered voltage-gated sodium channels more sensitive to lower concentration of the indoxacarb metabolite DCJW. Based on these findings, in vivo studies were performed on the cockroach Periplaneta americana and mortality rates were measured at 24 h, 48 h and 72 h after treatments. Comparative studies of the toxicity between indoxacarb alone and indoxacarb combined with deltamethrin or nanoencapsulated deltamethrin (LNC-deltamethrin), indicated that LNC-deltamethrin potentiated the effect of indoxacarb. We also demonstrated that nanoencapsulation protected deltamethrin from esterase-induced enzymatic degradation and led to optimize indoxacarb efficacy while reducing doses. Moreover, our results clearly showed the benefit of using LNC-deltamethrin rather than piperonyl butoxide and deltamethrin in combination commonly used in formulation. This innovative strategy offers promise for increasing insecticide efficacy while reducing both doses and side effects on non-target organisms.

Influence of Cellular and Molecular Factors on Membrane Target Sensitivity to Insecticides.

The effective control of insect pests is based on the use of insecticides. Most of these compounds act on molecular targets in the insect nervous system. However, the largescale deployment of insecticide treatment has led to the development of resistance, which decreases insecticide efficacy. Although the resistance mechanisms are largely studied today, this review aims to point out new insights on the less-known cellular and molecular factors involved in the modulation of the sensitivity of the targets to insecticides. This review will focus on the phosphorylation/dephosphorylation process, the post-transcriptional events such as editing and alternative splicing and the influence of the association with auxiliary proteins of the receptors and/or ion channels targeted by insecticides. In addition, the involvement of calcium-dependent signaling pathways in the modulation of the sensitivity of the target to insecticides will also be considered and discussed. Finally, this review will insist on different strategies proposed to optimize the efficacy of insecticide treatment while reducing doses to decrease side effects on environment and on non-target organisms by combining two different chemical insecticides or a given active ingredient associated with biological and/or chemical synergistic agents. This review is part of the special issue "Insecticide Mode of Action: From Insect to Mammalian Toxicity".

Injection of insect membrane in Xenopus oocyte: An original method for the pharmacological characterization of neonicotinoid insecticides.

INTRODUCTION: Insect nicotinic acetylcholine receptors (nAChRs) represent a major target of insecticides, belonging to the neonicotinoid family. However, the pharmacological profile of native nAChRs is poorly documented, mainly because of a lack of knowledge of their subunit stoichiometry, their tissue distribution and the weak access to nAChR-expressing cells. In addition, the expression of insect nAChRs in heterologous systems remains hard to achieve. Therefore, the structure-activity characterization of nAChR-targeting insecticides is made difficult. The objective of the present study was to characterize insect nAChRs by an electrophysiological approach in a heterologous system naturally devoid of these receptors to allow a molecular/cellular investigation of the mode of action of neonicotinoids. Methods To overcome impediments linked to the expression of insect nAChR mRNA or cDNA, we chose to inject insect membranes from the pea aphid (Acyrthosiphon pisum) into Xenopus oocytes. This microtransplantation technique was designed to gain access to native nAChRs embedded in their membrane, through direct stimulation with nicotinic agonists. Results We provide evidence that an enriched-nAChR membrane allows us to characterize native receptors. The presence of such receptors was confirmed with fluorescent α-BgTX labeling. Electrophysiological recordings of nicotine-induced inward currents allowed us to challenge the presence of functional nAChR. We compared the effect of nicotine (NIC) with clothianidin (CLO) and we assessed the effect of thiamethoxam (TMX). Discussion This technique has been recently highlighted with mammalian and human material as a powerful functional approach, but has, to our knowledge, never been used with insect membrane. In addition, the use of the insect membrane microtransplantation opens a new and original way for pharmacological screening of neurotoxic insecticides, including neonicotinoids. Moreover, it might also be a powerful tool to investigate the pharmacological properties of insect nAChR.

Brief sensory experience differentially affects the volume of olfactory brain centres in a moth.

