Discrimination between edible and contaminated foods is crucial for the survival of animals. In Drosophila, a family of gustatory receptors (GRs) expressed in taste neurons is thought to mediate the recognition of sugars and bitter compounds, thereby controlling feeding behavior. Using the Gal4/UAS system, we have characterized the expression of Gr genes in detail, and find that gene expression falls into two distinct groups. The first group, which make up the majority of the genes analyzed, are co-expressed with Gr66a, and functional ablation of taste neurons expressing these genes demonstrates that these neurons mediate sensitivity to bitter substrates. A second, distinct group of taste neurons, express Gr5a and mediate sensitivity to trehalose. We find that these two sub-populations of taste neurons - attractive-type and avoidance-type - project afferents to discrete areas of the primary taste center in the CNS. These results demonstrate how bitter and sweet taste are coded for in the periphery and indicates that information about different taste modalities is initially segregated in the CNS. We have also used the Gal4/UAS system to thoroughly characterize the expression profile of a cluster of six Grs - Gr28a and Gr28b.a-b.e. We find that these receptors are expressed not only in taste neurons, but other sensory neurons, as well as neurons in the CNS. RNA in situ hybridization confirms this unusual expression pattern. In order to explore the function of these Grs, these genes have been deleted using ends-out homologous recombination, to produce Gr28 mutant flies. Initial behavioral experiments with Gr28 mutant flies suggest that these receptors may play a role in detecting aversive substrates and/or modulate avoidance responses to these substrates. / Dissertation

Philosophiae Doctor - PhD / Tsetse flies are the sole vectors of trypanosomes that cause nagana and sleeping sickness in animals and humans respectively in tropical Africa. Tsetse are unique: both sexes adults are exclusive blood-feeders, females are mated young and give birth to a single mature larva in sheltered habitats per pregnancy. Tsetse use chemoreception to detect and respond to chemical stimuli, helping them to locate hosts, mates, larviposition and resting sites. The detection is facilitated by chemoreceptors expressed on sensory neurons to cause specific responses. Specific molecular factors that mediate these responses are poorly understood in tsetse flies. This study aimed to identify and characterize genes that potentially mediate chemoreception in Glossina morsitans morsitans tsetse flies. These genes included sensory odorant (OR), gustatory (GR), ionotropic (IR), and related genes for odorant-binding (OBP), chemosensory (CSP) and sensory neuron membrane (SNMP) proteins. Synaptic transmission in higher brain sites may involve ionotropic glutamate-gated (iGluR) and metabotropic glutamate-gated (mGluR) receptors. The genes were annotated in G. m. morsitans genome scaffold assembly GMOY1.1 Yale strain using orthologs from D. melanogaster as query via TBLASTX algorithm at e-value below 1e-03. Positive blast hits were seeded as gene constructs in their respective scaffolds, and used as genomic reference onto which female fly-derived RNA sequence reads were mapped using CLC Genomics workbench suite. Seeded gene models were modified using RNA-Seq reads then viewed and re-edited using Artemis genome viewer tool. The genome was iteratively searched using the G. m. morsitans gene model sequences to recover additional similar hit sequences. The gene models were confirmed through comparisons against the NCBI conserved domains database (CDD) and non-redundant Swiss-Prot database. Trans-membrane domains and secretory peptides were predicted using TMHMM and SignalP tools respectively. Putative functions of the genes were confirmed via Blast2GO searches against gene ontology database. Evolutionary relationships amongst and between the genes were established using maximum likelihood estimates using best fitting amino acid model test in MEGA5 suite and PhyML tool. Expression profiles of genes were estimated using the RNA-seq data via CLCGenomics RNA-sequences analysis pipeline. Overall, 46 ORs, 14 GRs, and 19 IRs were identified, of which 21, 6 and 4 were manually identified for ORs, GRs, and IRs respectively. Additionally, 15 iGluRs, 6 mGluRs, 5 CSPs, 15 CD36-like, and 32 OBPs were identified. Six copies of OR genes (GmmOR41-46) were homologous to DmelOr67d, a single copy cis vacenyl acetate (cVA) receptor . Genes whose receptor homologs are associated with responses to CO2, GmmGR1-4, had higher expression profiles from amongst glossina GR genes. Known core-receptor homologs OR1, IR8a, IR25a and IR64a were conserved, and three species-specific divergent IRs (IR10a, IR56b and IR56d) were identified. Homologs of GluRIID, IR93a, and sweet taste receptors (Gr5a and Gr64a) were not identified in the genome. Homolog for LUSH protein, GmmOBP26, and sensory neuron membrane receptors SNMP1 and SNMP2 were conserved in the genome. Results indicate reduced repertoire of the chemosensory genes, and suggest reduced host range of the tsetse flies compared to other Diptera. Genes in multiple copies suggest their prioritization in chemoreception, which in turn may be tied to high specificity in host selection. Genes with high sequence conservation and expression profiles probably relate to their broad expression and utility within the fly nervous system. These results lay foundation for future comparative studies with other insects, provide opportunities for functional studies, and form the basis for re-examining new approaches for improving tsetse control tools and possible drug targets based on chemoreception.

