A Drosophila Gustatory Receptor Essential for Aversive Taste and Inhibiting Male-to-Male Courtship

Contact chemosensation is required for several behaviors that promote insect survival. These include evasive behaviors such as suppression of feeding on repellent compounds, known as antifeedants, and inhibition of male-to-male courtship. However, the gustatory receptors (GRs) required for responding to nonvolatile avoidance chemicals are largely unknown. Exceptions include Drosophila GR66a and GR93a, which are required to prevent ingestion of caffeine [1] and [2], and GR32a, which is necessary for inhibiting male-to-male courtship [3]. However, GR32a is dispensable for normal taste. Thus, distinct GRs may function in sensing avoidance pheromones and antifeedants. Here, we describe the requirements for GR33a, which is expressed widely in gustatory receptor neurons (GRNs) that respond to aversive chemicals. Gr33a mutant flies were impaired in avoiding all nonvolatile repellents tested, ranging from quinine to denatonium, lobeline, and caffeine. Gr33a mutant males also displayed increased male-to-male courtship, implying that it functioned in the detection of a repulsive male pheromone. In contrast to the broadly required olfactory receptor (OR) OR83b, which is essential for trafficking other ORs [4], GR66a and GR93a are localized normally in Gr33a mutant GRNs. Thus, rather than regulating GR trafficking, GR33a may be a coreceptor required for sensing all nonvolatile repulsive chemicals, including tastants and pheromones.     

Two Gr genes underlie sugar reception in Drosophila

We have analyzed the molecular basis of sugar reception in Drosophila. We define the response spectrum, concentration dependence, and temporal dynamics of sugar-sensing neurons. Using in situ hybridization and reporter gene expression, we identify members of the Gr5a-related taste receptor subfamily that are coexpressed in sugar neurons. Neurons expressing reporters of different Gr5a-related genes send overlapping but distinct projections to the brain and thoracic ganglia. Genetic analysis of receptor genes shows that Gr5a is required for response to one subset of sugars and Gr64a for response to a complementary subset. A Gr5a;Gr64a double mutant shows no physiological or behavioral responses to any tested sugar. The simplest interpretation of our results is that Gr5a and Gr64a are each capable of functioning independently of each other within individual sugar neurons and that they are the primary receptors used in the labellum to detect sugars.     

Taste perception and coding in Drosophila

BACKGROUND: 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. RESULTS: We have characterized in detail the expression of eight Gr genes in the labial palps, the fly's main taste organ. These genes fall into two distinct groups: seven of them, including Gr66a, are expressed in 22 or fewer taste neurons in each labial palp. Additional experiments show that many of these genes are coexpressed in partially overlapping sets of neurons. In contrast, Gr5a, which encodes a receptor for trehalose, is expressed in a distinct and larger set of taste neurons associated with most chemosensory sensilla, including taste pegs. Mapping the axonal targets of cells expressing Gr66a and Gr5a reveals distinct projection patterns for these two groups of neurons in the brain. Moreover, tetanus toxin-mediated inactivation of Gr66a- or Gr5a-expressing cells shows that these two sets of neurons mediate distinct taste modalities-the perception of bitter (caffeine) and sweet (trehalose) taste, respectively. CONCLUSION: Discrimination between two taste modalities-sweet and bitter-requires specific sets of gustatory receptor neurons that express different Gr genes. Unlike the Drosophila olfactory system, where each neuron expresses a single olfactory receptor gene, taste neurons can express multiple receptors and do so in a complex Gr gene code that is unique for small sets of neurons.     

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 fly's 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.     

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 fly''s 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.     

A chemosensory gene family encoding candidate gustatory and olfactory receptors in Drosophila

A novel family of candidate gustatory receptors (GRs) was recently identified in searches of the Drosophila genome. We have performed in situ hybridization and transgene experiments that reveal expression of these genes in both gustatory and olfactory neurons in adult flies and larvae. This gene family is likely to encode both odorant and taste receptors. We have visualized the projections of chemosensory neurons in the larval brain and observe that neurons expressing different GRs project to discrete loci in the antennal lobe and subesophageal ganglion. These data provide insight into the diversity of chemosensory recognition and an initial view of the representation of gustatory information in the fly brain.     