Experience modifies behaviour in animals so that they adapt to their environment. In male noctuid moths, Spodoptera littoralis, brief pre-exposure to various behaviourally relevant sensory signals modifies subsequent behaviour towards the same or different sensory modalities. Correlated with a behavioural increase in responses of male moths to the female-emitted sex pheromone after pre-exposure to olfactory, acoustic or gustatory stimuli, an increase in sensitivity of olfactory neurons within the primary olfactory centre, the antennal lobe, is found for olfactory and acoustic stimuli, but not for gustatory stimuli. Here, we investigated whether anatomical changes occurring in the antennal lobes and in the mushroom bodies (the secondary olfactory centres) possibly correlated with the changes observed in behaviour and in olfactory neuron physiology. Our results showed that significant volume changes occurred in glomeruli (olfactory units) responsive to sex pheromone following exposure to both pheromone and predator sounds. The volume of the mushroom body input region (calyx) also increased significantly after pheromone and predator sound treatment. However, we found no changes in the volume of antennal lobe glomeruli or of the mushroom body calyx after pre-exposure to sucrose. These findings show a relationship of antennal lobe sensitivity changes to the pheromone with changes in the volume of the related glomeruli and the output area of antennal lobe projection neurons elicited by sensory cues causing a behavioural change. Behavioural changes observed after sucrose pre-exposure must originate from changes in higher integration centres in the brain.

The Repellent DEET Potentiates Carbamate Effects via Insect Muscarinic Receptor Interactions: An Alternative Strategy to Control Insect Vector-Borne Diseases.

Insect vector-borne diseases remain one of the principal causes of human mortality. In addition to conventional measures of insect control, repellents continue to be the mainstay for personal protection. Because of the increasing pyrethroid-resistant mosquito populations, alternative strategies to reconstitute pyrethroid repellency and knock-down effects have been proposed by mixing the repellent DEET (N,N-Diethyl-3-methylbenzamide) with non-pyrethroid insecticide to better control resistant insect vector-borne diseases. By using electrophysiological, biochemichal, in vivo toxicological techniques together with calcium imaging, binding studies and in silico docking, we have shown that DEET, at low concentrations, interacts with high affinity with insect M1/M3 mAChR allosteric site potentiating agonist effects on mAChRs coupled to phospholipase C second messenger pathway. This increases the anticholinesterase activity of the carbamate propoxur through calcium-dependent regulation of acetylcholinesterase. At high concentrations, DEET interacts with low affinity on distinct M1/M3 mAChR site, counteracting the potentiation. Similar dose-dependent dual effects of DEET have also been observed at synaptic mAChR level. Additionally, binding and in silico docking studies performed on human M1 and M3 mAChR subtypes indicate that DEET only displays a low affinity antagonist profile on these M1/M3 mAChRs. These results reveal a selective high affinity positive allosteric site for DEET in insect mAChRs. Finally, bioassays conducted on Aedes aegypti confirm the synergistic interaction between DEET and propoxur observed in vitro, resulting in a higher mortality of mosquitoes. Our findings reveal an unusual allosterically potentiating action of the repellent DEET, which involves a selective site in insect. These results open exciting research areas in public health particularly in the control of the pyrethroid-resistant insect-vector borne diseases. Mixing low doses of DEET and a non-pyrethroid insecticide will lead to improvement in the efficiency treatments thus reducing both the concentration of active ingredients and side effects for non-target organisms. The discovery of this insect specific site may pave the way for the development of new strategies essential in the management of chemical use against resistant mosquitoes.

Inhibition of PaCaMKII-E isoform in the dorsal unpaired median neurosecretory cells of cockroach reduces nicotine- and clothianidin-induced currents.

Cellular responses to Ca(2+) 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 Ca(2+) 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.

A novel method using Autographa californica multiple nucleopolyhedrovirus for increasing the sensitivity of insecticide through calcium influx in insect cell line.

