Anupama Dahanukar - US grants

In Drosophila, as in vertebrates and caenorhabditis elegans, a large family of odorant receptor genes is responsible for the remarkable discriminatory power of the olfactory system. For this capability, it is essential that the brain discern which of the numerous receptors has been activated by an odorant. Several lines of evidence suggest that odorant receptors themselves may provide a link between the odorant recognition properties of a neuron and its central pattern of connectivity, which may specify perception and behavioral response to a particular odor. While recent results of experiments in the mouse do validate a role for odorant receptors in olfactory system development, the mechanisms by which odorant receptors dictate neuronal connectivity are not understood. The experiments proposed here aim to address this problem using Drosophila as the model system. The experimental approach will be to first characterize the developmental expression of odorant receptors and next to examine the consequences of varying the levels of receptor proteins on development of the olfactory nervous system. These studies will contribute towards the understanding of basic principles of neurobiology.

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.

Animals regulate the quality and quantity of their food intake to maintain nutrient homeostasis. Although studies on regulation of feeding behavior have focused on central feedback mechanisms that control appetite, some evidence suggests that peripheral sensory activity is modulated under certain conditions. That such modulation can be specific - carbohydrate deprivation causes increased sensitivity to sugars and protein deprivation causes increased sensitivity to amino acids - argues for the existence of mechanisms that target the activity of receptors or cells that recognize specific categories of taste chemicals. In insects, the levels of macronutrients in the hemolymph are known to sway the responsiveness of taste neurons, however the molecular mechanisms by which this occurs are poorly understood. This project investigates the hypothesis that members of a large, divergent family of Gustatory receptor (Gr) genes are regulatory targets of pathways that operate to sense internal nutritional needs. In addition, genome-wide approaches will be applied to identify novel genes that are differentially regulated by changes in nutrient demand. These studies will determine the importance of Gr regulation under different physiological conditions and establish a foundation for inquiry into the molecular underpinnings of compensatory food selection and intake.

The proposed studies will generate broader impact through an educational plan that provides hands-on research experience to several early-career undergraduate students from under-represented groups through a summer research program. The project will also provide training for a graduate student and a postdoctoral scientist. Each year two undergraduate students will be selected to participate in a follow-up research training program during which time they will engage in genetic screens to identify loci involved in regulating feeding behaviors. All participants will engage in near-peer mentoring and annual assessment will be used to develop effective ways with which to integrate research training with science education and career guidance. The data generated from this project will be made available through scientific publications and deposition in public databases.

DESCRIPTION (provided by applicant): The mechanisms by which various classes of chemical stimuli are detected and how that translates into appropriate behaviors is a central problem in gustatory neurobiology. The overarching objective of this proposal is to investigate the molecular and cellular mechanisms of carboxylic acid taste in Drosophila, and to identify neurophysiological responses to carboxylic acids in the mosquito, Aedes aegypti. Drosophila is an excellent model to study the fundamental logic by which the taste system is organized in insects. While a lot of progress has been made in deciphering the molecular and cellular mechanisms of certain categories of tastants including sweet and bitter compounds, little is known about the fly's taste responses to acids. Based on preliminary studies, the central hypothesis of this proposal is that acid stimuli can activate and inhibit different categories of taste neurons, and acid-mediated inhibition of taste neurons is dependent on the function of a highly conserved member of the Ionotropic receptor family, Ir25a. The approach to test this hypothesis will be to: 1) undertake electrophysiological analyses to characterize activation of taste neurons by carboxylic acids, 2) validate the identity of taste neurons that are activated in response to acid stimuli and determine their contribution in acid aversion behavior, 3) characterize acid-mediated inhibition of sweet neurons, 4) link the function of Ir25a to acid-mediated inhibition of sugar neurons, and 5) initiate an analysis of excitatory and inhibitory responses to acids in taste neurons of Aedes aegypti. The proposed research is innovative because it represents a departure from previous studies of chemosensory responses to carboxylic acids that focused on the role of the olfactory system, and because the research plan encompasses a multidisciplinary approach spanning genetic, electrophysiological and behavioral analyses. The proposed research is significant because it will provide insight into the molecular and cellular basis of acid taste in a valuable model organism, which will aid in studying fundamental problems of chemosensory coding and behavior. Notably, carboxylic acids of low volatility are components of human sweat and their detection by taste neurons may be important for behaviors of human blood-feeding insects such as Aedes aegypti and Anopheles gambiae, which transmit deadly diseases. An understanding of the function of evolutionarily conserved receptors in the detection of carboxylic acids may lead to novel strategies for control of arthropod disease vectors.

