The evolution of borneol repellency in culicine mosquitoes.

Insects have developed remarkable adaptations to effectively interact with plant secondary metabolites and utilize them as cues to identify suitable hosts. Consequently, humans have used aromatic plants for centuries to repel mosquitoes. The repellent effects of plant volatile compounds are mediated through olfactory structures present in the antennae, and maxillary palps of mosquitoes. Mosquito maxillary palps contain capitate-peg sensilla, which house three olfactory sensory neurons, of which two are mainly tuned to either carbon dioxide or octenol - two animal host odorants. However, the third neuron, which expresses the OR49 receptor, has remained without a known ecologically-relevant odorant since its initial discovery. In this study, we used odorant mixtures and terpenoid-rich Cannabis essential oils to investigate the activation of OR49. Our results demonstrate that two monoterpenoids, borneol and camphor, selectively activate OR49, and OR9-expressing neurons, as well as the MD3 glomerulus in the antennal lobe. We confirm that borneol repels female mosquitoes, and knocking out the gene encoding the OR49 receptor suppresses the response of the corresponding olfactory sensory neuron. Importantly, this molecular mechanism of action is conserved across culicine mosquito species, underscoring its significance in their olfactory systems.

A sucrose-specific receptor in Bemisia tabaci and its putative role in phloem feeding.

In insects, specialized feeding on the phloem sap (containing mainly the sugar sucrose) has evolved only in some hemipteran lineages. This feeding behavior requires an ability to locate feeding sites buried deeply within the plant tissue. To determine the molecular mechanism involved, we hypothesized that the phloem-feeding whitefly Bemisia tabaci relies on gustatory receptor (GR)-mediated sugar sensing. We first conducted choice assays, which indicated that B. tabaci adults consistently choose diets containing higher sucrose concentrations. Next, we identified four GR genes in the B. tabaci genome. One of them, BtabGR1, displayed significant sucrose specificity when expressed in Xenopus oocytes. Silencing of BtabGR1 significantly interfered with the ability of B. tabaci adults to discriminate between non-phloem and phloem concentrations of sucrose. These findings suggest that in phloem feeders, sugar sensing by sugar receptors might allow tracking an increasing gradient of sucrose concentrations in the leaf, leading eventually to the location of the feeding site.

The ovipositor cue indole inhibits animal host attraction in Aedes aegypti (Diptera: Culicidae) mosquitoes.

BACKGROUND: Mosquitoes are responsible for disease transmission worldwide. They possess the ability to discriminate between different ecological resources, including nectar sources, animal hosts and oviposition sites, a feature mediated by their olfactory system. Insect repellents, such as N,N-diethyl-meta-toluamide (also called DEET), have been shown to activate and inhibit mosquito odorant receptors, resulting in behavioral modulation. This and other repellents currently available for personal protection against mosquitoes are topically applied to the skin and operate at a short range. In our search for potential long-range inhibitors of attractants to human hosts, we have hypothesized that the shared chemical similarities between indole and DEET may confer the former with the ability to block odorant receptor function and inhibit human host attraction in a similar way as DEET. METHODS: We used the two-electrode voltage clamp system to assay Xenopus laevis oocytes as a platform to compare the pharmacological effect of commercially available insect repellents and indole on the Aedes aegypti (R)-1-octen-3-ol receptor, OR8, a receptor involved in the decision-making of female mosquitoes to identify human hosts. We also conducted arm-in-a-cage and wind-tunnel bioassays to explore the effect of indole on human host-seeking female Aedes aegypti mosquitoes. RESULTS: Our results demonstrate that indole inhibited the Aedes aegypti (R)-1-octen-3-ol receptor OR8. In our arm-in-a-cage assay, 1 M of DEET reduced mosquito visits on average by 69.3% while the same indole concentration achieved 97.8% inhibition. This effect of indole on flight visits was dose-dependent and disappeared at 1 µM. In the flight tunnel, indole elicited on average 27.5% lower speed, 42.3% lower upwind velocity and 30.4% higher tortuosity compared to the control. CONCLUSIONS: Indole significantly inhibits OR8 activation by (R)-1-octen-3-ol, mosquito visits to a human hand and long-range human host-seeking. The volatility of indole may be leveraged to develop a novel insect repellent in the context of personal mosquito protection.

