The mode of action for most mosquito repellents is unknown. This is primarily due to the difficulty in monitoring how the mosquito olfactory system responds to repellent odors. Here, we used the Q-system of binary expression to enable activity-dependent Ca 2+ imaging in olfactory neurons of the African malaria mosquito Anopheles coluzzii. This system allows neuronal responses to common insect repellents to be directly visualized in living mosquitoes from all olfactory organs, including the antenna. The synthetic repellents N,N-diethyl-meta-toluamide (DEET) and IR3535 did not activate Anopheles odorant receptor co-receptor (Orco)-expressing olfactory receptor neurons (ORNs) at any concentration, and picaridin weakly activated ORNs only at high concentrations. In contrast, natural repellents (i.e. lemongrass oil and eugenol) strongly activated small numbers of ORNs in the Anopheles mosquito antennae at low concentrations. We determined that DEET, IR3535, and picaridin decrease the response of Orco-expressing ORNs when these repellents are physically mixed with activating human-derived odorants. We present evidence that synthetic repellents may primarily exert their olfactory mode of action by decreasing the amount of volatile odorants reaching ORNs. These results suggest that synthetic repellents disruptively change the chemical profile of host scent signatures on the skin surface, rendering humans invisible to Anopheles mosquitoes.

Gravid mosquitoes use chemosensory (olfactory, gustatory, or both) cues to select oviposi tion sites suitable for their offspring. In nature, these cues originate from plant infusions, microbes, mosquito immature stages, and predators. While attractants and stimulants are cues that could show the availability of food (plant infusions and microbes) and suitable conditions (the presence of conspecifics), repellents and deterrents show the risk of predation, infection with pathogens, or strong competition. Many studies have addressed the question of which substances can act as positive or negative cues in different mosquito species, with sometimes apparently contradicting results. These studies often differ in species, substance concentration, and other experimental details, making it difficult to compare the results. In this review, we compiled the available information for a wide range of species and substances, with particular attention to cues originating from larval food, immature stages, predators, and to syn thetic compounds. We note that the effect of many substances differs between species, and that many substances have been tested in few species only, revealing that the information is scattered across species, substances, and experimental conditions.

Drosophila melanogaster olfactory neurons have long been thought to express only one chemosensory receptor gene family. There are two main olfactory receptor gene families in Drosophila, the odorant receptors (ORs) and the ionotropic receptors (IRs). The dozens of odorant-binding receptors in each family require at least one co-receptor gene in order to function: Orco for ORs, and Ir25a, Ir8a, and Ir76b for IRs. Using a new genetic knock-in strategy, we targeted the four co-receptors representing the main chemosensory families in D. melanogaster (Orco, Ir8a, Ir76b, Ir25a). Co-receptor knock-in expression patterns were verified as accurate representations of endogenous expression. We find extensive overlap in expression among the different co-receptors. As defined by innervation into antennal lobe glomeruli, Ir25a is broadly expressed in 88% of all olfactory sensory neuron classes and is co-expressed in 82% of Orco+ neuron classes, including all neuron classes in the maxillary palp. Orco, Ir8a, and Ir76b expression patterns are also more expansive than previously assumed. Single sensillum recordings from Orco-expressing Ir25a mutant antennal and palpal neurons identify changes in olfactory responses. We also find co-expression of Orco and Ir25a in Drosophila sechellia and Anopheles coluzzii olfactory neurons. These results suggest that co-expression of chemosensory receptors is common in insect olfactory neurons. Together, our data present the first comprehensive map of chemosensory co-receptor expression and reveal their unexpected widespread co-expression in the fly olfactory system.