<p>Gustation, also known as taste perception, is critical for the survival of most animal species. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of sugars, bitter or toxic compounds, and pheromones. However, with a few notable exceptions, the functions of most GRs involved in feeding are unknown. Our research has focused on a cluster of highly-related Drosophila Grs, known as the Gr64 family, that have been shown to be critical for the perception of multiple sugars. Furthermore, we have demonstrated that another gene related to the Gr64 genes, Gr61a, is a sugar receptor that is narrowly tuned to a subset of pyranose sugars and may (along with the Gr64 genes) be indispensable for early fly development. </p><p>As a complementary approach to our behavioral analysis, we have examined the expression pattern of the Drosophila sugar receptors using knock-in driver alleles created by homologous recombination. As expected, most of these drivers have shown strong expression in various taste tissues. Intriguingly, some of these knock-in alleles also show expression in the maxillary palp and antenna, tissues previously thought to be involved only in olfaction. These expression patterns raise interesting questions about the true range of function of these chemosensory receptors and whether or not they might be involved in olfaction as well as gustation.</p> / Dissertation

Les capacités cognitives pour assurer l’exploration et la découverte de nouvelles niches écologiques sont au cœur des processus d’adaptation et de survie des espèces vertébrés et invertébrés. A cet égard, les systèmes neuronaux chimio-sensoriels composés des organes olfactifs et gustatifs permettent le guidage et repérage des sources de nourritures et/ou des partenaires sexuels. Un fait marquant chez les insectes et en particulier la drosophile réside dans le fait que les organes gustatifs sont disséminés sur le corps. La bordure antérieure de l’aile est tapissée avec des sensilles gustatives alternées avec des sensilles mécaniques. La fonctionnalité et le rôle des cellules gustatives au niveau de l’aile de la drosophile reste énigmatique et à ce jour largement inconnue (Stocker, 1994). Notre travail a consisté à explorer la signalisation et le mécanisme de transduction de ces récepteurs et à questionner leur importance dans l’adaptation des insectes à leur écosystème. Nos résultats portent sur trois volets. Nous avons vérifié que l’expression des récepteurs du goût est effective dans les ailes des trois insectes différents (drosophiles, pucerons et abeilles) par RT-PCR. Nous avons ensuite étudié la fonctionnalité de ces récepteurs vis-à-vis des molécules sucrées et amères à l’aide d’une souche transgénique (G-CaMP), qui exhibe une forte fluorescence provoquée par des piques de calcium cytosolique. Enfin, des tests comportementaux ont été réalisé avec une souche transgénique (Poxn*) dans laquelle les sensilles chimio-sensorielles de l’aile sont spécifiquement invalidés sans altérer les autres structures olfactives et/ou gustatives. Les résultats montrent un effet significatif des cellules chimio-sensorielles de l’aile quant à l’orientation dans l’espace et à l’apprentissage Bayesien. Nos résultats sur ces trois volets nous ont permis d’élaborer des hypothèses au regard de l’évolution neuroanatomique de l’aile des insectes depuis les organismes ancestraux d’origine marine desquels ils dérivent. Des experts en aérodynamiques proposent la création d’un vortex durant le vol qui forme une spirale de courant d’air le long de la bordure antérieur de l’aile. La parfaite superposition entre ce vortex et le nerf costal de l’aile nous permet de déduire que les vibrations de l’aile entre 50 et 1.000 Hertz chez les insectes sont en mesure de nébuliser des matériaux (micro poussières, micro gouttelettes, molécules faiblement volatiles) lesquels vont être captés/entrainés dans le vortex et adressés aux sensilles gustatives. Notre hypothèse est que ce mécanisme permettrait aux insectes pollinisateurs de gouter les fleurs sans se poser et sans mettre à contribution la trompe buccale (proboscis). Ce scénario permettrait de dissocier le goût de l’ingestion digestive en évitant les empoisonnements par des molécules toxiques émises par les plantes et d’autre part il rend l’exploration plus efficace, en minimisant le temps de recherche. / Cognitive capacities used to ensure the exploration and discovery of new ecological niches are at the heart of the process of adaptation and survival of vertebrate and invertebrate species. In this respect, the neural chemosensory systems, composed of the olfactory and gustatory organs, allow the guidance and finding of food sources and/or sexual partners. A striking feature in insects and particularly in