Trehalose sensitivity in Drosophila correlates with mutations in and expression of the gustatory receptor gene Gr5a

Drosophila taste gene Tre is located on the distal X chromosome and controls gustatory sensitivity to a subset of sugars [1, 2]. Two adjacent, seven-transmembrane domain genes near the Tre locus are candidate genes for Tre. One (CG3171) encodes a rhodopsin family G protein receptor [3, 4], and the other (Gr5a) is a member of a chemosensory gene family encoding a putative gustatory receptor [5-7]. We carried out molecular analyses of mutations in Tre to elucidate their involvement in the gustatory phenotype. Here, we show that Tre mutations induced by P element-mediated genomic deletions disrupt Gr5a gene organization and the expression of Gr5a mRNA, while disruption of the CG3171 gene or its expression was not always associated with mutations in Tre. In flies with the spontaneous mutation Tre(01), both CG3171 and Gr5a mRNAs are transcribed. Coding sequences of these two candidate genes were compared among various strains. A total of three polymorphic sites leading to amino acid changes in CG3171 were not correlated with the gustatory phenotype. Among four nonsynonymous sites in Gr5a, a single nucleotide polymorphism leading to an Ala218Thr substitution in the predicted second intracellular loop cosegregated with Tre(01). Taken together, the mutation analyses support that Gr5a is allelic to Tre.     

Comparing the effects of five naturally occurring monosaccharide and oligosaccharide sugars on longevity and carbohydrate nutrient levels of a parasitic phorid fly, Pseudacteon tricuspis

Abstract.  The longevity and nutrient levels of Pseudacteon tricuspis provided with 1  m solutions of five naturally occurring sugars, fructose, glucose, sucrose, trehalose and melezitose, are compared. All but melezitose, result in significant increases in the longevity of P. tricuspis in comparison with sugar-starved flies (flies provided with water only). Sugar-starved female and male P. tricuspis have an average longevity of 3.3 and 4.1 days, respectively. Provision of free water in addition to sugar solution is necessary for optimum longevity by female and male flies. Longevity is increased by 2.4–2.6-fold by the two monosaccharides, fructose and glucose, and by 2.6–2.8-fold by the disaccharides, sucrose and trehalose. Phorid flies provided with the trisaccharide sugar, melezitose, had a marginal increase in lifespan (approximately 1 day), but this is not significantly different from the longevity of sugar-starved flies. Significantly greater levels of total sugars are detected in P. tricuspis fed the disaccharide sugars (sucrose, trehalose) or the monosaccharide sugars (fructose, glucose), compared with flies provided with melezitose (trisaccharide), or to sugar-starved flies. Fructose is not detected in sugar-starved flies, or in flies fed glucose or trehalose. However, high levels of fructose are detected in flies fed sucrose or fructose, whereas levels of fructose in melezitose-fed flies are intermediate. In general, significantly greater glycogen levels are detected in P. tricuspis fed sucrose, glucose, trehalose or fructose, compared with melezitose-fed or sugar-starved flies. Levels of total sugars and glycogen in sugar-fed flies are positively correlated with wing length, possibly indicating a higher accumulation of storage sugars by larger flies. These results are discussed in relation to the nutritional ecology of the phorid fly.     

Go α is involved in sugar perception in Drosophila

Detection of chemical compounds in food sources is based on the activation of 7 transmembrane gustatory receptors (GRs) in mammals and in insects such as Drosophila, although the receptors are not conserved between the classes. Different combinations of Drosophila GRs are involved in the detection of sugars, but the activated signaling cascades are largely unknown. Because 7 transmembrane receptors usually couple to G-proteins, we tried to unravel the intracellular signaling cascade in taste neurons by screening heterotrimeric G-protein mutant flies for gustatory deficits. We found the subunit Goα to be involved in feeding behavior and cell excitability by different transgenic and pharmacological approaches. Goα is involved in the detection of sucrose, glucose, and fructose, but not with trehalose and maltose. Our studies reveal that Goα plays an important role in the perception of some sweet tastants. Because the perception of other sweet stimuli was not affected by mutations in Goα, we also found strong indication for the existence of multiple signaling pathways in the insect gustatory system.     