Identification and functional characterization of olfactory indolergic receptors in Musca domestica.

In mosquitoes, indolic compounds are detected by a group of olfactory indolergic Odorant Receptors (indolORs). The ancient origin of indole and 3-methylindole as chemical signals suggest that they may be detected by insects outside the Culicidae clade. To test this hypothesis, we have identified potential indolOR genes in brachyceran flies based on sequence homology. Because of the crucial roles of indolic compounds in oviposition and foraging, we have focused our attention on the housefly Musca domestica. Using a heterologous expression system, we have identified indolOR transcript expression in the female antennae, and have characterized MdomOR30a and MdomOR49b as 3-methylindole and indole receptors, respectively. We have identified a set of 92 putative indolOR genes encoded in the genomes of Culicoidea, Psychodidae and brachycera, described their phylogenetic relationships, and exon/intron structures. Further characterization of indolORs will impact our understanding of insect chemical ecology and will provide targets for the development of novel odor-based tools that can be integrated into existing vector surveillance and control programs.

Identification and functional characterization of olfactory indolergic receptors in Drosophila melanogaster.

Indole-sensitive odorant receptors or indolORs belong to a mosquito-specific expansion as ancient as the Culicidae lineage. Brachyceran flies appeared to lack representative members of this group despite the importance of indolics in this important group of dipterans. To explore whether indolORs occur in other brachyceran species, we searched for candidate indolORs in Drosophila melanogaster. Using phylogenetic tools, we show that D. melanogaster OR30a, OR43a, and OR49b form a distinct monophyletic lineage with mosquito indolORs. To explore a potential functional orthology with indolORs, we expressed these three Drosophila ORs in Xenopus laevis oocytes and measured their responses to a panel of indolic compounds. We provide evidence that OR30a, OR43a, and OR49b exhibit high sensitivity to indoles. Along with the recent discovery of indolORs in the housefly Musca domestica, our findings suggest that indolORs are a widespread feature of the peripheral olfactory systems of Diptera.

Volatile allosteric antagonists of mosquito odorant receptors inhibit human-host attraction.

Odorant-dependent behaviors in insects are triggered by the binding of odorant ligands to the variable subunits of heteromeric olfactory receptors. Previous studies have shown, however, that specific odor binding to ORco, the common subunit of odorant receptor heteromers, may allosterically alter olfactory receptor function and profoundly affect subsequent behavioral responses. Using an insect cell-based screening platform, we identified and characterized several antagonists of the odorant receptor coreceptor of the African malaria vector Anopheles gambiae (AgamORco) in a small collection of natural volatile organic compounds. Because some of the identified antagonists were previously shown to strongly repel Anopheles and Culex mosquitoes, we examined the bioactivities of the identified antagonists against Aedes, the third major genus of the Culicidae family. The tested antagonists inhibited the function of Ae. aegypti ORco ex vivo and repelled adult Asian tiger mosquitoes (Ae. albopictus). Binary mixtures of specific antagonists elicited higher repellency than single antagonists, and binding competition assays suggested that this enhanced repellence is due to antagonist interaction with distinct ORco sites. Our results also suggest that the enhanced mosquito repellency by antagonist mixtures is due to additive rather than synergistic effects of the specific antagonist combinations on ORco function. Taken together, these findings provide novel insights concerning the molecular aspects of odorant receptor function. Moreover, our results demonstrate that a simple screening assay may be used for the identification of allosteric modifiers of olfactory-driven behaviors capable of providing enhanced personal protection against multiple mosquito-borne infectious diseases.

The Emergence of Insect Odorant Receptor-Based Biosensors.