Drosophila is that gustatory organs are disseminated in the body. The anterior margin of the wing is lined with gustatory sensilla alternated with mechanosensory sensilla. The function of gustatory cells in the wing of Drosophila remains enigmatic and actually quite unknown (Stocker, 1994). Our work consisted in exploring the signaling and the transduction mechanisms of these receptors and in questioning their importance in the adaptation of insects to their ecosystem. Our results are based on three components. We have verified that the expression of gustatory receptors occurs in the wings of three different insects (Drosophila, aphid and honey bee) by RT-PCR. We have studied the function of these receptors vis-à-vis of sweet and bitter molecules using a transgenic line (G-CaMP) that exhibits a strong fluorescence provoked by cytosolic calcium picks. Finally, behavioral assays have been realized with a transgenic line (Poxn*) in which the chemosensory sensilla have been invalidated without altering the other olfactory and gustatory structures. Our results show a significant effect of wing chemosensory cells as far as orientation is space and Bayesian learning and have permitted us to elaborate hypothesis regarding the neuroanatomical evolution of the wing of insects since ancestral organisms of marine origin from which they derive. Experts in aerodynamics propose the creation of a vortex during flight that forms a spiral of air along the anterior border of the wing. The perfect superposition between this vortex and the costal nerve of the wing allows us to deduce that the vibrations of the insect wing between 50 and 1.000 Hertz are able to nebulize materials (microdust, microdrops, weakly volatile molecules), which are captured/trapped in the vortex and addressed to the gustatory sensilla. Our hypothesis is that this mechanism would let pollinator insects taste flowers without landing and without involving the proboscis. In this scenario insects would dissociate taste from ingestion, avoiding poisoning by toxic molecules emitted by plants and rending exploration more efficient by minimizing searching time.

<p dir="ltr">Given the relatively poor visual acuity of compound eyes, many insects have developed alternative means for navigating their environment. For example, insects often rely on chemosensation to find food, mates, and inter- and intraspecific communication. Eusocial insects in particular have developed complex systems of pheromone communication to organize their colonies, enabling them to partition labor for foraging, brood care, and colony defense tasks to different portions of the colony. A variety of genes coding for proteins are involved in detecting these chemicals, including gustatory receptors, ionotropic receptors, and odorant receptors (ORs). Eusocial insects, and especially ants, have evolved an expanded clade of ORs in their genome, likely due to an increased reliance on pheromones compared to other insects. The ability to recognize nestmates from non-nestmates is one of the vital functions performed by these ORs, which detect hydrocarbons present on the cuticle to distinguish friend from foe. However, research into the details of nestmate recognition has been stymied due to difficulties in manipulating OR genes. Despite advances in genetic sequencing and manipulation technologies, strict reproductive divisions within most ant lineages make generating transgenic ants nearly impossible, and so we have been left with limited options to further investigate these receptors. To narrow down the ORs that could be involved in nestmate recognition in the Argentine ant (Mayr, 1868), I took a multi-pronged approach of generating tissue transcriptomes to identify ORs that are selectively upregulated in the antennae, as well as conducting a phylostratigraphic analysis to identify which OR genes arose more recently in the Argentine ant genome. While conducting these analyses, it became necessary to reannotate the set of Argentine ant OR genes, due to current published annotations not containing the full breadth of <i>L. humile</i> ORs. Finally, I orally administered fluorescently-labelled dsRNA to workers, and tracked the extent to which ingested dsRNA is capable of traversing the tissues of ant workers, to investigate whether RNAi is a viable method for investigating gene function for genes showing tissue-selective expression. I discovered a subset of OR genes that are highly expressed in the antennae and confirmed that dsRNA is able to reach the antennae and knock down OR gene expression through ingestion, meaning that RNA interference is a viable method for the practical study of ant OR genes and can be used to further explore how individual ORs regulate nestmate recognition.</p>