Non-Invasive Screening for Alzheimer’s Disease by Sensing Salivary Sugar Using Drosophila Cells Expressing Gustatory Receptor (Gr5a) Immobilized on an Extended Gate Ion-Sensitive Field-Effect Transistor (EG-ISFET) Biosensor

Body fluids are often used as specimens for medical diagnosis. With the advent of advanced analytical techniques in biotechnology, the diagnostic potential of saliva has been the focus of many studies. We recently reported the presence of excess salivary sugars, in patients with Alzheimer’s disease (AD). In the present study, we developed a highly sensitive, cell-based biosensor to detect trehalose levels in patient saliva. The developed biosensor relies on the overexpression of sugar sensitive gustatory receptors (Gr5a) in Drosophila cells to detect the salivary trehalose. The cell-based biosensor was built on the foundation of an improved extended gate ion-sensitive field-effect transistor (EG-ISFET). Using an EG-ISFET, instead of a traditional ion-sensitive field-effect transistor (ISFET), resulted in an increase in the sensitivity and reliability of detection. The biosensor was designed with the gate terminals segregated from the conventional ISFET device. This design allows the construction of an independent reference and sensing region for simultaneous and accurate measurements of samples from controls and patients respectively. To investigate the efficacy of the cell-based biosensor for AD screening, we collected 20 saliva samples from each of the following groups: participants diagnosed with AD, participants diagnosed with Parkinson’s disease (PD), and a control group composed of healthy individuals. We then studied the response generated from the interaction of the salivary trehalose of the saliva samples and the Gr5a in the immobilized cells on an EG-ISFET sensor. The cell-based biosensor significantly distinguished salivary sugar, trehalose of the AD group from the PD and control groups. Based on these findings, we propose that salivary trehalose, might be a potential biomarker for AD and could be detected using our cell-based EG-ISFET biosensor. The cell-based EG-ISFET biosensor provides a sensitive and direct approach for salivary sugar detection and may be used in the future as a screening method for AD.     

Inositol 1,4,5-trisphosphate transduction cascade in taste reception of the fleshfly, Boettcherisca peregrina

The role of an inositol 1,4,5-trisphosphate (IP3)-mediated transduction cascade in the response of taste receptor cells of the fleshfly Boettcherisca peregrina was investigated by using the following reagents: neomycin (an inhibitor of IP3 production), U73122 (an inhibitor of phospholipase C), adenophostin A (an agonist of the IP3-gated channel), IP3, ruthenium red (a blocker of the IP3-gated channel), and 2-aminoethoxydiphenylborate (2-APB; an antagonist of the IP3-gated channel). For introduction into the receptor cell, the reagents were mixed with a detergent, deoxycholate (DOC). After treatment with neomycin + DOC or U73122 + DOC, the response of the sugar receptor cell to sugars was depressed compared with responses after treatment with DOC alone. During the treatment of adenophostin A + DOC, the response of the sugar receptor cell was elicited. After treatment with IP3 + DOC, the response of the sugar receptor cell to sugars and to amino acids was apparently enhanced. When taste stimuli were administered in the presence of ruthenium red or 2-APB, the response of the sugar receptor cell to glucose were inhibited. The expression of genes for substances involved in the IP3 transduction cascade, such as G protein alpha subunit (dGqalpha), phospholipase C (norpA), and IP3 receptor (itpr), were examined in the taste receptor cell of the fruitfly Drosophila melanogaster by using the pox-neuro70 mutant (poxn70), which lacks taste receptor cells. The expressed levels of dGqalpha and itpr in the tarsus of poxn70 mutant flies were reduced compared with those of wild-type flies. These results suggest that the IP3 transduction cascade is involved in the response of the sugar receptor cell of the fly.     