The olfactory receptor neurons of insects and vertebrates are gated by odorant receptor (OR) proteins of which several members have been shown to exhibit remarkable sensitivity and selectivity towards volatile organic compounds of significant importance in the fields of medicine, agriculture and public health. Insect ORs offer intrinsic amplification where a single binding event is transduced into a measurable ionic current. Consequently, insect ORs have great potential as biorecognition elements in many sensor configurations. However, integrating these sensing components onto electronic transducers for the development of biosensors has been marginal due to several drawbacks, including their lipophilic nature, signal transduction mechanism and the limited number of known cognate receptor-ligand pairs. We review the current state of research in this emerging field and highlight the use of a group of indole-sensitive ORs (indolORs) from unexpected sources for the development of biosensors.

Corrigendum: Supersensitive Odorant Receptor Underscores Pleiotropic Roles of Indoles in Mosquito Ecology.

[This corrects the article DOI: 10.3389/fncel.2018.00533.].

The sulcatone receptor of the strict nectar-feeding mosquito Toxorhynchites amboinensis.

Controlling Ae. aegypti populations and the prevention of mosquito bites includes the development of monitoring, repelling and attract-and-kill strategies that are based on understanding the chemical ecology of these pests. Olfactory-mediated attraction to mammals has recently been linked to the mosquito Aedes aegypti odorant receptor Or4, which is activated by animal-released 6-Methyl-5-hepten-2-one (sulcatone). This odorant is also a major component of flower scents and may play a role outside animal-host seeking. To explore the role of this chemical cue, we looked at the interaction between sulcatone and an Or4 homolog expressed in the antennae of the strict nectar-feeding mosquito Toxorhynchites amboinensis. Using the two-electrode voltage clamp of Xenopus oocytes as a heterologous expression system, we show that this receptor is a high intensity sulcatone receptor comparable to its Aedes counterparts. We also show that OR4 is activated by other aliphatic ketones and is inhibited by DEET. This pharmacological characterization suggests that sulcatone may be operating in more than one context in the Culicidae family.

The evolutionarily conserved indolergic receptors of the non-hematophagous elephant mosquito Toxorhynchites amboinensis.

The conservation of the mosquito indolergic receptors across the Culicinae and Anophelinae mosquito lineages, which spans 200 million years of evolution, is a testament to the central role of indolic compounds in the biology of these insects. Indole and skatole have been associated with the detection of oviposition sites and animal hosts. To evaluate the potential ecological role of these two compounds, we have used a pharmacological approach to characterize homologs of the indolergic receptors Or2 and Or10 in the non-hematophagous elephant mosquito Toxorhynchites amboinensis. We provide evidence that both receptors are narrowly tuned to indole and skatole like their counterparts from hematophagous mosquitoes. These findings indicate that Toxorhynchites detects indole and skatole in an ecological context to be determined and underscore the importance of understanding the role of these compounds in mosquitoes.

Supersensitive Odorant Receptor Underscores Pleiotropic Roles of Indoles in Mosquito Ecology.

Mosquitoes exhibit highly diverse and fast evolving odorant receptors (ORs). The indole-sensitive OR gene clade, comprised of Or2 and Or10 is a notable exception on account of its conservation in both mosquito subfamilies. This group of paralogous genes exhibits a complex developmental expression pattern in Aedes aegypti: AaegOr2 is expressed in both adults and larvae, AaegOr10 is adult-specific and a third member named AaegOr9 is larva-specific. OR2 and OR10 have been deorphanized and are selectively activated by indole and skatole, respectively. Using the two-electrode voltage clamp of Xenopus oocytes expressing Ae. aegypti ORs, we show that AaegOR9 is supersensitive and narrowly tuned to skatole. Our findings suggest that Ae. aegypti has evolved two distinct molecular strategies to detect skatole in aquatic and terrestrial environments, highlighting the central ecological roles of indolic compounds in the evolutionary and life histories of these insects.

