Date Published: May 11, 2015
Publisher: Public Library of Science
Author(s): Aly Abd-Ella, Maria Stankiewicz, Karolina Mikulska, Wieslaw Nowak, Cédric Pennetier, Mathilde Goulu, Carole Fruchart-Gaillard, Patricia Licznar, Véronique Apaire-Marchais, Olivier List, Vincent Corbel, Denis Servent, Bruno Lapied, Israel Silman.
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.
In recent years, because of the increasing pyrethroid-resistant mosquito populations [1,2], repellents and particularly DEET, considered as the standard product against mosquitoes, have gained increasing interest in public health for protecting people. Previous data have indicated that DEET displays a complex broad-spectrum action. It affects various types of insect sensory receptor neurons [3–12] such as olfactory receptor neurons, odorant receptors, and gustatory receptor neurons conditioning insect avoidance behavior and it alters fine-tuning of functionally olfactory receptor neurons. Additionally, it has been reported that DEET is not only a behaviour-modifying agent. It blocks, at peripheral nervous system level, insect neuromuscular junction and affects central octopaminergic synapses to induce neuroexcitation and toxicity . Furthermore, DEET is also considered as a reversible inhibitor of insect acetylcholinesterase (AChE), an enzyme involved in the rapid hydrolysis of the neurotransmitter acetylcholine at cholinergic synapses in the central nervous system and is able to strengthen the toxicity of anticholinesterase insecticides such as carbamates . According to these data, alternative strategies to reconstitute pyrethroid repellency and knock-down effects have been proposed by mixing DEET with non-pyrethroid insecticide to better control resistant insect vector-borne diseases [15–17]. However, except few findings, which report that cytochrome-P450 monooxygenases are responsible for the enhanced toxicity observed between DEET and the carbamate propoxur in mosquitoes Aedes aegypti , the underlying cellular mechanisms involved in the synergism remain unknown. In the cockroaches Periplaneta americana central nervous system, pacemaker neurosecretory cells, named the Dorsal Unpaired Median (DUM) neurons [19,20] are known neuronal model used for electro-pharmacological studies . Because adult DUM neuron cell bodies express different cholinergic receptors including nAChR resistant to α-bungarotoxin, muscarinic AChR subtypes (mAChR) and AChR with “mixed” nicotinic-muscarinic pharmacology activated by ACh, which is regulated by AChE [22–24], they represent a suitable cellular model to investigate the mode of action of both repellents and insecticides on the insect cholinergic system. Consequently, the following study has been designed to bring new insights on the neurophysiological action of DEET and to identify new molecular targets underlying the synergistic effect.
We report here that mixing low doses of the repellent DEET with the carbamate propoxur increases mortality rate of female Ae. aegypti. The first attractive aspect of this study is that in vitro studies performed in insect neurons, showing synergism between these two compounds, correlate well with the increased lethal effect observed in vivo. The molecular events underlying the synergism are summarized as shown (Fig 3C). DEET interacts with positive cooperativity and high affinity on M1/M3 mAChR allosteric site, potentiating the effect of cholinergic agonist on mAChRs. This leads to activation of PLC causing IP3 production. The latter produces the release of calcium from internal stores, which results in the calcium/CaM complex formation reducing the PI-PLC activity involved in the regulation of AChE. This finally renders AChE more sensitive to propoxur. At the opposite, high concentrations of DEET binds with low affinity on distinct mAChR interaction site inducing antagonism function, which decreases AChE sensitivity to propoxur.