DESCRIPTION (provided by applicant): The ability to detect various chemicals and to use this information to make feeding decisions is a defining feature of the taste system from humans to insects. In Drosophila, a large family of Gustatory receptor (Gr) genes encodes divergent receptors that are expressed in taste neurons. Despite ongoing efforts, we know little about the ligand response profiles of individual Gr proteins in any insect. Gr proteins represent a novel family, distantly related only to insect Odorant receptor (Or) proteins, and thus lack the advantage of structural or functional comparisons with equivalent groups. The same feature of exclusivity in insects and arthropods, however, makes Grs attractive targets for developing novel strategies of insect control. In preliminary studies, we have developed an in vivo functional expression system in which to identify the response properties of individual insect Grs. The overarching objective of this proposal is to elucidate functional properties of individual taste receptors in Drosophila and the malaria mosquito Anopheles gambiae, and their role in instructing feeding behavior. We propose to systematically characterize the tuning profiles of selected taste receptors of the fruit fly and the mosquito by taking advantage of our ectopic expression system. The experiments are designed to reveal fundamental features of Gr function including the activation and inhibition profiles of individual receptors, the extent of functional overlap between receptors, and the relationship between receptor sequence and function. The approach to address these questions will be to: 1) Identify the tuning profiles of internally expressed Drosophila taste receptors, and evaluate their roles in feeding behavior by genetic analysis, 2) Characterize novel cross-modality interactions between Drosophila sweet taste receptors and bitter tastants, and examine the functional role of sweet taste neuron inhibition in feeding behavior, and 3) Identify activators and inhibitors of putative sweet taste receptors of An. gambiae and characterize the response profiles of endogenous sweet taste neurons in the mosquito proboscis. The proposed research is innovative because it employs a novel in vivo ectopic expression system for decoding Gr function and combines it with molecular genetics, electrophysiological, and behavioral analyses of gustatory function. The research is significant because functional analysis of Gr genes will contribute to our understanding of the fundamental principles by which tastants are encoded across a diverse repertoire of receptors and how mechanisms of tastant detection translate to food selection. Additionally, our studies will shed light on molecular and cellular mechanisms of sugar detection in mosquitoes, which may be applicable for improving sugar-baited traps that are used in mosquito surveillance and control strategies.

This award will enable a team of computer scientists and entomologists at the University of California-Riverside to develop sensors and software that will allow the classification of flying insects. The ability to automatically and accurately classify flying insects has the potential to have significant impact human affairs, because insects spread disease, feed on crops and livestock, and ruin food stores, at a combined annual cost of billions of dollars and incalculable human suffering.
 
The intellectual merit of the project is in producing algorithms, devices, and procedures that will radically expand the ability to conduct insect surveillance. Recent advances in sensor technology and machine learning and the ongoing revolution in Big Data are just beginning to enable development of advanced algorithms that will help usher in a new era of computational entomology. The investigators will build inexpensive devices that can detect and classify flying insects. For at least some genera of insects the resulting classification labels will go beyond species-level to predict sex and physiological states (such as virgin vs. gravid and newly emerged vs. mature) of individual insects. The investigators will create computational devices that can determine the origin of selected insects, and selectively capture targeted insects for downstream molecular diagnostic analysis. Producing such information will both accelerate basic research in entomology and will allow more effective vector control. The broader impacts of the project are inherent in the potential to significantly improve the quality and volume of insect surveillance, thus allowing more effective Integrated Vector Management. In the case of mosquitoes, more effective interventions are known to directly save lives. Resulting algorithms will allow the creation of systems to provide actionable information on multiple scales, from informing a policy committee to instructing an agricultural robot to open a valve. The projects comprehensive educational and outreach activities have already been piloted on a small scale and include detailed plans to reach out to underserved communities at the K-12 and college levels.

This National Science Foundation Research Traineeship (NRT) award to the University of California, Riverside (UCR) will enable a team of investigators from Computer Science/Engineering and Entomology/Life Sciences to prepare the next generation of scientists and engineers to exploit the unreasonable effectiveness of data to understand insects by integrating the disciplines of computer science and entomological biology. The NRT in Integrated Computational Entomology (NICE) will train students to be at the forefront of science in computing for biological domains, providing biological scientists a foundation in computing techniques and engineers an understanding of critical entomological and ecological issues. The project anticipates training at least forty (40) MS and PhD students, including twenty (20) funded PhD trainees from the life sciences, computer science and engineering.