Due to an intensive use of chemical insecticides, resistance mechanisms to insecticides together with adverse effects on non-target organisms have been largely reported. Improvement in pest control strategy represents an urgent need to optimize efficiency in the control of pest insects. In this context, a novel method based on the use of insect specific virus applied in combination with chemical insecticide, which could lead to sensitization of the insect target to insecticides is described. Insect virus, the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), applied onto Sf9 cells induces an increase of intracellular calcium concentration via extracellular calcium influx. Co-application of AcMNPV with chlorpyrifos-ethyl onto Sf9 cells expressing the key enzyme acetylcholinesterase (AChE), known to be targeted by organophosphate insecticides, increases 1.5-fold the sensitivity of AChE to the insecticide. This effect is correlated with intracellular calcium concentration rise since AcMNPV-induced potentiating insecticide effect is counteracted by pretreatment with the calcium channel blocker, cadmium chloride. Increasing insecticide target sensitivity through intracellular calcium modulation by using insect virus co-applied with a chemical insecticide is a very promising strategy allowing optimization of insecticide treatment while reducing the concentration of insecticides used.

Calcium pathways such as cAMP modulate clothianidin action through activation of α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors.

Clothianidin is a neonicotinoid insecticide developed in the early 2000s. We have recently demonstrated that it was a full agonist of α-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors expressed in the cockroach dorsal unpaired median neurons. Clothianidin was able to act as an agonist of imidacloprid-insensitive nAChR2 receptor and internal regulation of cAMP concentration modulated nAChR2 sensitivity to clothianidin. In the present study, we demonstrated that cAMP modulated the agonist action of clothianidin via α-bungarotoxin-sensitive and insensitive receptors. Clothianidin-induced current-voltage curves were dependent to clothianidin concentrations. At 10 µM clothianidin, increasing cAMP concentration induced a linear current-voltage curve. Clothianidin effects were blocked by 0.5 µM α-bungarotoxin suggesting that cAMP modulation occurred through α-bungarotoxin-sensitive receptors. At 1 mM clothianidin, cAMP effects were associated to α-bungarotoxin-insensitive receptors because clothianidin-induced currents were blocked by 5 µM mecamylamine and 20 µM d-tubocurarine. In addition, we found that application of 1mM clothianidin induced a strong increase of intracellular calcium concentration. These data reinforced the finding that calcium pathways including cAMP modulated clothianidin action on insect nicotinic acetylcholine receptors. We proposed that intracellular calcium pathways such as cAMP could be a target to modulate the mode of action of neonicotinoid insecticides.

Multiple spectral inputs improve motion discrimination in the Drosophila visual system.

Color and motion information are thought to be channeled through separate neural pathways, but it remains unclear whether and how these pathways interact to improve motion perception. In insects, such as Drosophila, it has long been believed that motion information is fed exclusively by one spectral class of photoreceptor, so-called R1 to R6 cells; whereas R7 and R8 photoreceptors, which exist in multiple spectral classes, subserve color vision. Here, we report that R7 and R8 also contribute to the motion pathway. By using electrophysiological, optical, and behavioral assays, we found that R7/R8 information converge with and shape the motion pathway output, explaining flies' broadly tuned optomotor behavior by its composite responses. Our results demonstrate that inputs from photoreceptors of different spectral sensitivities improve motion discrimination, increasing robustness of perception.

Nornicotine application on cockroach dorsal unpaired median neurons induces two distinct ionic currents: implications of different nicotinic acetylcholine receptors.

The goal of the present study is to examine the agonist action of nornicotine on insect nicotinic acetylcholine receptors. Using patch-clamp techniques on cockroach dorsal unpaired median neurons, we demonstrated that nornicotine induced two distinct ionic currents named types 1 and 2. We found that alpha-bungarotoxin induced a rapid desensitization of type 1 currents whereas type 2 was completely blocked. Interestingly, types 1 and 2 currents were not blocked by the muscarinic antagonist, pirenzepine but by co-application of 1 µM pirenzepine and 0.5 µM alpha-bungarotoxin, suggesting that muscarinic receptors modulated nornicotine-induced current amplitudes. In addition, type 1 current amplitudes were strongly reduced by 20 µM d-tubocurarine and 5 µM mecamylamine which blocked the previously identified alpha-bungarotoxin-insensitive nAChR1 and nAChR2 receptors. Co-application of alpha-bungarotoxin with d-tubocurarine or mecamylamine completely blocked all ionic currents. We propose that types 1 and 2 currents are associated to several nicotinic receptors subtypes, including nAChR1 and nAChR2 receptors. Finally, we conclude that nornicotine could be used as an agonist to identify distinct insect nicotinic receptors.

