Targeting a dual detector of skin and CO2 to modify mosquito host seeking.

Female mosquitoes that transmit deadly diseases locate human hosts by detecting exhaled CO2 and skin odor. The identities of olfactory neurons and receptors required for attraction to skin odor remain a mystery. Here, we show that the CO2-sensitive olfactory neuron is also a sensitive detector of human skin odorants in both Aedes aegypti and Anopheles gambiae. We demonstrate that activity of this neuron is important for attraction to skin odor, establishing it as a key target for intervention. We screen ~0.5 million compounds in silico and identify several CO2 receptor ligands, including an antagonist that reduces attraction to skin and an agonist that lures mosquitoes to traps as effectively as CO2. Analysis of the CO2 receptor ligand space provides a foundation for understanding mosquito host-seeking behavior and identifies odors that are potentially safe, pleasant, and affordable for use in a new generation of mosquito control strategies worldwide.

Receptors underlying an odorant's valence across concentrations in Drosophila larvae.

Odorants interact with receptors expressed in specialized olfactory neurons, and neurons of the same class send their axons to distinct glomeruli in the brain. The stereotypic spatial glomerular activity map generates recognition and the behavioral response for the odorant. The valence of an odorant changes with concentration, typically becoming aversive at higher concentrations. Interestingly, in Drosophila larvae, the odorant (E)-2-hexenal is aversive at low concentrations and attractive at higher concentrations. We investigated the molecular and neural basis of this phenomenon, focusing on how activities of different olfactory neurons conveying opposing effects dictate behaviors. We identified the repellant neuron in the larvae as one expressing the olfactory receptor Or7a, whose activation alone at low concentrations of (E)-2-hexenal elicits an avoidance response in an Or7a-dependent manner. We demonstrate that avoidance can be overcome at higher concentrations by activation of additional neurons that are known to be attractive, most notably odorants that are known activators of Or42a and Or85c. These findings suggest that in the larval stage, the attraction-conveying neurons can overcome the aversion-conveying channels for (E)-2-hexenal.

A System-Wide Understanding of the Human Olfactory Percept Chemical Space.

The fundamental units of olfactory perception are discrete 3D structures of volatile chemicals that each interact with specific subsets of a very large family of hundreds of odorant receptor proteins, in turn activating complex neural circuitry and posing a challenge to understand. We have applied computational approaches to analyze olfactory perceptual space from the perspective of odorant chemical features. We identify physicochemical features associated with ~150 different perceptual descriptors and develop machine-learning models. Validation of predictions shows a high success rate for test set chemicals within a study, as well as across studies more than 30 years apart in time. Due to the high success rates, we are able to map ~150 percepts onto a chemical space of nearly 0.5 million compounds, predicting numerous percept-structure combinations. The chemical structure-to-percept prediction provides a system-level view of human olfaction and opens the door for comprehensive computational discovery of fragrances and flavors.

Functional characterization of odorant receptors in the ponerine ant, Harpegnathos saltator.

Animals use a variety of sensory modalities-including visual, acoustic, and chemical-to sense their environment and interact with both conspecifics and other species. Such communication is especially critical in eusocial insects such as honey bees and ants, where cooperation is critical for survival and reproductive success. Various classes of chemoreceptors have been hypothesized to play essential roles in the origin and evolution of eusociality in ants, through their functional roles in pheromone detection that characterizes reproductive status and colony membership. To better understand the molecular mechanisms by which chemoreceptors regulate social behaviors, we investigated the roles of a critical class of chemoreceptors, the odorant receptors (ORs), from the ponerine ant Harpegnathos saltator in detecting cuticular hydrocarbon pheromones. In light of the massive OR expansion in ants (∼400 genes per species), a representative survey based on phylogenetic and transcriptomic criteria was carried out across discrete odorant receptor subfamilies. Responses to several classes of semiochemicals are described, including cuticular hydrocarbons and mandibular gland components that act as H. saltator pheromones, and a range of more traditional general odorants. When viewed through the prism of caste-specific OR enrichment and distinctive OR subfamily odorant response profiles, our findings suggest that whereas individual HsOrs appear to be narrowly tuned, there is no apparent segregation of tuning responses within any discrete HsOr subfamily. Instead, the HsOR gene family as a whole responds to a broad array of compounds, including both cuticular hydrocarbons and general odorants that are likely to mediate distinct behaviors.

