Anandasankar Ray - US grants

DESCRIPTION (provided by applicant): Aedes aegypti mosquitoes transmit Dengue and Yellow Fever virus, and Culex quinquefasciatus mosquitoes transmit the West Nile virus and filarial parasite Wuchereria bancrofti to hundreds of millions of people. Methods for inhibiting vector- human contact, particularly in conjunction with other existing programs of vector control, may play a critical role in controlling the spread of these diseases. Female mosquitoes, like most other blood-feeding insects, identify their human hosts primarily using olfactory cues like carbon dioxide in exhaled breath, as well as other odors present in skin emanations. The goal of this research is to modify odor-guided host-seeking behavior of Aedes and Culex mosquitoes using cheap and environmentally safe odor molecules that can be used in very small quantities as effective trapping agents, masking agents and repellents. To achieve these goals we will focus on functional analyses of the CO2 sensing neuron. CO2 is detected in this neuron using a receptor comprising three members of the Gustatory receptor family that encode a highly conserved heteromeric CO2 receptor. We plan to use an integrated platform of neurophysiology, chemical- informatics, molecular, genetic and behavioral methods to;1) identify effective volatile agonists and antagonists for the CO2 receptors, 2) test the effects of these molecules on behavior of female Aedes and Culex mosquitoes, in particular, for the ability of antagonists in masking agents or repellents, and 3) agonists to improve trapping efficiency. In preliminary studies, we have identified a few volatile inhibitors and activators of the CO2 receptor in Aedes and Culex. We now propose to conduct further studies to identify more effective chemicals that inhibit or activate physiological and behavioral responses to CO2 in female mosquitoes. Successful completion of the proposed studies will lead to the development of a new generation of effective and safe insect repellents and lures that act by modifying CO2 receptor function. PUBLIC HEALTH RELEVANCE: Aedes and Culex mosquitoes transmit several diseases including Dengue and lymphatic filariasis, and it is estimated that there are more than a hundred million people worldwide that are infected. Mosquitoes find their human hosts using the sense of smell. The goal of the proposed research is to modify odor-guided host-seeking behavior of Aedes and Culex mosquitoes using cheap and environmentally safe odor molecules that can be used in very small quantities as effective trapping agents, masking agents and repellents. Successful completion of the proposed studies will lead to the development of a new generation of effective and safe insect repellents and lures that act by modifying odor receptor function.

DESCRIPTION (provided by applicant): Genes that are transcribed in response to neuronal activity, named immediate early genes (IEGs), comprise members that are involved in important biological processes such as synaptic plasticity, learning and memory, and oncogenesis. A few genes have been identified as IEGs in mammals, but very little is known about IEGs in insects such as Drosophila melanogaster, which is used as a model to study these biological process. Furthermore, little is understood about genes that are downregulated by neuronal activity. We propose to undertake a genome-wide transcriptome analysis to identify genes that are both up-regulated and down- regulated in response to sensory neuron activity in three species of insects including Drosophila melanogaster, Drosophila pseudoobscura and the malaria vector Anopheles gambiae. By performing this analysis for three different species, we will be able to identify candidate IEGs for each species, as well as identify evolutionarily conserved IEGs that are regulated similarly across the three species. We propose to validate the identified candidate IEGs using a well-established quantitative RT-PCR.approach. Finally, we propose to map specificity of activity-induced transcription of IEGs to appropriate neurons using two alternative strategies, RNA in situ analysis and transgenic IEG-promoter analysis. For the transgenic strategy we propose to identify candidate regulatory regions of IEGs and test these promoter sequences in transgenic flies to determine whether they can drive reporter gene expression in response to neuronal activity. Successful completion of the proposed studies will provide a genome-wide catalog of genes that are regulated in an activity-dependent fashion in Drosophila and Anopheles. A successful method to transcriptionally report neuronal activity in vivo in insects would be a tremendous advance and could be applicable for the investigation of complex neurobiological problems including higher order processing of chemosensory information, chemsosensory coding, sensory integration and memory formation. PUBLIC HEALTH RELEVANCE: We propose to identify evolutionarily conserved genes that are regulated by activation of neurons in Drosophila and Anopheles using a genome-wide approach. In mammals activity-regulated genes have been associated with a number of important physiological processes such as nervous system function and cancer biology, and the ability to study them in the model insect Drosophila melanogaster will shed light on the fundamental mechanisms of their regulation and function. The potential use of these genes and/or their DNA regulatory elements as tracers of nervous system activity will be invaluable in dissecting neural circuits that are involved in processing and integrating sensory information in Drosophila. Furthermore, in disease vectors such as Anopheles gambiae that use chemical cues to identify their hosts, an understanding of evolutionarily conserved genes that function in neural circuits that guide host-seeking behaviors may lead to novel strategies for insect control.

