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.

The insect repellent IR3535 is one of the important alternative in the fight against mosquito-borne disease such as malaria, dengue, chikungunya, yellow fever and Zika. Using a multidisciplinary approach, we propose the development of an innovative insecticide-based vector control strategy using an unexplored property of IR3535. We have demonstrated that in insect neurosecretory cells, very low concentration of IR3535 induces intracellular calcium rise through cellular mechanisms involving orthosteric/allosteric sites of the M1-muscarinic receptor subtype, G protein βγ subunits, background potassium channel inhibition generating depolarization, which induces voltage-gated calcium channel activation. The resulting internal calcium concentration elevation increases nicotinic receptor sensitivity to the neonicotinoid insecticide thiacloprid. The synergistic interaction between IR3535 and thiacloprid contributes to significantly increase the efficacy of the treatment while reducing concentrations. In this context, IR3535, used as a synergistic agent, seems to promise a new approach in the optimization of the integrated vector management for vector control.Mosquito-biting rates represent a major concern in overall vector capacity. It is possible to drastically lower the spread of mosquito-borne disease by disrupting host-seeking and feeding 1,2 . Therefore, repellents represent an important alternative in the fight against mosquito-borne disease such as malaria, dengue, chikungunya, yellow fever and Zika 3 . Modes of action of the most commonly used insect repellents such as DEET, IR3535, picaridine characterized so far are diverse. They can i) elicit deterrent feeding behavior, ii) modulate mosquito behavior through gustatory mechanism effect via gustatory receptor neurons 4,5 and iii) affect olfactory mechanism of action involving transmembrane odorant receptor proteins 6-8 located in olfactory receptor neurons [9][10][11][12][13][14][15][16][17] .Recent studies indicate that some repellent chemicals, such as DEET can also directly act on both insect peripheral and central nervous systems. They induce locomotor activity disruption, neuroexcitation (via octopamine receptors), cholinergic system alterations (e.g., acetylcholinesterase inhibition and M1/M3 muscarinic acetylcholine receptor subtype interactions) and monooxygenase regulation [17][18][19][20][21][22] . This demonstrates that repellents can modulate multiple physiological functions through complex mechanisms. Unfortunately, the precise mechanisms of how these chemicals modulate the specific molecular targets in insects still remain elusive, contested and/or misunderstood. Exploring precisely the mode of action of such compounds may lead to new more effective alternatives in the Insect Resistance Management for preventing the spread of mosquito-borne diseases. In this context, we commonly use cockroach neurosecretory cells identified as dorsal unpaired median (DUM) neurons to explore the "non-classical" effects of repellents 18,22 . Because DUM neurons are ...