Véronique Apaire-Marchais

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.

Anopheles gambiae mosquito isolated neurons: A new biological model for optimizing insecticide/repellent efficacy

To understand better the mode of action of insecticides and repellents used in vector-borne diseases control, we developed a new biological model based on mosquito neurons isolated from adults Anopheles gambiae heads. This cellular model is well adapted to multidisciplinary approaches: electrophysiology, pharmacology, molecular biology and biochemical assays. Using RT-PCR, we demonstrated that isolated neurons express the nicotinic acetylcholine receptor subunit α1 (Agα1 nAchR), two acetylcholinesterases (AChE-1 and AChE-2) and three voltage-gated ion channels required for membrane excitability (AgCav1, AgNav1 and AgKv1). In order to correlate the expression of the different transcripts, encoding functional AgNav channel, nAChR receptor and AChE enzymes detected by RT-PCR, with electrophysiological activity we used patch-clamp technique. We revealed that AgNav and AChE which are targeted by insecticide and/or repellent were sensitive to the pyrethroid permethrin and to the repellent DEET, respectively. In addition, using colorimetric method, we also showed that AChE was sensitive to the carbamate propoxur. These results indicated that this novel neuronal mosquito model will lead to molecular and functional characterization of insecticide/repellent targets and appears as a powerful tool to investigate the development of highly specific and effective strategies for disease vector control.

The insect repellent N,N-diethyl-m-toluamide (DEET) induces angiogenesis via allosteric modulation of the M3 muscarinic receptor in endothelial cells

The insect repellent N,N-diethyl-m-toluamide (DEET) has been reported to inhibit AChE (acetylcholinesterase) and to possess potential carcinogenic properties with excessive vascularization. In the present paper, we demonstrate that DEET specifically stimulates endothelial cells that promote angiogenesis which increases tumor growth. DEET activates cellular processes that lead to angiogenesis including proliferation, migration and adhesion. This is associated with an enhancement of NO production and VEGF expression in endothelial cells. M3 silencing or the use of a pharmacological M3 inhibitor abrogates all of these effects which reveals that DEET-induced angiogenesis is M3 sensitive. The experiments involving calcium signals in both endothelial and HEK cells overexpressing M3 receptors, as well as binding and docking studies demonstrate that DEET acts as an allosteric modulator of the M3 receptor. In addition, DEET inhibited AChE which increased acetylcholine bioavailability and binding to M3 receptors and also strengthened proangiogenic effects by an allosteric modulation.

Virus and calcium: an unexpected tandem to optimize insecticide efficacy.

The effective control of insect pests is based on the rational use of the most efficient and safe insecticide treatments. To increase the effects of classical insecticides and to avoid the ability of certain pest insects to develop resistance, it is essential to propose novel strategies. Previous studies have shown that calcium-dependent phosphorylation/dephosphorylation is now considered as a new cellular mechanism for increasing the target sensitivity to insecticides. Because it is known that virus entry is correlated with intracellular calcium concentration rise, this report attempts to present the most important data relevant to the feasibility of combining an insect virus such as baculovirus or densovirus with an insecticide. In this case, the insect virus is not used as a bioinsecticide but acts as a synergistic agent able to trigger calcium rise and to activate calcium-dependent intracellular signalling pathways involved in the increase of the membrane receptors and/or ion channels sensitivity to insecticides. This virus-insecticide mixture represents a promising alternative to optimize the efficacy of insecticides against insect pests while reducing the doses.

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.

Unusual modes of action of the repellent DEET in insects highlight some human side effects

Abstract N,N‐diethyl‐m‐toluamide (DEET) induces favorable repellency against insects by acting on the sensory nervous system. According to emerging literature reports, DEET side effects in humans involve new molecular targets including the cholinergic system, acetylcholinesterase (AChE), muscarinic M1 and M3 receptor and the participation of the second messenger nitric oxide (NO). Most of these molecular events targeted by DEET have previously been characterized in insects while they have been considered as marginal compared to classical repellent properties. Despite these uncommon actions in insects, there is no consensus on the effects in human. Based on these data, this review provides new insights on side effects in human and more largely in mammals by identifying the unusual properties of DEET in insects, which seem to be correlated with adverse effects in mammals. These data will be very helpful to understand better the toxicological effects observed in order to protect non‐target organisms from the toxicity.

Synergistic agent and intracellular calcium, a successful partnership in the optimization of insecticide efficacy

Integrated Pest Management and Integrated Vector Management worldwide are developed in agriculture and public health to counteract and limit the exponential increasing development of insect resistance to insecticides. However, facing the predominance of some resistant populations, new strategies are urgently needed to target resistant insects. An innovative approach lies in the optimization of commonly used insecticides when combined with chemical or biological synergistic agents. By an increase of intracellular calcium concentration followed by activation of calcium-dependant signalling pathways, the synergistic agents are able to indirectly increase target sites sensitivity to insecticide by inducing conformational change. The synergistic agents are of great interest in optimizing the efficacy of insecticides and in overcoming resistance mechanisms.

L’impact de la vaccination contre les papillomavirus chez les adolescents : enquête dans le Maine-et-Loire

En 2006, la vaccination contre certains papillomavirus genitaux humains a haut risque oncogene, recommandee mais non obligatoire, a ete introduite dans le calendrier vaccinal francais pour les jeunes filles de 14 ans, avec un rattrapage possible jusqu’a 23 ans. Pour evaluer les connaissances des jeunes concernes, leurs opinions et leurs sources d’information sur les papillomavirus, le cancer du col et la vaccination, une enquete a ete realisee dans le Maine-et-Loire aupres de 179 collegiens en classe de troisieme.