Jesse Slone

A fructose receptor functions as a nutrient sensor in the Drosophila brain.

Internal nutrient sensors play important roles in feeding behavior, yet their molecular structure and mechanism of action are poorly understood. Using Ca(2+) imaging and behavioral assays, we show that the gustatory receptor 43a (Gr43a) functions as a narrowly tuned fructose receptor in taste neurons. Remarkably, Gr43a also functions as a fructose receptor in the brain. Interestingly, hemolymph fructose levels are tightly linked to feeding status: after nutritious carbohydrate consumption, fructose levels rise several fold and reach a concentration sufficient to activate Gr43a in the brain. By using different feeding paradigms and artificial activation of Gr43a-expressing brain neurons, we show that Gr43a is both necessary and sufficient to sense hemolymph fructose and promote feeding in hungry flies but suppress feeding in satiated flies. Thus, our studies indicate that the Gr43a-expressing brain neurons function as a nutrient sensor for hemolymph fructose and assign opposing valence to feeding experiences in a satiation-dependent manner.

Sugar Receptors in Drosophila

The detection and discrimination of chemical compounds in potential foods are essential sensory processes when animals feed. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of mostly nonvolatile chemicals that include sugars, a diverse group of toxic compounds present in many inedible plants and spoiled foods, and pheromones [1-6]. With the exception of a trehalose (GR5a) and a caffeine (GR66a) receptor [7-9], the functions of GRs involved in feeding are unknown. Here, we show that the Gr64 genes encode receptors for numerous sugars. We generated a fly strain that contained a deletion for all six Gr64 genes (DeltaGr64) and showed that these flies exhibit no or a significantly diminished proboscis extension reflex (PER) response when stimulated with glucose, maltose, sucrose, and several other sugars. The only considerable response was detected when Gr64 mutant flies were stimulated with fructose. Interestingly, response to trehalose is also abolished in these flies, even though they contain a functional Gr5a gene, which has been previously shown to encode a receptor for this sugar [8, 9]. This observation indicates that two or more Gr genes are necessary for trehalose detection, suggesting that GRs function as multimeric receptor complexes. Finally, we present evidence that some members of the Gr64 gene family are transcribed as a polycistronic mRNA, providing a mechanism for the coexpression of multiple sugar receptors in the same taste neurons.

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.

Drosophila Sugar Receptors in Sweet Taste Perception, Olfaction, and Internal Nutrient Sensing

Identification of nutritious compounds is dependent on expression of specific taste receptors in appropriate taste-cell types [1]. In contrast to mammals, which rely on a single, broadly tuned heterodimeric sugar receptor [2], the Drosophila genome harbors a small subfamily of eight, closely related gustatory receptor (Gr) genes, Gr5a, Gr61a, and Gr64a-Gr64f, of which three have been proposed to mediate sweet taste [3-6]. However, expression and function of several of these putative sugar Gr genes are not known. Here, we present a comprehensive expression and functional analysis using Gr(LEXA/GAL4) alleles that were generated through homologous recombination. We show that sugar Gr genes are expressed in a combinatorial manner to yield at least eight sets of sweet-sensing neurons. Behavioral investigations show that most sugar Gr mutations affect taste responses to only a small number of sugars and that effective detection of most sugars is dependent on more than one Gr gene. Surprisingly, Gr64a, one of three Gr genes previously proposed to play a major role in sweet taste [3, 4], is not expressed in labellar taste neurons, and Gr64a mutant flies exhibit normal sugar responses elicited from the labellum. Our analysis provides a molecular rationale for distinct tuning profiles of sweet taste neurons, and it favors a model whereby all sugar Grs contribute to sweet taste. Furthermore, expression in olfactory organs and the brain implies novel roles for sugar Gr genes in olfaction and internal nutrient sensing, respectively. Thus, sugar receptors may contribute to feeding behavior via multiple sensory systems.

Identification of a Drosophila Glucose Receptor Using Ca2+ Imaging of Single Chemosensory Neurons

Evaluation of food compounds by chemosensory cells is essential for animals to make appropriate feeding decisions. In the fruit fly Drosophila melanogaster, structurally diverse chemicals are detected by multimeric receptors composed of members of a large family of Gustatory receptor (Gr) proteins. Putative sugar and bitter receptors are expressed in distinct subsets of Gustatory Receptor Neurons (GRN) of taste sensilla, thereby assigning distinct taste qualities to sugars and bitter tasting compounds, respectively. Here we report a Ca2+ imaging method that allows association of ligand-mediated responses to a single GRN. We find that different sweet neurons exhibit distinct response profiles when stimulated with various sugars, and likewise, different bitter neurons exhibit distinct response profiles when stimulated with a set of bitter chemicals. These observations suggest that individual neurons within a taste modality are represented by distinct repertoires of sweet and bitter taste receptors, respectively. Furthermore, we employed this novel method to identify glucose as the primary ligand for the sugar receptor Gr61a, which is not only expressed in sweet sensing neurons of classical chemosensory sensilla, but also in two supersensitive neurons of atypical taste sensilla. Thus, single cell Ca2+ imaging can be employed as a powerful tool to identify ligands for orphan Gr proteins.