Transmembrane potential polarization, calcium influx, and receptor conformational state modulate the sensitivity of the imidacloprid-insensitive neuronal insect nicotinic acetylcholine receptor to neonicotinoid insecticides.

Neonicotinoid insecticides act selectively on insect nicotinic acetylcholine receptors (nAChRs). Recent studies revealed that their efficiency was altered by the phosphorylation/dephosphorylation process and the intracellular signaling pathway involved in the regulation of nAChRs. Using whole-cell patch-clamp electrophysiology adapted for dissociated cockroach dorsal unpaired median (DUM) neurons, we demonstrated that intracellular factors involved in the regulation of nAChR function modulated neonicotinoid sensitivity. DUM neurons were known to express two α-bungarotoxin-insensitive nAChR subtypes: nAChR1 and nAChR2. Whereas nAChR1 was sensitive to imidacloprid, nAChR2 was insensitive to this insecticide. Here, we demonstrated that, like nicotine, acetamiprid and clothianidin, other types of neonicotinoid insecticides, acted as agonists on the nAChR2 subtype. Using acetamiprid, we revealed that both steady-state depolarization and hyperpolarization affected nAChR2 sensitivity. The measurement of the input membrane resistance indicated that change in the acetamiprid-induced agonist activity was related to the receptor conformational state. Using cadmium chloride, ω-conotoxin GVIA, and (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-acetamide (LOE 908), we found that inhibition of calcium influx through high voltage-activated calcium channels and transient receptor potential γ (TRPγ) activated by both depolarization and hyperpolarization increased nAChR2 sensitivity to acetamiprid. Finally, using N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7), forskolin, and cAMP, we demonstrated that adenylyl cyclase sensitive to the calcium/calmodulin complex regulated internal cAMP concentration, which in turn modulated TRPγ function and nAChR2 sensitivity to acetamiprid. Similar TRPγ-induced modulatory effects were also obtained when clothianidin was tested. These findings bring insights into the signaling pathway modulating neonicotinoid efficiency and open novel strategies for optimizing insect pest control.

Overexpression of grappa encoding a histone methyltransferase enhances stress resistance in Drosophila.

Histone deacetylases, such as silent information regulator 2 (Sir2) and Rpd3 are involved in chromatin silencing and implicated in lifespan determination in several organisms. The yeast Dot1 gene encoding a histone methyltransferase affects localization of silencing proteins including Sir2, and plays an essential role in the repair of damaged DNA. However, it is not known whether an alteration of a histone methyltransferase activity influences lifespan or stress resistance, which is often associated with extended lifespan. Here we investigated whether the Drosophilagrappa (gpp) gene, a Dot1 homolog influences lifespan and stress resistance using transgenic flies overexpressing gpp and those bearing a partial loss-of-function mutation. Overexpression of gpp throughout the adult stage did not extend the lifespan, but significantly enhanced resistances when they were kept on medium containing 1% H(2)O(2), or those with poor nutrients. As well, gpp-overexpressing flies were behaviourally more active than control flies. We investigated whether gpp overexpression induced anti-oxidant genes, Catalase, Sod, Sod2, GstD2, dhd, TrxT and Trx-2. However, none of these genes was induced. A partial loss-of-function mutations in gpp dramatically reduced the lifespan under oxidative and caloric stresses. Taken together, these results demonstrated that gpp is required for normal lifespan and stress resistance, and that its overexpression increases stress resistance in Drosophila, without obvious induction of representative anti-oxidant genes.