Odorant and Gustatory Receptors in the Tsetse Fly Glossina morsitans morsitans

Tsetse flies use olfactory and gustatory responses, through odorant and gustatory receptors (ORs and GRs), to interact with their environment. Glossina morsitans morsitans genome ORs and GRs were annotated using homologs of these genes in Drosophila melanogaster and an ab initio approach based on OR and GR specific motifs in G. m. morsitans gene models coupled to gene ontology (GO). Phylogenetic relationships among the ORs or GRs and the homologs were determined using Maximum Likelihood estimates. Relative expression levels among the G. m. morsitans ORs or GRs were established using RNA-seq data derived from adult female fly. Overall, 46 and 14 putative G. m. morsitans ORs and GRs respectively were recovered. These were reduced by 12 and 59 ORs and GRs respectively compared to D. melanogaster. Six of the ORs were homologous to a single D. melanogaster OR (DmOr67d) associated with mating deterrence in females. Sweet taste GRs, present in all the other Diptera, were not recovered in G. m. morsitans. The GRs associated with detection of CO₂ were conserved in G. m. morsitans relative to D. melanogaster. RNA-sequence data analysis revealed expression of GmmOR15 locus represented over 90% of expression profiles for the ORs. The G. m. morsitans ORs or GRs were phylogenetically closer to those in D. melanogaster than to other insects assessed. We found the chemoreceptor repertoire in G. m. morsitans smaller than other Diptera, and we postulate that this may be related to the restricted diet of blood-meal for both sexes of tsetse flies. However, the clade of some specific receptors has been expanded, indicative of their potential importance in chemoreception in the tsetse.     

Evolution of the sugar receptors in insects

Background  

Perception of sugars is an invaluable ability for insects which often derive quickly accessible energy from these molecules. A distinctive subfamily of eight proteins within the gustatory receptor (Gr) family has been identified as sugar receptors (SRs) in Drosophila melanogaster (Gr5a, Gr61a, and Gr64a-f). We examined the evolution of these SRs within the 12 available Drosophila genome sequences, as well as three mosquito, two moth, and beetle, bee, and wasp genome sequences.     

Differentiated response to sugars among labellar chemosensilla in Drosophila

Recent findings have indicated that the Gr genes for putative gustatory receptors of Drosophila melanogaster are expressed in a spatially restricted pattern among chemosensilla on the labellum. However, evidence for a functional segregation among the chemosensilla is lacking. In this work, labellar chemosensilla were classified and numbered into three groups, L-, I- and S-type, based on their morphology. Electrophysiological responses to sugars and salt were recorded from all the accessible labellar chemosensilla by the tip-recording method. All the L-type sensilla gave good responses to sugars in terms of action potential firing rates, while the probability for successful recordings from the I-type and S-type sensilla was lower. No differences were found in the responses to sugars between chemosensilla belonging to the same type; however, dose-response curves for several different sugars varied among the sensilla types. The L-type sensilla gave the highest frequency of nerve responses to all the sugars. The I-type sensilla also responded to all the sugars but with a lower magnitude of firing rate than the L-type sensilla. The S-type sensilla gave a good response to sucrose, and lower responses to the other sugars. These results suggest that there might be variations in the expression level or pattern of multiple receptors for sugars among the three types of chemosensilla. The expression pattern of six Gr genes was examined using the Gal4/UAS-GFP system, and sensilla were identified according to the innervation pattern of each GFP-expressing taste cell. None of the spatial expression patterns of the six Gr genes corresponded to the sugar sensitivity differences we observed.     

Sex-specific non-pheromonal taste receptors in Drosophila

Taste receptors have recently been reported in Drosophila [1,2], but little is known of the relation between receptor and response. Morphological studies of the distribution of chemosensory sensilla indicate that the fruit fly has two major sites of gustation: the proboscis and the legs [3]. The taste sensilla on both these sites are similar in structure and each sensillum generally houses four gustatory neurons [4]. Early anatomical observations have demonstrated a sexual dimorphism in the number of tarsal sensilla [5] and in their central projections [6]. We measured the electrophysiological responses of the prothoracic taste sensilla to non-pheromonal substances--salts, sugars and water--and found a clear sexual dimorphism. From the response profile of individual sensilla, we were able to distinguish three types of tarsal sensilla in females as against only two types in males. The female-specific type, which responded specifically to sugar, was absent in males except when male gustatory neurons were genetically feminised. The fact that tarsal gustatory hairs exhibit a sexual dimorphism that affects the perception of non-pheromonal compounds suggests that sexual identity is more complex than has previously been thought [7,8].     