Epigenetic regulation of olfactory receptor gene expression by the Myb-MuvB/dREAM complex.

In both mammals and insects, an olfactory neuron will usually select a single olfactory receptor and repress remaining members of large receptor families. Here we show that a conserved multiprotein complex, Myb-MuvB (MMB)/dREAM, plays an important role in mediating neuron-specific expression of the carbon dioxide (CO(2)) receptor genes (Gr63a/Gr21a) in Drosophila. Activity of Myb in the complex is required for expression of Gr63a/Gr21a and acts in opposition to the histone methyltransferase Su(var)3-9. Consistent with this, we observed repressive dimethylated H3K9 modifications at the receptor gene loci, suggesting a mechanism for silencing receptor gene expression. Conversely, other complex members, Mip120 (Myb-interacting protein 120) and E2F2, are required for repression of Gr63a in inappropriate neurons. Misexpression in mutants is accompanied by an increase in the H3K4me3 mark of active chromatin at the receptor gene locus. Nuclei of CO(2) receptor-expressing neurons contain reduced levels of the repressive subunit Mip120 compared with surrounding neurons and increased levels of Myb, suggesting that activity of the complex can be regulated in a cell-specific manner. Our evidence suggests a model in which olfactory receptors are regulated epigenetically and the MMB/dREAM complex plays a critical role in specifying, maintaining, and modulating the receptor-to-neuron map.

Odour receptors and neurons for DEET and new insect repellents.

There are major impediments to finding improved DEET alternatives because the receptors causing olfactory repellency are unknown, and new chemicals require exorbitant costs to determine safety for human use. Here we identify DEET-sensitive neurons in a pit-like structure in the Drosophila melanogaster antenna called the sacculus. They express a highly conserved receptor, Ir40a, and flies in which these neurons are silenced or Ir40a is knocked down lose avoidance to DEET. We used a computational structure-activity screen of >400,000 compounds that identified >100 natural compounds as candidate repellents. We tested several and found that most activate Ir40a(+) neurons and are repellents for Drosophila. These compounds are also strong repellents for mosquitoes. The candidates contain chemicals that do not dissolve plastic, are affordable and smell mildly like grapes, with three considered safe in human foods. Our findings pave the way to discover new generations of repellents that will help fight deadly insect-borne diseases worldwide.

Antennal Olfactory Physiology and Behavior of Males of the Ponerine Ant Harpegnathos saltator.

In comparison to the large amount of study on the communication abilities of females in ant societies and their associated chemical ecology and sensory physiology, such study of male ants has been largely ignored; accordingly, little is known about their olfactory sensory capabilities. To address this, we explored peripheral odor sensitivities in male Harpegnathos saltator by measuring the electrophysiological activity of olfactory sensory neurons within antennal trichoid and coeloconic sensilla using an extracellular recording technique. In an initial trial of 46 compounds, sensilla trichodea responded strongly to two alarm pheromone components, while a limited number of non-hydrocarbon odorants elicited strong responses in sensilla coeloconica. Both sensillar types responded indifferently to 31 cuticular hydrocarbons (CHCs) and synthetic long-chain hydrocarbons (HCs) typically found on insect cuticle. In a search for sensilla responding to CHCs and other compounds, we found some sensilla that responded to synthetic HCs and CHCs from virgin queen postpharyngeal glands that are potentially used in close range mate recognition. Olfactometer bioassays of male ants to 15 non-HCs correlated sensory responsiveness to the respective behavioral responses. Comparing olfactory responses between H. saltator males and females, we found that sensilla coeloconica and basiconica of workers showed greater responses and broader selectivity to all compounds. The rarity of CHC-responding trichoid sensilla in Harpegnathos males suggests a more specific role in sexual communication compared to that in females, which use CHCs in a broader communication context.

Ultra-prolonged activation of CO2-sensing neurons disorients mosquitoes.

