Anandasankar Ray

Modification of CO2 avoidance behaviour in Drosophila by inhibitory odorants

The fruitfly Drosophila melanogaster exhibits a robust and innate olfactory-based avoidance behaviour to CO2, a component of odour emitted from stressed flies. Specialized neurons in the antenna and a dedicated neuronal circuit in the higher olfactory system mediate CO2 detection and avoidance. However, fruitflies need to overcome this avoidance response in some environments that contain CO2 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 CO2-sensitive neurons in the antenna. Using an in vivo expression system we establish that the odorants act on the Gr21a/Gr63a CO2 receptor. The presence of these odorants significantly and specifically reduces CO2-mediated avoidance behaviour, as well as avoidance mediated by ‘Drosophila stress odour’. We propose a model in which behavioural avoidance to CO2 is directly influenced by inhibitory interactions of the novel odours with CO2 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 CO2 avoidance cue even after the odorant is no longer present. We also show that related odorants are effective inhibitors of the CO2 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 CO2-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.

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

Carbon dioxide (CO2) 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 CO2 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 CO2-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 CO2 for several minutes. We also identify odours that strongly inhibit CO2-sensitive neurons as candidates for use in disruption of host-seeking behaviour, as well as an odour that evokes CO2-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 CO2-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 CO2-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 CO2-mediated hut entry behaviour of Culex mosquitoes. The three classes of CO2-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.

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.

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.

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.

Epigenetic (re)programming of caste-specific behavior in the ant Camponotus floridanus

Epigenetic control of caste-specific foraging In carpenter ants, separate behavioral classes, known as castes, are determined by the epigenetic regulation of genes. Simola et al. treated ants of different castes with drugs that affected histone acetylation. Reducing histone acetylation stimulated scouting and foraging behavior. The foraging and scouting behaviors of young ants were permanently changed by directly injecting their brains with histone acetylation inhibitors. Science, this issue p. 10.1126/science.aac6633 Changes in histone acetylation explain differences in the foraging and scouting patterns of different castes of carpenter ants. INTRODUCTION Eusocial insects, such as ants, live a communal lifestyle within colonies of close genetic relatives. Colony members are organized into castes defined by behavioral and, in some species, morphological traits. This caste system allows for colonial division of labor and is a key adaptation of eusocial insects. However, there is limited understanding of the molecular regulation of caste-specific behavior and the principles underlying division of labor. In the carpenter ant Camponotus floridanus, morphologically distinct worker castes called minors and majors exhibit unique patterns of histone posttranslational modifications, including lysine acetylation regulated by CBP [cyclic adenosine monophosphate response element–binding protein (CREB) binding protein], a conserved histone acetyltransferase (HAT). RATIONALE Because chromatin regulators such as CBP have been associated with caste-specific traits, we tested whether caste-specific behavioral states in eusocial insects are functionally regulated via epigenetic mechanisms. We assessed innate differences in foraging and scouting (by the lead forager), classic altruistic behaviors of eusocial systems, between minor and major worker castes in C. floridanus. We examined whether CBP and histone deacetylases (HDACs) functionally regulate caste-specific foraging and scouting behaviors. Further, we tested whether caste-specific behavioral states may be reprogrammed through epigenomic manipulations. RESULTS C. floridanus minors and majors exhibited innate differences in foraging and scouting behaviors, with minors performing the bulk of both activities. Treatments with small-molecule inhibitors of class I and II HDAC activity (HDACi) enhanced foraging and scouting. This gain of function was suppressed by treatment with a small-molecule HAT inhibitor of CBP (HATi). Transcriptome and chromatin analyses in the brains of minors treated with HATi and HDACi revealed changes in genes linked to regions of hyperacetylated histone H3 Lys27 (a lysine targeted by CBP) near CBP binding sites. Although untreated majors rarely foraged, suppression of histone deacetylation by injection of HDACi or small interfering RNAs (siRNAs) against the HDAC-encoding gene Rpd3 into young major brains was sufficient to induce and sustain minor-like foraging and scouting for up to 50 days. Strikingly, coinjection of CBP HATi suppressed HDACi-induced foraging and scouting in majors. CONCLUSION Caste-specific foraging and scouting behaviors are tightly linked to morphology and are likely regulated epigenetically by the balance between CBP-mediated acetylation and HDAC-mediated deacetylation of histones in the ant central brain. Thus, behavioral plasticity can be manipulated in the ant C. floridanus by pharmacological and genetic tools that target chromatin regulatory enzymes to stimulate, inhibit, and reprogram behavior. These findings reveal the epigenome as a likely substrate underlying caste-based division of labor in eusocial insects. Furthermore, in light of the conserved role of CBP in learning and memory in both invertebrates and mammals, these data suggest that CBP-mediated histone acetylation may similarly facilitate the complex social interactions found in vertebrate species. An epigenetic model for division of labor. Left: Workers were injected at eclosion and tested for foraging activity. HDAC inhibition (HDACi) with chromatin drugs or siRNA enhanced foraging; HATi suppressed foraging. Right: Minor and major workers express distinct behavioral ontogenies. Minors forage earlier in life and with greater intensity than majors. HDACi in majors stimulated minor-like foraging behavior, a gain of function suppressed by HATi treatment. Eusocial insects organize themselves into behavioral castes whose regulation has been proposed to involve epigenetic processes, including histone modification. In the carpenter ant Camponotus floridanus, morphologically distinct worker castes called minors and majors exhibit pronounced differences in foraging and scouting behaviors. We found that these behaviors are regulated by histone acetylation likely catalyzed by the conserved acetyltransferase CBP. Transcriptome and chromatin analysis in brains of scouting minors fed pharmacological inhibitors of CBP and histone deacetylases (HDACs) revealed hundreds of genes linked to hyperacetylated regions targeted by CBP. Majors rarely forage, but injection of a HDAC inhibitor or small interfering RNAs against the HDAC Rpd3 into young major brains induced and sustained foraging in a CBP-dependent manner. Our results suggest that behavioral plasticity in animals may be regulated in an epigenetic manner via histone modification.

