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 behavioral response for the odorant. The valence of an odorant changes with concentration, typically becoming aversive at higher concentrations. Interestingly in the Drosophila larvae, the odorant (E)-2-hexenal is aversive at low concentrations and attractive at higher concentrations. We investigate the molecular and neural basis of this phenomenon focusing on how activities of different olfactory neurons conveying opposing effects dictate behaviors. We identify 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 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.

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.

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.

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.

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.

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.

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.

Predicting novel drugs for SARS-CoV-2 using machine learning from a >10 million chemical space.

There is an urgent need for the identification of effective therapeutics for COVID-19 and we have developed a machine learning drug discovery pipeline to identify several drug candidates. First, we collect assay data for 65 target human proteins known to interact with the SARS-CoV-2 proteins, including the ACE2 receptor. Next, we train machine learning models to predict inhibitory activity and use them to screen FDA registered chemicals and approved drugs (~100,000) and ~14 million purchasable chemicals. We filter predictions according to estimated mammalian toxicity and vapor pressure. Prospective volatile candidates are proposed as novel inhaled therapeutics since the nasal cavity and respiratory tracts are early bottlenecks for infection. We also identify candidates that act across multiple targets as promising for future analyses. We anticipate that this theoretical study can accelerate testing of two categories of therapeutics: repurposed drugs suited for short-term approval, and novel efficacious drugs suitable for a long-term follow up.

Circadian Regulation of Light-Evoked Attraction and Avoidance Behaviors in Daytime- versus Nighttime-Biting Mosquitoes.

Mosquitoes pose widespread threats to humans and other animals as disease vectors [1]. Day- versus night-biting mosquitoes occupy distinct time-of-day niches [2, 3]. Here, we explore day- versus night-biting female and male mosquitoes' innate temporal attraction/avoidance behavioral responses to light and their regulation by circadian circuit and molecular mechanisms. Day-biting mosquitoes Aedes aegypti, particularly females, are attracted to light during the day regardless of spectra. In contrast, night-biting mosquitoes, Anopheles coluzzii, specifically avoid ultraviolet (UV) and blue light during the day. Behavioral attraction to/avoidance of light in both species change with time of day and show distinct sex and circadian neural circuit differences. Males of both diurnal and nocturnal mosquito species show reduced UV light avoidance in anticipation of evening onset relative to females. The circadian neural circuits of diurnal/day- and nocturnal/night-biting mosquitoes based on PERIOD (PER) and pigment-dispersing factor (PDF) expression show similar but distinct circuit organizations between species. The basis of diurnal versus nocturnal behaviors is driven by molecular clock timing, which cycles in anti-phase between day- versus night-biting mosquitoes. Observed differences at the neural circuit and protein levels provide insight into the fundamental basis underlying diurnality versus nocturnality. Molecular disruption of the circadian clock severely interferes with light-evoked attraction/avoidance behaviors in mosquitoes. In summary, attraction/avoidance behaviors show marked differences between day- versus night-biting mosquitoes, but both classes of mosquitoes are circadian and light regulated, which may be applied toward species-specific control of harmful mosquitoes.

Predicting Human Olfactory Perception from Activities of Odorant Receptors.

Odor perception in humans is initiated by activation of odorant receptors (ORs) in the nose. However, the ORs linked to specific olfactory percepts are unknown, unlike in vision or taste where receptors are linked to perception of different colors and tastes. The large family of ORs (~400) and multiple receptors activated by an odorant present serious challenges. Here, we first use machine learning to screen ~0.5 million compounds for new ligands and identify enriched structural motifs for ligands of 34 human ORs. We next demonstrate that the activity of ORs successfully predicts many of the 146 different perceptual qualities of chemicals. Although chemical features have been used to model odor percepts, we show that biologically relevant OR activity is often superior. Interestingly, each odor percept could be predicted with very few ORs, implying they contribute more to each olfactory percept. A similar model is observed in Drosophila where comprehensive OR-neuron data are available.