The Drosophila NR4A nuclear receptor DHR38 regulates carbohydrate metabolism and glycogen storage

Animals balance nutrient storage and mobilization to maintain metabolic homeostasis, a process that is disrupted in metabolic diseases like obesity and diabetes. Here, we show that DHR38, the single fly ortholog of the mammalian nuclear receptor 4A family of nuclear receptors, regulates glycogen storage during the larval stages of Drosophila melanogaster. DHR38 is expressed and active in the gut and body wall of larvae, and its expression levels change in response to nutritional status. DHR38 null mutants have normal levels of glucose, trehalose (the major circulating form of sugar), and triacylglycerol but display reduced levels of glycogen in the body wall muscles, which constitute the primary storage site for carbohydrates. Microarray analysis reveals that many metabolic genes are mis-regulated in DHR38 mutants. These include phosphoglucomutase, which is required for glycogen synthesis, and the two genes that encode the digestive enzyme amylase, accounting for the reduced amylase enzyme activity seen in DHR38 mutant larvae. These studies demonstrate that a critical role of nuclear receptor 4A receptors in carbohydrate metabolism has been conserved through evolution and that nutritional regulation of DHR38 expression maintains the proper uptake and storage of glycogen during the growing larval stage of development.     

Diverse roles for the Drosophila fructose sensor Gr43a

The detection of nutrients, both in food and within the body, is crucial for the regulation of feeding behavior, growth, and metabolism. While the molecular basis for sensing food chemicals by the taste system has been firmly linked to specific taste receptors, relatively little is known about the molecular nature of the sensors that monitor nutrients internally. Recent reports of taste receptors expressed in other organ systems, foremost in the gastrointestinal tract of mammals and insects, has led to the proposition that some taste receptors may also be used as sensors of internal nutrients. Indeed, we provided direct evidence that the Drosophila gustatory receptor 43a (Gr43a) plays a critical role in sensing internal fructose levels in the fly brain. In addition to the brain and the taste system, Gr43a is also expressed in neurons of the proventricular ganglion and the uterus. Here, we discuss the multiple potential roles of Gr43a in the fly. We also provide evidence that its activation in the brain is likely mediated by the neuropeptide Corazonin. Finally, we posit that Gr43a may represent only a precedent for other taste receptors that sense internal nutrients, not only in flies but, quite possibly, in other animals, including mammals.     

Diverse roles for the Drosophila fructose sensor Gr43a

The detection of nutrients, both in food and within the body, is crucial for the regulation of feeding behavior, growth, and metabolism. While the molecular basis for sensing food chemicals by the taste system has been firmly linked to specific taste receptors, relatively little is known about the molecular nature of the sensors that monitor nutrients internally. Recent reports of taste receptors expressed in other organ systems, foremost in the gastrointestinal tract of mammals and insects, has led to the proposition that some taste receptors may also be used as sensors of internal nutrients. Indeed, we provided direct evidence that the Drosophila gustatory receptor 43a (Gr43a) plays a critical role in sensing internal fructose levels in the fly brain. In addition to the brain and the taste system, Gr43a is also expressed in neurons of the proventricular ganglion and the uterus. Here, we discuss the multiple potential roles of Gr43a in the fly. We also provide evidence that its activation in the brain is likely mediated by the neuropeptide Corazonin. Finally, we posit that Gr43a may represent only a precedent for other taste receptors that sense internal nutrients, not only in flies but, quite possibly, in other animals, including mammals.     

Gustatory receptor 22e is essential for sensing chloroquine and strychnine in Drosophila melanogaster

Chloroquine, an amino quinolone derivative commonly used as an anti-malarial drug, is known to impart an unpleasant taste. Little research has been done to study chloroquine taste in insects, therefore, we examined both the deterrant properties and mechanisms underlying chloroquine perception in fruit flies. We identified the antifeedant effect of chloroquine by screening 21 gustatory receptor (Grs) mutants through behavioral feeding assays and electrophysiology experiments. We discovered that two molecular sensors, GR22e and GR33a, act as chloroquine receptors, and found that chloroquine-mediated activation of GRNs occurs through S-type sensilla. At the same time, we successfully recapitulated the chloroquine receptor by expressing GR22e in ectopic gustatory receptor neurons. We also found that GR22e forms a part of the strychnine receptor. We suggest that the Drosophila strychnine receptor might have a very complex structure since five different GRs are required for strychnine-induced action potentials.