The Molecular Basis of Sugar Sensing in Drosophila Larvae

Evaluation of food chemicals is essential to make appropriate feeding decisions. The molecular genetic analysis of Gustatory receptor (Gr) genes and the characterization of the neural circuits that they engage has led to a broad understanding of taste perception in adult Drosophila [1, 2]. For example, eight relatively highly conserved members of the Gr gene family (Gr5a, Gr61a, and Gr64a-f), referred to as sugar Gr genes, are thought to be involved in sugar taste in adult flies [3-8], while the majority of the remaining Gr genes are likely to encode bitter taste receptors [9-11], albeit some function as pheromone [12-14] and carbon dioxide [15, 16] receptors. In contrast to the adult fly, relatively little is known about the cellular and molecular basis of taste perception in larvae. Here, we identify Gr43a, which was recently shown to function as a hemolymph fructose sensor in adult flies [17], as the major larval sugar receptor. We show that it is expressed in taste neurons, proventricular neurons, as well as sensory neurons of the brain. Larvae lacking Gr43a fail to sense sugars, while larvae mutant for all eight sugar Gr genes exhibit no obvious defect. Finally, we show that brain neurons are necessary and sufficient for sensing all main dietary sugars, which probably involves a postingestive mechanism of converting carbohydrates into fructose.

An Engineered orco Mutation Produces Aberrant Social Behavior and Defective Neural Development in Ants

Ants exhibit cooperative behaviors and advanced forms of sociality that depend on pheromone-mediated communication. Odorant receptor neurons (ORNs) express specific odorant receptors (ORs) encoded by a dramatically expanded gene family in ants. In most eusocial insects, only the queen can transmit genetic information, restricting genetic studies. In contrast, workers in Harpegnathos saltator ants can be converted into gamergates (pseudoqueens) that can found entire colonies. This feature facilitated CRISPR-Cas9 generation of germline mutations in orco, the gene that encodes the obligate co-receptor of all ORs. orco mutations should significantly impact olfaction. We demonstrate striking functions of Orco in odorant perception, reproductive physiology, and social behavior plasticity. Surprisingly, unlike in other insects, loss of OR functionality also dramatically impairs development of the antennal lobe to which ORNs project. Therefore, the development of genetics in Harpegnathos establishes this ant species as a model organism to study the complexity of eusociality.

Functional characterization of odorant receptors in the ponerine ant, Harpegnathos saltator

Significance The tuning of odorant receptors to their particular odorants is crucial for better understanding of how olfactory cues mediate ant social interactions. To help decode the olfactory system of ants, a selection of odorant receptors (ORs) from several phylogenetically distinct subfamilies from the ponerine ant Harpegnathos saltator were tested against a panel of ant semiochemicals. Responses were observed to both cuticular hydrocarbon components, some of which are known pheromones, and “general odorants,” demonstrating broad coverage of these odor spaces across several subfamilies of receptors. These results do not align with currently held hypotheses of OR subfamily odor coding and provide further insight into the evolution of pheromone perception within ant clades and the role this plays in complex social behaviors. 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.

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.

A genetic tool kit for cellular and behavioral analyses of insect sugar receptors

Arthropods employ a large family of up to 100 putative taste or gustatory receptors (Grs) for the recognition of a wide range of non-volatile chemicals. In Drosophila melanogaster, a small subfamily of 8 Gr genes is thought to mediate the detection of sugars, the flys major nutritional source. However, the specific roles for most sugar Gr genes are not known. Here, we report the generation of a series of mutant sugar Gr knock-in alleles and several composite sugar Gr mutant strains, including a sugar blind strain, which will facilitate the characterization of this gene family. Using Ca2+ imaging experiments, we show that most gustatory receptor neurons (GRNs) of sugar blind flies (lacking all 8 sugar Gr genes) fail to respond to any sugar tested. Moreover, expression of single sugar Gr genes in most sweet GRNs of sugar-blind flies does not restore sugar responses. However, when pair-wise combinations of sugar Gr genes are introduced to sweet GRNs, responses to select sugars are restored. We also examined the cellular phenotype of flies homozygous mutant for Gr64a, a Gr gene previously reported to be a major contributor for the detection of many sugars. In contrast to these claims, we find that sweet GRNs of Gr64a homozygous mutant flies show normal responses to most sugars, and only modestly reduced responses to maltose and maltotriose. Thus, the precisely engineered genetic mutations of single Gr genes and construction of a sugar-blind strain provide powerful analytical tools for examining the roles of Drosophila and other insect sugar Gr genes in sweet taste.