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 olfactory neurons have long been thought to express only one chemosensory receptor modality. Using a new genetic knock-in strategy, we targeted the four co-receptors representing the chemosensory modes in Drosophila (Orco, Ir25a, Ir8a, Ir76b). 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 olfactory neuron classes and is co-expressed in 64% of Orco+ neuron classes, including all neuron classes in the maxillary palp. Orco, Ir8a, and Ir76b expression patterns are also expanded. Single sensillum recordings from Orco-expressing Ir25a mutant antennal and palpal neurons identify significant changes in olfactory responses. These results suggest polymodal expression and function of chemosensory receptors is common in olfactory neurons. We present a new map of the olfactory system reflecting this polymodal expression.

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.

Methyl N,N-dimethyl anthranilate (MDA), ethyl anthranilate (EA) and butyl anthranilate (BA) were previously shown to repel Aedes aegypti mosquitoes from landing on human skin. However, the effect of these compounds on the orientation of flying mosquitoes in a choice situation and their effect on mosquito oviposition are not yet known. Here, we used a modified Y-tube olfactometer to test the effect of these compounds on the orientation of Aedes aegypti flying towards skin odor (human fingers), and we tested their effect on Aedes aegypti oviposition choice in a cage assay. In both behavioral situations we compared the effect to the well-documented repellent N,N-diethyl-meta-toluamide (DEET). MDA, EA, and DEET inhibited Aedes aegypti from flying towards skin odor while BA had no such effect. Conversely, MDA had no effect on oviposition while EA, BA, and DEET deterred oviposition, with the strongest effect observed for BA. Thus, we confirm that EA and DEET are generally repellent, while MDA is repellent only in a host-seeking context, and BA is deterrent only in an oviposition context. These compounds appear of potential use in mosquito control programs.

As vectors for disease, mosquitoes are a global threat to human health. The Anopheles mosquito is the deadliest mosquito species as the insect vector for the malaria-causing parasite, which kills hundreds of thousands every year. These mosquitoes are reliant on their sense of smell (olfaction) to guide most of their behaviors, and a better understanding of Anopheles olfaction identifies opportunities for reducing the spread of malaria. This review takes a detailed look at Anopheles olfaction. We explore a range of topics from chemosensory receptors, olfactory neurons, and sensory appendages to behaviors guided by olfaction (including host-seeking, foraging, oviposition, and mating), to vector management strategies that target mosquito olfaction. We identify many research areas that remain to be addressed.

Background: The species-specific mode of action for DEET and many other mosquito repellents is often unclear. Confusion may arise for many reasons. First, the response of a single mosquito species is often used to represent all mosquito species. Second, behavioural studies usually test the effect of repellents on mosquito attraction towards human odorants, rather than their direct repulsive effect on mosquitoes. Third, the mosquito sensory neuron responses towards repellents are often not directly examined. Methods: A close proximity response assay was used to test the direct repulsive effect of six mosquito repellents on Anopheles coluzzii, Aedes aegypti and Culex quinquefasciatus mosquitoes. Additionally, the behavioural assay and calcium imaging recordings of antennae were used to test the response of An. coluzzii mosquitoes towards two human odorants (1-octen-3-ol and benzaldehyde) at different concentrations, and mixtures of the repellents lemongrass oil and p-menthane-3,8-diol (PMD) with DEET. Results: Anopheles coluzzii mosquitoes were repelled by lemongrass oil and PMD, while Ae. aegypti and Cx. quinquefasciatus mosquitoes were repelled by lemongrass oil, PMD, eugenol, and DEET. In addition, high concentrations of 1-octen-3-ol and benzaldehyde were repellent, and activated more olfactory receptor neurons on the An. coluzzii antennae than lower concentrations. Finally, changes in olfactory responses to repellent mixtures reflected changes in repulsive behaviours. Conclusions: The findings described here suggest that different species of mosquitoes have different behavioural responses to repellents. The data further suggest that high-odour concentrations may recruit repellent-sensing neurons, or generally excite many olfactory neurons, yielding repellent behavioural responses. Finally, DEET can decrease the neuronal and behavioural response of An. coluzzii mosquitoes towards PMD but not towards lemongrass oil. Overall, these studies can help inform mosquito repellent choice by species, guide decisions on effective repellent blends, and could ultimately identify the olfactory neurons and receptors in mosquitoes that mediate repellency.

18The mode of action for most mosquito repellents is unknown. This is primarily due to the 19 difficulty in monitoring how the mosquito olfactory system responds to repellent odors. 20Here, we used the Q-system of binary expression to enable activity-dependent Ca 2+ 21 imaging in olfactory neurons of the African malaria mosquito Anopheles coluzzii. This 22 disruptively change the chemical profile of host scent signatures on the skin surface 34 rendering humans invisible to mosquitoes. 35 36 Introduction 37Mosquitoes are vectors for many debilitating diseases such as malaria, Zika, dengue 38 fever, and yellow fever. Malaria alone caused an estimated 435000 deaths in 2017 1 . 39Mosquitoes primarily depend on olfaction, in combination with other senses, to locate 40 their hosts 2,3 . Therefore, targeting the mosquito's sense of smell using repellent 41 odorants is an effective strategy to prevent them from biting humans. The synthetic 42 compound N,N-diethyl-meta-toluamide (DEET) is the most widely used mosquito 43 repellent in public use since 1957 4,5 . However, DEET has some drawbacks, including 44 high concentrations (~>30%) are required for it to be effective, an unpleasant odor and 45 oily feeling to some people, and the ability to dissolve some plastics and synthetic 46 rubber 4 . Commercially synthetized alternatives to DEET have been developed (IR3535, 47 picaridin), but these too have similar drawbacks, such as also requiring high 48 concentrations to be effective. In order to improve or identify new repellents, a better 49 understanding of how insect repellents affect a mosquito's olfactory system is needed. 50 However, the olfactory mode of action of synthetic insect repellents such as DEET, 51 IR3535, and picaridin, as well as natural insect repellents such as lemongrass oil and 52 eugenol, is surprisingly not well understood. 53The olfactory system of the Anopheles gambiae species of mosquitoes consists 54 of three organs: the antennae, maxillary palps, and labella 2,6,7 . Each of these organs is 55 covered with sensory hairs called sensilla, and each sensillum houses olfactory sensory 56 neurons that may contain one of three types of chemoreceptors: odorant receptors 57 (ORs), gustatory receptors (Grs), and/or ionotropic receptors (IRs). ORs are expressed 58 in the majority of olfactory neurons, and each OR is expressed along with the Odorant 59Receptor Co-receptor (Orco) to form a receptor complex that is either narrowly or 60 broadly tuned to a variety of host-derived odors 2,6,8 . Grs, specifically Gr22, Gr23, and 61Gr24, are expressed in sensilla on the mosquito maxillary palps and respond to carbon 62 dioxide 9 . IRs are likely activated by acids and amines 10,11 . 63A consensus for how DEET affects the mosquito olfactory system and alters host 64 seeking behavior has not yet emerged. Currently, there are three hypotheses of how 65 DEET affects mosquitoes: 1) DEET directly activates chemoreceptors (ORs, Grs, and/or 66 IRs) on the mosquito antennae, maxillary palps, or the labella to drive repe...