Contributions of the Conserved Insect Carbon Dioxide Receptor Subunits to Odor Detection.

The CO2 receptor in mosquitoes is broadly tuned to detect many diverse odorants. The receptor consists of three subunits (Gr1, Gr2, and Gr3) in mosquitoes but only two subunits in Drosophila: Gr21a (Gr1 ortholog) and Gr63a (Gr3 ortholog). We demonstrate that Gr21a is required for CO2 responses in Drosophila, as has been shown for Gr63a. Next, we generate a Drosophila double mutant for Gr21a and Gr63a, and in this background, we functionally express combinations of Aedes Gr1, Gr2, and Gr3 genes in the CO2 empty neuron. Only two subunits, Gr2 and Gr3, suffice for response to CO2. Addition of Gr1 increases sensitivity to CO2, whereas it decreases the response to pyridine. The inhibitory effect of the antagonist isobutyric acid is observed upon addition of Gr1. Gr1 therefore increases the diversity of ligands of the receptor and modulates the response of the receptor complex.

Repellency Assessment of Nepeta cataria Essential Oils and Isolated Nepetalactones on Aedes aegypti.

There is an increased need for improved and affordable insect repellents to reduce transmission of rapidly spreading diseases with high mortality rates. Natural products are often used when DEET cannot be afforded or accessed and when consumers choose not to use a synthetic repellent. The essential oils from two newly bred Nepeta cataria (catnip) plants representing two different chemotypes and their respective isolated nepetalactone isomers were evaluated as mosquito repellents against Aedes aegypti mosquitoes that transmit the Zika and Dengue virus in a one choice landing rate inhibition assay. A dose response curve was generated for each treatment and a time course analysis of repellency was performed over 24 hours with a N. cataria essential oil sample. The results indicate that all essential oil samples and their respective purified nepetalactone isomers were able to achieve greater than 95% repellency. Between two and four hours, the ability to repel more than 95% of the mosquitoes diminished. At the lowest concentrations tested, the nepetalactones and crude essential oil samples were more effective than DEET at reducing the number of mosquito landings.

Odorant ligands for the CO2 receptor in two Anopheles vectors of malaria.

Exhaled CO2 is an important host-seeking cue for Anopheles mosquitoes, which is detected by a highly conserved heteromeric receptor consisting of three 7-transmembrane proteins Gr22, Gr23, and Gr24. The CO2 receptor neuron has been shown to also respond sensitively to a variety of odorants in Aedes aegypti. The detection of CO2 is important for upwind navigation and for enhancing the attraction to body heat as well as to skin odorants. The orthologs of the CO2 receptor proteins are present in malaria-transmitting mosquitoes like Anopheles coluzzii and Anopheles sinensis. Activators and inhibitors of the CO2-neuron were tested on the maxillary palps in these two species by single-sensillum electrophysiology. The electrophysiological testing of three prolonged-activator odorants identified originally in Aedes aegypti also showed varying ability to reduce the CO2-ellicited increase in spikes. These findings provide a foundation for comparing the functional conservation with the evolutionary conservation of an important class of odorant receptor. The identification of a suite of natural odorants that can be used to modify the CO2-detection pathway may also contribute to odor-blends that can alter the behavior of these disease transmitting mosquitoes.

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.

Signaling Mode of the Broad-Spectrum Conserved CO2 Receptor Is One of the Important Determinants of Odor Valence in Drosophila.