Carbon dioxide (CO(2)) present in exhaled air is the most important sensory cue for female blood-feeding mosquitoes, causing activation of long-distance host-seeking flight, navigation towards the vertebrate host and, in the case of Aedes aegypti, increased sensitivity to skin odours. The CO(2) detection machinery is therefore an ideal target to disrupt host seeking. Here we use electrophysiological assays to identify a volatile odorant that causes an unusual, ultra-prolonged activation of CO(2)-detecting neurons in three major disease-transmitting mosquitoes: Anopheles gambiae, Culex quinquefasciatus and A. aegypti. Importantly, ultra-prolonged activation of these neurons severely compromises their ability subsequently to detect CO(2) for several minutes. We also identify odours that strongly inhibit CO(2)-sensitive neurons as candidates for use in disruption of host-seeking behaviour, as well as an odour that evokes CO(2)-like activity and thus has potential use as a lure in trapping devices. Analysis of responses to panels of structurally related odours across the three mosquitoes and Drosophila, which have related CO(2)-receptor proteins, reveals a pattern of inhibition that is often conserved. We use video tracking in wind-tunnel experiments to demonstrate that the novel ultra-prolonged activators can completely disrupt CO(2)-mediated activation as well as source-finding behaviour in Aedes mosquitoes, even after the odour is no longer present. Lastly, semi-field studies demonstrate that use of ultra-prolonged activators disrupts CO(2)-mediated hut entry behaviour of Culex mosquitoes. The three classes of CO(2)-response-modifying odours offer powerful instruments for developing new generations of insect repellents and lures, which even in small quantities can interfere with the ability of mosquitoes to seek humans.

The Drosophila melanogaster phospholipid flippase dATP8B is required for odorant receptor function.

The olfactory systems of insects are fundamental to all aspects of their behaviour, and insect olfactory receptor neurons (ORNs) exhibit exquisite specificity and sensitivity to a wide range of environmental cues. In Drosophila melanogaster, ORN responses are determined by three different receptor families, the odorant (Or), ionotropic-like (IR) and gustatory (Gr) receptors. However, the precise mechanisms of signalling by these different receptor families are not fully understood. Here we report the unexpected finding that the type 4 P-type ATPase phospholipid transporter dATP8B, the homologue of a protein associated with intrahepatic cholestasis and hearing loss in humans, is crucial for Drosophila olfactory responses. Mutations in dATP8B severely attenuate sensitivity of odorant detection specifically in Or-expressing ORNs, but do not affect responses mediated by IR or Gr receptors. Accordingly, we find dATP8B to be expressed in ORNs and localised to the dendritic membrane of the olfactory neurons where signal transduction occurs. Localisation of Or proteins to the dendrites is unaffected in dATP8B mutants, as is dendrite morphology, suggesting instead that dATP8B is critical for Or signalling. As dATP8B is a member of the phospholipid flippase family of ATPases, which function to determine asymmetry in phospholipid composition between the outer and inner leaflets of plasma membranes, our findings suggest a requirement for phospholipid asymmetry in the signalling of a specific family of chemoreceptor proteins.

Olfactory Mechanisms for Discovery of Odorants to Reduce Insect-Host Contact.

Insects have developed highly sophisticated and sensitive olfactory systems to find animal or plant hosts for feeding. Some insects vector pathogens that cause diseases in hundreds of millions of people and destroy billions of dollars of food products every year. There is great interest, therefore, in understanding how the insect olfactory system can be manipulated to reduce their contact with hosts. Here, we review recent advances in our understanding of insect olfactory detection mechanisms, which may serve as a foundation for designing insect control programs based on manipulation of their behaviors by using odorants. Because every insect species has a unique set of olfactory receptors and olfactory-mediated behaviors, we focus primarily on general principles of odor detection that potentially apply to most insects. While these mechanisms have emerged from studies on model systems for study of insect olfaction, such as Drosophila melanogaster, they provide a foundation for discovery of odorants to repel vector insects or reduce their host-seeking behavior.

Modification of CO2 avoidance behaviour in Drosophila by inhibitory odorants.