The project will be the first program of its kind, anywhere in the world, and will meet high standards for innovation while offering a structure for demanding training in entomology/life sciences integrated with computational techniques in machine learning, data mining, and statistics. The NICE program recognizes and advances Computational Entomology as an emerging interdisciplinary field. Computational Entomology as a discipline recognizes that entomological and ecological problems generate enormous amounts of data, and that fully exploiting this data will require individuals whose knowledge spans two otherwise disparate fields. The training and research structure of the proposed project seeks to bridge large gaps in training, language, approach, perspective and knowledge that continue to divide the engineering/informatics and life sciences disciplines. Through coursework and joint projects with government agencies and companies, trainees will experience the translation of research outcomes into implemented public policy or agricultural/medical products and services. This project will scale to include graduate student trainees at UCR receiving NRT support and those not receiving funding, and will be sustainable at UCR as the new curriculum will become incorporated across the participating departments and degree programs. This project will also serve as a replicable Computational Entomology education and training model for other institutions.

The NSF Research Traineeship (NRT) Program is designed to encourage the development and implementation of bold, new potentially transformative scalable models for STEM graduate education training. The Traineeship Track is dedicated to effective training of STEM graduate students in high priority interdisciplinary research areas, through the comprehensive traineeship model that is innovative, evidence-based, and aligned with changing workforce and research needs.

PROJECT SUMMARY Although male mosquitoes are obligate carbohydrate feeders, female mosquitoes that transmit pathogens to humans feed on nectar as well as blood, which is required to complete cycles of egg development. Carbohydrate and blood meals are dispatched to different compartments of the digestive tract. Sugars enter the crop, a specialized foregut organ in the Diptera, access to which is controlled by a sphincter valve. Blood meals, on the other hand, are directed to the midgut, which is equipped with specialized structures such as the peritrophic membrane to protect the mosquito from effects of pathogens and imbalances from chemical constituents of the blood, as well as to aid digestion. Previous investigations of mechanisms that underlie ?switching? of meal destination in various dipterans implicate physical factors such as meal temperature and osmotic pressure, as well as the chemical nature of the meal. However, the literature describes a lot of variability across species, and this problem has not received much attention for the past couple of decades. We have conducted preliminary experiments to first establish the robustness of the switching mechanism in the Aedes aegypti mosquito, and to test the involvement of physical and chemical factors in affecting meal destination. We use these results to set up conditions that disrupt meal destination, and determine the consequences of diverting a blood meal to both the crop and midgut on fecundity. Based on our results, our hypothesis is that in A. aegypti, the crop valve opens for a carbohydrate meal but remains closed for a blood meal, which thus ends up in the midgut. Furthermore, our findings show that shunting blood to the crop yields a dramatic reduction in the number of blood-fed females that lay eggs as well as the number of eggs laid by individual females. Our overall goals are to build upon these initial findings to investigate meal factors and sensory mechanisms that underlie the meal destination switching mechanism. Our experimental approach will be 1) to further characterize the biological importance of the switching mechanism, 2) to identify meal properties (physical and chemical) that influence meal destination, and 3) to test the roles of candidate genes, selected by the elucidation of critical meal properties, in controlling meal destination. Completion of the proposed experiments will provide an understanding of fundamental principles of feeding and digestive tract function that may be shared across many hematophagous insects that spread deadly diseases.

Project Summary Fat represents a calorically potent food source that yields approximately twice the amount of energy as carbohydrates or proteins per unit of mass. Dietary lipids are comprised of both triacylglycerides and fatty acids, and growing evidence suggests that it is the free fatty acids that are detected by the gustatory system. The highly palatable taste of fatty acids promotes food consumption, activates reward centers in mammals, and contributes to hedonic feeding that underlies many metabolism-related disorders. Despite a role in the etiology of metabolic diseases, little is known about how dietary fats are detected by the gustatory system to promote feeding. We have shown that fatty acids function as appetitive tastants and identified the first putative fatty acid receptor in the fly. This proposal seeks to identify the cellular and neural circuit principles that underlie the detection and processing of fatty acids. In addition, these experiments will investigate whether flies are capable of discriminating between different appetitive tastants, and whether fatty acids and sugars are processed by shared or independent neuronal circuits. The completion of the proposed experiments will provide a detailed understanding of fat taste, and inform fundamental principles of sensory coding that may be shared across phyla.