Chemosensory Responses to the Repellent Nepeta Essential Oil and Its Major Component Nepetalactone by Aedes aegypti (Diptera: Culicidae), a Vector of Zika Virus.

Nepeta essential oil (Neo; catnip) and its major component, nepetalactone, have long been known to repel insects including mosquitoes. However, the neural mechanisms through which these repellents are detected by mosquitoes, including the yellow fever mosquito Aedes aegypti (L.), an important vector of Zika virus, were poorly understood. Here we show that Neo volatiles activate olfactory receptor neurons within the basiconic sensilla on the maxillary palps of female Ae. aegypti. A gustatory receptor neuron sensitive to the feeding deterrent quinine and housed within sensilla on the labella of females was activated by both Neo and nepetalactone. Activity of a second gustatory receptor neuron sensitive to the feeding stimulant sucrose was suppressed by both repellents. Our results provide neural pathways for the reported spatial repellency and feeding deterrence of these repellents. A better understanding of the neural input through which female mosquitoes make decisions to feed will facilitate design of new repellents and management strategies involving their use.

Evolutionarily conserved odorant receptor function questions ecological context of octenol role in mosquitoes.

Olfaction is a key insect adaptation to a wide range of habitats. In the last thirty years, the detection of octenol by blood-feeding insects has been primarily understood in the context of animal host-seeking. The recent discovery of a conserved octenol receptor gene in the strictly nectar-feeding elephant mosquito Toxorhynchites amboinensis (TaOr8) suggests a different biological role. Here, we show that TaOR8 is a functional ortholog of its counterparts in blood-feeding mosquitoes displaying selectivity towards the (R)-enantiomer of octenol and susceptibility to the insect repellent DEET. These findings suggest that while the function of OR8 has been maintained throughout mosquito evolution, the context in which this receptor is operating has diverged in blood and nectar-feeding mosquitoes.

Olfactory disruption: toward controlling important insect vectors of disease.

Chemical repellents are used to decrease contacts between insect disease vectors and their hosts, thus reducing the probability of disease transmission. The molecular mechanisms by which repellents have their effects are poorly understood and remain a controversial topic. Here, we present recent results of studies aimed at a more thorough understanding of the mode of action of repellents and discuss the implications of these findings for future research and development of novel or improved repellents.

The genetics of chemoreception in the labella and tarsi of Aedes aegypti.

The yellow-fever mosquito Aedes aegypti is a major vector of human diseases, such as dengue, yellow fever, chikungunya and West Nile viruses. Chemoreceptor organs on the labella and tarsi are involved in human host evaluation and thus serve as potential foci for the disruption of blood feeding behavior. In addition to host detection, these contact chemoreceptors mediate feeding, oviposition and conspecific recognition; however, the molecular landscape of chemoreception in these tissues remains mostly uncharacterized. Here we report the expression profile of all putative chemoreception genes in the labella and tarsi of both sexes of adult Ae. aegypti and discuss their possible roles in the physiology and behavior of this important disease vector.

The maxillary palp of Aedes aegypti, a model of multisensory integration.

Female yellow-fever mosquitoes, Aedes aegypti, are obligate blood-feeders and vectors of the pathogens that cause dengue fever, yellow fever and Chikungunya. This feeding behavior concludes a series of multisensory events guiding the mosquito to its host from a distance. The antennae and maxillary palps play a major role in host detection and other sensory-mediated behaviors. Compared to the antennae, the maxillary palps are a relatively simple organ and thus an attractive model for exploration of the neuromolecular networks underlying chemo- and mechanosensation. In this study, we surveyed the expressed genetic components and examined their potential involvement with these sensory modalities. Using Illumina sequencing, we identified the transcriptome of the maxillary palps of physiologically mature female Ae. aegypti. Genes expressed in the maxillary palps included those involved in sensory reception, signal transduction and neuromodulation. In addition to previously reported chemosensory genes, we identified candidate transcripts potentially involved in mechanosensation and thermosensation. This survey lays the groundwork to explore sensory networks in an insect appendage. The identification of genes involved in thermosensation provides prospective molecular targets for the development of chemicals aimed at disrupting the behavior of this medically important insect.