DESCRIPTION (provided by applicant): The female Anopheles gambiae mosquito is an extremely efficient vector for the malarial parasite Plasmodium falciparum and transmits this deadly disease to nearly 500 million people, causing nearly 750,000 deaths every year. Since the mosquito relies primarily on the sense of smell to find a human host, the olfactory system is a prime target for development of strategies to disrupt contact between mosquitoes and humans. Current methods of control that exploit the mosquito olfactory system such as repellents applied to the skin like DEET and CO2-traps are not useful for the population at risk due to high costs, inconvenience of direct skin applications, and bulk. The goal of this proposal is to identify affordable, convenient, and safe odor-based applications that can be used in small quantities to protect a spatial area such as an abode from Anopheles mosquitoes, and design low-cost lures for traps. One of the primary cues that mosquitoes use from a distance is CO2 present in exhaled breath. As the mosquito navigates closer using the CO2 plumes, it is also thought to be attracted to skin and sweat associated odors. In this proposal we focus on the CO2-sensing neuron of Anopheles gambiae as a target for development of odor-mediated behavior disruption strategies. For this purpose we have developed an array of powerful technologies that involve neurophysiology, chemical- informatics, and behavioral analysis. First, we plan to investigate a novel observation that the CO2-sensing neuron is also involved in detecting human-skin odorants, presumably causing close-range attraction. Since little is understood about attraction towards skin, we expect our analyses to reveal mechanisms underlying this important behavioral process. Second, we plan to identify odorants that can mimic the olfactory activity of CO2, and test their utility in acting as efficient lures for traps.And third, we propose to identify odorants that can block detection of CO2 by either inhibiting the CO2-sensing neuron, or by ultra-prolonged activation of the neuron. These CO2-detection masking odors will be tested for the ability to protect a spatial area, such as a small abode, from host-seeking Anopheles mosquitoes. Successful completion of this proposal will provide safe and affordable odorants that can reduce contact between humans and Anopheles gambiae mosquitoes.

DESCRIPTION (provided by applicant): The ability to acquire, store and recall memories is a defining feature of the brain that is conserved from humans to Drosophila. Immediate Early Genes (IEGs) are genes whose expression is stimulated by neuronal activity, and is often transient, a feature that makes them prime candidates for modifying neural plasticity. Yet very little is understood about the regulation and function of IEGs in any organism. Aberrations in neural plasticity, learning and memory can cause severe neurological conditions such as Alzheimer's, dementia, post-traumatic stress and autism, underscoring the need for elucidating molecular mechanisms that underlie memory. We have identified a suite of 288 IEGs in the Drosophila brain using RNASeq analysis after exposure to sensory stimuli of various durations (10, 20, 30, 45 mins), 65% of which have homologs in humans. Based on the kinetics of expression modulation the IEGs cluster into 5 groups suggesting differences in regulatory mechanisms. Using an unbiased bioinformatics approach we were able to identify over-represented DNA sequence motifs in the upstream regions of each cluster of IEGs. The motifs for cluster 1 and 2 are remarkably alike, and match the binding site of the GATA family of transcription factors. Genetic and molecular analysis showed that a GATA factor, grain, was indeed required for expression of IEGs. The goal of this proposal is to test the hypothesis that grain and other GATA factors play a role in activity-dependent modulation of IEG expression and therefore a central role in acquisition and retention of memory. The proposal is highly significant since this represents one of the first major IEG regulatory pathways since CREB and could present an opportunity to understand the link between neuronal activity, IEG expression and memory. In addition, the proposal will use IEG promoters to provide transgenic tools to map activated neural circuits in brains of live behaving flies. The approach for analysis of IEG regulation by GATA factors will be accomplished in two specific aims outlined here. (1) The first aim will validate the role of grain in IEG regulation, identify its genome-wide targets, map changes in its localization in the brain and investigate its role in memory. (2) The second aim will investigate the regulatory DNA sequences of the GATA- dependent IEGs to test for direct regulation. In addition it will develop transgenic flies where the IEG promoter will be used to encode a genetic fluorescent reporter that marks activated neurons. Successful completion of the proposed studies will uncover transcription factors and neuronal circuits underlying fundamental mechanisms of learning and memory and establish tools that will be widely useful for anatomical studies of neural circuits.

? DESCRIPTION (provided by applicant): Insects such as mosquitoes use their olfactory and gustatory systems to find their hosts and in the process can transmit deadly diseases to hundreds of millions of people worldwide causing substantial mortality and morbidity. The chemosensory receptors therefore provide excellent targets to design behavior disruption strategies. The insect repellent DEET is effective against a variety of insects including mosquitoes; however it is rarely used by the population at risk in tropical countries due to high costs relative to incomes, and the inconvenience of continuous application on skin. The effectiveness of DEET is due in part to it being detected by aversive receptors in both the olfactory and the gustatory system; however nothing was known about their identity. In a recent breakthrough using the model system Drosophila melanogaster we have identified a DEET-detecting neuron in the antenna that expresses Ir40a and we have shown that both the neuron and the gene are required for avoidance. We have also developed a cheminformatic method to predict new ligands for the DEET receptor that are from natural sources including compounds found in food and flavoring. The goal of this proposal is to identify the conserved DEET-sensing olfactory and gustatory receptors and utilize them as targets to create novel classes of powerful broad-spectrum insect repellents that are safe and affordable. In addition high-throughput assay platforms with the target receptors for DEET will be created, which will serve as a foundation for discovery of even better repellents in the future. For this proposal we plan to use an array of technologies that involve chemical-informatics, neurophysiology, and behavioral analysis. First, we plan to validate the role of the Ir40a/Ir93a/Ir25a in detection of DEET and use them to test the computationally predicted ligands. Second, we plan to identify gustatory receptors that detect DEET as bitter and then test the predicted ligands as bitter aversive compounds. Third, we propose to identify neuronal circuits in the higher brain centers that process the aversive behavior from the olfactory and gustatory system. And fourth, we will test the dual aversive compounds for repellency in Aedes aegypti mosquitoes transmits Dengue and Yellow fever. Successful completion of this proposal will provide safe, affordable and pleasant smelling odorants that are better than DEET in reducing contact between humans and mosquitoes.