The fruitfly Drosophila melanogaster exhibits a robust and innate olfactory-based avoidance behaviour to CO(2), a component of odour emitted from stressed flies. Specialized neurons in the antenna and a dedicated neuronal circuit in the higher olfactory system mediate CO(2) detection and avoidance. However, fruitflies need to overcome this avoidance response in some environments that contain CO(2) such as ripening fruits and fermenting yeast, which are essential food sources. Very little is known about the molecular and neuronal basis of this unique, context-dependent modification of innate olfactory avoidance behaviour. Here we identify a new class of odorants present in food that directly inhibit CO(2)-sensitive neurons in the antenna. Using an in vivo expression system we establish that the odorants act on the Gr21a/Gr63a CO(2) receptor. The presence of these odorants significantly and specifically reduces CO(2)-mediated avoidance behaviour, as well as avoidance mediated by 'Drosophila stress odour'. We propose a model in which behavioural avoidance to CO(2) is directly influenced by inhibitory interactions of the novel odours with CO(2) receptors. Furthermore, we observe differences in the temporal dynamics of inhibition: the effect of one of these odorants lasts several minutes beyond the initial exposure. Notably, animals that have been briefly pre-exposed to this odorant do not respond to the CO(2) avoidance cue even after the odorant is no longer present. We also show that related odorants are effective inhibitors of the CO(2) response in Culex mosquitoes that transmit West Nile fever and filariasis. Our findings have broader implications in highlighting the important role of inhibitory odorants in olfactory coding, and in their potential to disrupt CO(2)-mediated host-seeking behaviour in disease-carrying insects like mosquitoes.

Phylogenetic and transcriptomic analysis of chemosensory receptors in a pair of divergent ant species reveals sex-specific signatures of odor coding.

Ants are a highly successful family of insects that thrive in a variety of habitats across the world. Perhaps their best-known features are complex social organization and strict division of labor, separating reproduction from the day-to-day maintenance and care of the colony, as well as strict discrimination against foreign individuals. Since these social characteristics in ants are thought to be mediated by semiochemicals, a thorough analysis of these signals, and the receptors that detect them, is critical in revealing mechanisms that lead to stereotypic behaviors. To address these questions, we have defined and characterized the major chemoreceptor families in a pair of behaviorally and evolutionarily distinct ant species, Camponotus floridanus and Harpegnathos saltator. Through comprehensive re-annotation, we show that these ant species harbor some of the largest yet known repertoires of odorant receptors (Ors) among insects, as well as a more modest number of gustatory receptors (Grs) and variant ionotropic glutamate receptors (Irs). Our phylogenetic analyses further demonstrate remarkably rapid gains and losses of ant Ors, while Grs and Irs have also experienced birth-and-death evolution to different degrees. In addition, comparisons of antennal transcriptomes between sexes identify many chemoreceptors that are differentially expressed between males and females and between species. We have also revealed an agonist for a worker-enriched OR from C. floridanus, representing the first case of a heterologously characterized ant tuning Or. Collectively, our analysis reveals a large number of ant chemoreceptors exhibiting patterns of differential expression and evolution consistent with sex/species-specific functions. These differentially expressed genes are likely associated with sex-based differences, as well as the radically different social lifestyles observed between C. floridanus and H. saltator, and thus are targets for further functional characterization. Our findings represent an important advance toward understanding the molecular basis of social interactions and the differential chemical ecologies among ant species.

Odor coding in a disease-transmitting herbivorous insect, the Asian citrus psyllid.

Olfactory systems discriminate odorants very efficiently and herbivorous insects use them to find hosts in confounding and complex odor landscapes. The Asian citrus psyllid (ACP), Diaphorina citri, feeds on citrus flush and transmits Candidatus Liberibacter that causes citrus greening disease globally. Here, we perform a systematic analysis of odor detection in the ACP antenna using single-unit electrophysiology of rhinarial plate sensilla to a large panel of odorants from plants. We identify neurons that respond strongly to odorants found in the host citrus plants. Comparisons with the generalist yeast-feeding Drosophila melanogaster and specialist anthropophilic Anopheles gambiae reveal differences in odor-coding strategies for the citrus-seeking ACP. These findings provide a foundation for understanding host-odor coding in herbivorous insects.

