Remembering nutrient quality of sugar in Drosophila

Taste is an early stage in food and drink selection for most animals [1, 2]. Detecting sweetness indicates the presence of sugar and possible caloric content. However, sweet taste can be an unreliable predictor of nutrient value because some sugars cannot be metabolized. In addition, discrete sugars are detected by the same sensory neurons in the mammalian [3] and insect [4, 5] gustatory systems, making it difficult for animals to readily distinguish the identity of different sugars using taste alone [6-8]. Here we used an appetitive memory assay in Drosophila [9-11] to investigate the contribution of palatability and relative nutritional value of sugars to memory formation. We show that palatability and nutrient value both contribute to reinforcement of appetitive memory. Nonnutritious sugars formed less robust memory that could be augmented by supplementing with a tasteless but nutritious substance. Nutrient information is conveyed to the brain within minutes of training, when it can be used to guide expression of a sugar-preference memory. Therefore, flies can rapidly learn to discriminate between sugars using a postingestive reward evaluation system, and they preferentially remember nutritious sugars.     

Nutritional Value-Dependent and Nutritional Value-Independent Effects on Drosophila melanogaster Larval Behavior

Gustatory stimuli allow an organism not only to orient in its environment toward energy-rich food sources to maintain nutrition but also to avoid unpleasant or even poisonous substrates. For both mammals and insects, sugars-perceived as "sweet"-potentially predict nutritional benefit. Interestingly, even Drosophila adult flies are attracted to most high-potency sweeteners preferred by humans. However, the gustatory information of a sugar may be misleading as some sugars, although perceived as "sweet," cannot be metabolized. Accordingly, in adult Drosophila, a postingestive system that additionally evaluates the nutritional benefit of an ingested sugar has been shown to exist. By using a set of seven different sugars, which either offer (fructose, sucrose, glucose, maltodextrin, and sorbitol) or lack (xylose and arabinose) nutritional benefit, we show that Drosophila, at the larval stage, can perceive and evaluate sugars based on both nutrition-dependent and -independent qualities. In detail, we find that larval survival and feeding mainly depend on the nutritional value of a particular sugar. In contrast, larval choice behavior and learning are regulated in a more complex way by nutrition value-dependent and nutrition value-independent information. The simplicity of the larval neuronal circuits and their accessibility to genetic manipulation may ultimately allow one to identify the neuronal and molecular basis of the larval sugar perception systems described here behaviorally.     

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.     

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.     

Whole nerve chorda tympani responses to sweeteners in C57BL/6ByJ and 129P3/J mice

The C57BL/6ByJ (B6) strain of mice exhibits higher preferences than does the 129P3/J (129) strain for a variety of sweet tasting compounds. We measured gustatory afferent responses of the whole chorda tympani nerve in these two strains using a broad array of sweeteners and other taste stimuli. Neural responses were greater in B6 than in 129 mice to the sugars sucrose and maltose, the polyol D-sorbitol and the non-caloric sweeteners Na saccharin, acesulfame-K, SC-45647 and sucralose. Lower neural response thresholds were also observed in the B6 strain for most of these stimuli. The strains did not differ in their neural responses to amino acids that are thought to taste sweet to mice, with the exception of L-proline, which evoked larger responses in the B6 strain. Aspartame and thaumatin, which taste sweet to humans but are not strongly preferred by B6 or 129 mice, did not evoke neural responses that exceeded threshold in either strain. The strains generally did not differ in their neural responses to NaCl, quinine and HCl. Thus, variation between the B6 and 129 strains in the peripheral gustatory system may contribute to differences in their consumption of many sweeteners.     