Functional Development of the Octenol Response in Aedes aegypti.

Attraction of female Aedes aegypti mosquitoes to 1-octen-3-ol (octenol), CO2, lactic acid, or ammonia emitted by vertebrate hosts is not only contingent on the presence of odorants in the environment, but is also influenced by the insect's physiological state. For anautogenous mosquito species, like A. aegypti, newly emerged adult females neither respond to host odors nor engage in blood-feeding; the bases for these behaviors are poorly understood. Here we investigated detection of two components of an attractant blend emitted by vertebrate hosts, octenol, and CO2, by female A. aegypti mosquitoes using electrophysiological, behavioral, and molecular approaches. An increase in sensitivity of octenol olfactory receptor neurons (ORNs) was correlated with an increase in odorant receptor gene (Or) expression and octenol-mediated attractive behavior from day 1 to day 6 post-emergence. While the sensitivity of octenol ORNs was maintained through day 10, behavioral responses to octenol decreased as did the ability of females to discriminate between octenol and octenol + CO2. Our results show differing age-related roles for the peripheral receptors for octenol and higher order neural processing in the behavior of female mosquitoes.

Selectivity of odorant receptors in insects.

Insect olfactory receptors (ORs) detect chemicals, shape neuronal physiology, and regulate behavior. Although ORs have been categorized as "generalists" and "specialists" based on their ligand spectrum, both electrophysiological studies and recent pharmacological investigations show that ORs specifically recognize non-pheromonal compounds, and that our understanding of odorant-selectivity mirrors our knowledge of insect chemical ecology. As we are progressively becoming aware that ORs are activated through a variety of mechanisms, the molecular basis of odorant-selectivity and the corollary notion of broad-tuning need to be re-examined from a pharmacological and evolutionary perspective.

Odorant receptor modulation: ternary paradigm for mode of action of insect repellents.

The modulation of insect behavior for the purpose of controlling the spread of infectious diseases has been the task of a few insect repellents for which the mechanistic modes of action on odorant receptors (ORs) are unclear. Here, we study the effects of the repellents DEET and IR3535, and a novel OR co-receptor (Orco) agonist on odorant-evoked currents in Xenopus oocytes expressing two subtypes of Aedes aegypti ORs (AaORs). We show that DEET and IR3535 behave as insurmountable antagonists of ORs, and that modulation of OR activity is not restricted to antagonism and agonism, but also includes synergism. This knowledge of the molecular mechanisms underlying OR blockade, activation and hyperactivation will be fundamental to the development of novel strategies for the control of mosquito behavior.

Conservation of indole responsive odorant receptors in mosquitoes reveals an ancient olfactory trait.

Aedes aegypti and Anopheles gambiae are among the best-characterized mosquito species within the Culicinae and Anophelinae mosquito clades which diverged ∼150 million years ago. Despite this evolutionary distance, the olfactory systems of these mosquitoes exhibit similar morphological and physiological adaptations. Paradoxically, mosquito odorant receptors, which lie at the heart of chemosensory signal transduction pathways, belong to a large and highly divergent gene family. We have used 2 heterologous expression systems to investigate the functional characteristics of a highly conserved subset of Ors between Ae. aegypti and An. gambiae to investigate whether protein homology correlates with odorant-induced activation. We find that these receptors share similar odorant response profiles and that indole, a common and ecologically relevant olfactory cue, elicits strong responses from these homologous receptors. The identification of other highly conserved members of this Or clade from mosquito species of varying phylogenetic relatedness supports a model in which high sensitivity to indole represents an ancient ecological adaptation that has been preserved as a result of its life cycle importance. These results provide an understanding of how similarities and disparities among homologous OR proteins relate to olfactory function, which can lead to greater insights into the design of successful strategies for the control of mosquito-borne diseases.