Plasticity of gene expression in the nervous system by exposure to environmental odorants that inhibit HDACs.

Eukaryotes respond to secreted metabolites from the microbiome. However, little is known about the effects of exposure to volatiles emitted by microbes or in the environment that we are exposed to over longer durations. Using Drosophila melanogaster, we evaluated a yeast-emitted volatile, diacetyl, found at high levels around fermenting fruits where they spend long periods of time. Exposure to the diacetyl molecules in headspace alters gene expression in the antenna. In vitro experiments demonstrated that diacetyl and structurally related volatiles inhibited conserved histone deacetylases (HDACs), increased histone-H3K9 acetylation in human cells, and caused changes in gene expression in both Drosophila and mice. Diacetyl crosses the blood-brain barrier and exposure caused modulation of gene expression in the mouse brain, therefore showing potential as a neuro-therapeutic. Using two separate disease models previously known to be responsive to HDAC inhibitors, we evaluated the physiological effects of volatile exposure. Diacetyl exposure halted proliferation of a neuroblastoma cell line in culture. Exposure to diacetyl vapors slowed progression of neurodegeneration in a Drosophila model for Huntington's disease. These changes strongly suggest that certain volatiles in the surroundings can have profound effects on histone acetylation, gene expression, and physiology in animals.

Chemosensory detection of aversive concentrations of ammonia and basic volatile amines in insects.

Basic volatiles like ammonia are found in insect environments, and at high concentrations cause an atypical action potential burst, followed by inhibition in multiple classes of olfactory receptor neurons (ORNs) in Drosophila melanogaster. During the period of inhibition, ORNs are unable to fire action potentials to their ligands but continue to display receptor potentials. An increase in calcium is also observed in antennal cells of Drosophila and Aedes aegypti. In the gustatory system, ammonia inhibits sugar and salt responses in a dose-dependent manner. Other amines show similar effects in both gustatory and olfactory neurons, correlated with basicity. The concentrations that inhibit neurons reduce proboscis extension to sucrose in Drosophila. In Aedes, a brief exposure to volatile ammonia abolishes attraction to human skin odor for several minutes. These findings reveal an effect that prevents detection of attractive ligands in the olfactory and gustatory systems and has potential in insect control.

Plasticity of gene expression in the nervous system by exposure to environmental odorants that inhibit HDACs.

Eukaryotes are often exposed to microbes and respond to their secreted metabolites, such as the microbiome in animals or commensal bacteria in roots. Little is known about the effects of long-term exposure to volatile chemicals emitted by microbes, or other volatiles that we are exposed to over a long duration. Using the model system Drosophila melanogaster, we evaluate a yeast emitted volatile, diacetyl, found in high levels around fermenting fruits where they spend long periods of time. We find that exposure to just the headspace containing the volatile molecules can alter gene expression in the antenna. Experiments showed that diacetyl and structurally related volatile compounds inhibited human histone-deacetylases (HDACs), increased histone-H3K9 acetylation in human cells, and caused wide changes in gene expression in both Drosophila and mice. Diacetyl crosses the blood-brain barrier and exposure causes modulation of gene expression in the brain, therefore has potential as a therapeutic. Using two separate disease models known to be responsive to HDAC-inhibitors, we evaluated physiological effects of volatile exposure. First, we find that the HDAC inhibitor also halts proliferation of a neuroblastoma cell line in culture as predicted. Next, exposure to vapors slows progression of neurodegeneration in a Drosophila model for Huntington's disease. These changes strongly suggest that unbeknown to us, certain volatiles in the surroundings can have profound effects on histone acetylation, gene expression and physiology in animals.

Pentylamine inhibits humidity detection in insect vectors of human and plant borne pathogens.