A New Drosophila POU Gene, pdm3, Acts in Odor Receptor Expression and Axon Targeting of Olfactory Neurons

Olfaction depends on the differential activation of olfactory receptor neurons (ORNs) and on the proper transmission of their activities to the brain. ORNs select individual receptors to express, and they send axons to particular targets in the brain. Little is known about the molecular mechanisms underlying either process. We have identified a new Drosophila POU gene, pdm3, that is expressed in ORNs. Genetic analysis shows that pdm3 is required for odor response in one class of ORNs. We find that pdm3 acts in odor receptor expression in this class, and that the odor response can be rescued by the receptor. Another POU gene, acj6, is required for receptor expression in the same class, and we find a genetic interaction between the two POU genes. The results support a role for a POU gene code in receptor gene choice. pdm3 is also expressed in other ORN classes in which it is not required for receptor expression. For two of these classes, pdm3 is required for normal axon targeting. Thus, this mutational analysis, the first for a POU class VI gene, demonstrates a role for pdm3 in both of the processes that define the functional organization of ORNs in the olfactory system.

Mechanisms of odorant receptor gene choice in Drosophila and vertebrates

Odorant receptors are encoded by extremely large and divergent families of genes. Each receptor is expressed in a small proportion of neurons in the olfactory organs, and each neuron in turn expresses just one odorant receptor gene. This fundamental property of the peripheral olfactory system is widely conserved across evolution, and observed in vertebrates, like mice, and invertebrates, like Drosophila, despite their olfactory receptor gene families being evolutionarily unrelated. Here we review the progress that has been made in these two systems to understand the intriguing and elusive question: how does a single neuron choose to express just one of many possible odorant receptors and exclude expression of all others?

Cuticular Hydrocarbon Pheromones for Social Behavior and Their Coding in the Ant Antenna

The sophisticated organization of eusocial insect societies is largely based on the regulation of complex behaviors by hydrocarbon pheromones present on the cuticle. We used electrophysiology to investigate the detection of cuticular hydrocarbons (CHCs) by female-specific olfactory sensilla basiconica on the antenna of Camponotus floridanus ants through the utilization of one of the largest family of odorant receptors characterized so far in insects. These sensilla, each of which contains multiple olfactory receptor neurons, are differentially sensitive to CHCs and allow them to be classified into three broad groups that collectively detect every hydrocarbon tested, including queen and worker-enriched CHCs. This broad-spectrum sensitivity is conserved in a related species, Camponotus laevigatus, allowing these ants to detect CHCs from both nestmates and non-nestmates. Behavioral assays demonstrate that these ants are excellent at discriminating CHCs detected by the antenna, including enantiomers of a candidate queen pheromone that regulates the reproductive division of labor.