Odor detection involves hundreds of olfactory receptors from diverse families, making modeling of hedonic valence of an odorant difficult, even in Drosophila melanogaster where most receptors have been deorphanised. We demonstrate that a broadly tuned heteromeric receptor that detects CO2 (Gr21a, Gr63a) and other odorants is a key determinant of valence along with a few members of the Odorant receptor family in a T-maze, but not in a trap assay. Gr21a and Gr63a have atypically high amino acid conservation in Dipteran insects, and they use both inhibition and activation to convey positive or negative valence for numerous odorants. Inhibitors elicit a robust Gr63a-dependent attraction, while activators, strong aversion. The attractiveness of inhibitory odorants increases with increasing background CO2 levels, providing a mechanism for behavior modulation in odor blends. In mosquitoes, valence is switched and activation of the orthologous receptor conveys attraction. Reverse chemical ecology enables the identification of inhibitory odorants to reduce attraction of mosquitoes to skin.

Specialized odorant receptors in social insects that detect cuticular hydrocarbon cues and candidate pheromones.

Eusocial insects use cuticular hydrocarbons as components of pheromones that mediate social behaviours, such as caste and nestmate recognition, and regulation of reproduction. In ants such as Harpegnathos saltator, the queen produces a pheromone which suppresses the development of workers' ovaries and if she is removed, workers can transition to a reproductive state known as gamergate. Here we functionally characterize a subfamily of odorant receptors (Ors) with a nine-exon gene structure that have undergone a massive expansion in ants and other eusocial insects. We deorphanize 22 representative members and find they can detect cuticular hydrocarbons from different ant castes, with one (HsOr263) that responds strongly to gamergate extract and a candidate queen pheromone component. After systematic testing with a diverse panel of hydrocarbons, we find that most Harpegnathos saltator Ors are narrowly tuned, suggesting that several receptors must contribute to detection and discrimination of different cuticular hydrocarbons important in mediating eusocial behaviour.Cuticular hydrocarbons (CHC) mediate the interactions between individuals in eusocial insects, but the sensory receptors for CHCs are unclear. Here the authors show that in ants such as H. saltator, the 9-exon subfamily of odorant receptors (HsOrs) responds to CHCs, and ectopic expression of HsOrs in Drosophila neurons imparts responsiveness to CHCs.

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.

Chemosensory sensitivity reflects reproductive status in the ant Harpegnathos saltator.

Insects communicate with pheromones using sensitive antennal sensilla. Although trace amounts of pheromones can be detected by many insects, context-dependent increased costs of high sensitivity might lead to plasticity in sensillum responsiveness. We have functionally characterized basiconic sensilla of the ant Harpegnathos saltator for responses to general odors in comparison to cuticular hydrocarbons which can act as fertility signals emitted by the principal reproductive(s) of a colony to inhibit reproduction by worker colony members. When released from inhibition workers may become reproductive gamergates. We observed plasticity in olfactory sensitivity after transition to reproductive status with significant reductions in electrophysiological responses to several long-chained cuticular hydrocarbons. Although gamergates lived on average five times longer than non-reproductive workers, the shift to reproductive status rather than age differences matched the pattern of changes in olfactory sensitivity. Decreasing sensillum responsiveness to cuticular hydrocarbons could potentially reduce mutually inhibitory or self-inhibitory effects on gamergate reproduction.

The Role of Histone Deacetylase 6 in Synaptic Plasticity and Memory.

Histone deacetylases (HDACs) have been extensively studied as drug targets in neurodegenerative diseases, but less is known about their role in healthy neurons. We tested zinc-dependent HDACs using RNAi in Drosophila melanogaster and found memory deficits with RPD3 and HDAC6. We demonstrate that HDAC6 is required in both the larval and adult stages for normal olfactory memory retention. Neuronal expression of HDAC6 rescued memory deficits, and we demonstrate that the N-terminal deacetylase (DAC) domain is required for this ability. This suggests that deacetylation of synaptic targets associated with the first DAC domain, such as the active-zone scaffold Bruchpilot, is required for memory retention. Finally, electrophysiological experiments at the neuromuscular junction reveal that HDAC6 mutants exhibit a partial block of homeostatic plasticity, suggesting that HDAC6 may be required for the stabilization of synaptic strength. The learning deficit we observe in HDAC6 mutants could be a behavioral consequence of these synaptic defects.

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.