Insects house humidity-sensing neurons in the antenna, which is presumed to be important for a variety of behaviors and survival since water is a crucial component of the environment. Here we use the simple olfactory system of the Asian Citrus Psyllid (ACP), a citrus pest that transmits a deadly bacterium, to identify volatile amines that significantly inhibited humidity-induced activation of antennal neurons. The inhibition of action potentials is observed by single sensillum recordings and mixing these odorants with humid air abolished the humidity avoidance behavior of ACP. The inhibition is conserved in the humidity-sensing coeloconic neurons of dipteran Drosophila melanogaster that are known to detect humidity, but it is not seen in other coeloconic neurons that are not sensitive to humidity. Dipteran mosquitoes Aedes aegypti and Anopheles gambiae oviposit in water, and the addition of the humidity-inhibiting odorants in a two-choice oviposition assay significantly reduces oviposition. Our results demonstrate that a naturally occurring volatile compound can effectively "mask" detection of an important environmental cue and modify behavior of important vectors of plant and human disease pathogens. Odorants targeting the conserved humidity sensing system of insects, therefore, offer a novel strategy for modifying their behavior.

Mechanisms of odor receptor gene choice in Drosophila.

A remarkable problem in neurobiology is how olfactory receptor neurons (ORNs) select, from among a large odor receptor repertoire, which receptors to express. We use computational algorithms and mutational analysis to define positive and negative regulatory elements that are required for selection of odor receptor (Or) genes in the proper olfactory organ of Drosophila, and we identify an element that is essential for selection in one ORN class. Two odor receptors are coexpressed by virtue of the alternative splicing of a single gene, and we identify dicistronic mRNAs that each encode two receptors. Systematic analysis reveals no evidence for negative feedback regulation, but provides evidence that the choices made by neighboring ORNs of a sensillum are coordinated via the asymmetric segregation of regulatory factors from a common progenitor. We show that receptor gene choice in Drosophila also depends on a combinatorial code of transcription factors to generate the receptor-to-neuron map.

A regulatory code for neuron-specific odor receptor expression.

Olfactory receptor neurons (ORNs) must select-from a large repertoire-which odor receptors to express. In Drosophila, most ORNs express one of 60 Or genes, and most Or genes are expressed in a single ORN class in a process that produces a stereotyped receptor-to-neuron map. The construction of this map poses a problem of receptor gene regulation that is remarkable in its dimension and about which little is known. By using a phylogenetic approach and the genome sequences of 12 Drosophila species, we systematically identified regulatory elements that are evolutionarily conserved and specific for individual Or genes of the maxillary palp. Genetic analysis of these elements supports a model in which each receptor gene contains a zip code, consisting of elements that act positively to promote expression in a subset of ORN classes, and elements that restrict expression to a single ORN class. We identified a transcription factor, Scalloped, that mediates repression. Some elements are used in other chemosensory organs, and some are conserved upstream of axon-guidance genes. Surprisingly, the odor response spectra and organization of maxillary palp ORNs have been extremely well-conserved for tens of millions of years, even though the amino acid sequences of the receptors are not highly conserved. These results, taken together, define the logic by which individual ORNs in the maxillary palp select which odor receptors to express.

Prolonged activation of carbon dioxide-sensitive neurons in mosquitoes.

Many insects can detect carbon dioxide (CO2) plumes using a conserved receptor made up of members of the gustatory receptor (Gr) family Gr1, Gr2 and Gr3. Mosquitoes are attracted to host animals for blood meals using plumes of CO2 in the exhaled breath using the receptor expressed in the A neuron of the capitate peg sensilla type on the maxillary palps. The receptor is known to also detect several other classes of odorants, including ones emitted from human skin. Here, we discover that a common skin odorant, butyric acid, can cause a phasic activation followed by an unusually prolonged tonic activity after the stimulus is over in the CO2 neurons of mosquitoes. The effect is conserved in both Aedes aegypti and Anopheles gambiae mosquitoes. This raises a question about its role in a mosquito's preference for the skin odour of different individuals. Butyric acid belongs to a small number of odorants known to cause the prolonged activation of the CO2 receptor. A chemical informatic analysis identifies a specific set of physico-chemical features that can be used in a machine learning predictive model for the prolonged activators. Interestingly, this set is different from physico-chemical features selected for activators or inhibitors, indicating that each has a distinct structural basis. The structural understanding opens up an opportunity to find novel ligands to manipulate the CO2 receptor and mosquito behaviour.