Consumption of dietary sugar by gut bacteria determines Drosophila lipid content

Gut microorganisms are essential for the nutritional health of many animals, but the underlying mechanisms are poorly understood. This study investigated how lipid accumulation by adult Drosophila melanogaster is reduced in flies associated with the bacterium Acetobacter tropicalis which displays oral–faecal cycling between the gut and food. We demonstrate that the lower lipid content of A. tropicalis-colonized flies relative to bacteria-free flies is linked with a parallel bacterial-mediated reduction in food glucose content; and can be accounted for quantitatively by the amount of glucose acquired by the flies, as determined from the feeding rate and assimilation efficiency of bacteria-free and A. tropicalis-colonized flies. We recommend that nutritional studies on Drosophila include empirical quantification of food nutrient content, to account for likely microbial-mediated effects on diet composition. More broadly, this study demonstrates that selective consumption of dietary constituents by microorganisms can alter the nutritional balance of food and, thereby, influence the nutritional status of the animal host.     

Peripheral, central and behavioral responses to the cuticular pheromone bouquet in Drosophila melanogaster males

Pheromonal communication is crucial with regard to mate choice in many animals including insects. Drosophila melanogaster flies produce a pheromonal bouquet with many cuticular hydrocarbons some of which diverge between the sexes and differently affect male courtship behavior. Cuticular pheromones have a relatively high weight and are thought to be -- mostly but not only -- detected by gustatory contact. However, the response of the peripheral and central gustatory systems to these substances remains poorly explored. We measured the effect induced by pheromonal cuticular mixtures on (i) the electrophysiological response of peripheral gustatory receptor neurons, (ii) the calcium variation in brain centers receiving these gustatory inputs and (iii) the behavioral reaction induced in control males and in mutant desat1 males, which show abnormal pheromone production and perception. While male and female pheromones induced inhibitory-like effects on taste receptor neurons, the contact of male pheromones on male fore-tarsi elicits a long-lasting response of higher intensity in the dedicated gustatory brain center. We found that the behavior of control males was more strongly inhibited by male pheromones than by female pheromones, but this difference disappeared in anosmic males. Mutant desat1 males showed an increased sensitivity of their peripheral gustatory neurons to contact pheromones and a behavioral incapacity to discriminate sex pheromones. Together our data indicate that cuticular hydrocarbons induce long-lasting inhibitory effects on the relevant taste pathway which may interact with the olfactory pathway to modulate pheromonal perception.     

Drosophila Fatty Acid Taste Signals through the PLC Pathway in Sugar-Sensing Neurons

Taste is the primary sensory system for detecting food quality and palatability. Drosophila detects five distinct taste modalities that include sweet, bitter, salt, water, and the taste of carbonation. Of these, sweet-sensing neurons appear to have utility for the detection of nutritionally rich food while bitter-sensing neurons signal toxicity and confer repulsion. Growing evidence in mammals suggests that taste for fatty acids (FAs) signals the presence of dietary lipids and promotes feeding. While flies appear to be attracted to fatty acids, the neural basis for fatty acid detection and attraction are unclear. Here, we demonstrate that a range of FAs are detected by the fly gustatory system and elicit a robust feeding response. Flies lacking olfactory organs respond robustly to FAs, confirming that FA attraction is mediated through the gustatory system. Furthermore, flies detect FAs independent of pH, suggesting the molecular basis for FA taste is not due to acidity. We show that low and medium concentrations of FAs serve as an appetitive signal and they are detected exclusively through the same subset of neurons that sense appetitive sweet substances, including most sugars. In mammals, taste perception of sweet and bitter substances is dependent on phospholipase C (PLC) signaling in specialized taste buds. We find that flies mutant for norpA, a Drosophila ortholog of PLC, fail to respond to FAs. Intriguingly, norpA mutants respond normally to other tastants, including sucrose and yeast. The defect of norpA mutants can be rescued by selectively restoring norpA expression in sweet-sensing neurons, corroborating that FAs signal through sweet-sensing neurons, and suggesting PLC signaling in the gustatory system is specifically involved in FA taste. Taken together, these findings reveal that PLC function in Drosophila sweet-sensing neurons is a conserved molecular signaling pathway that confers attraction to fatty acids.     

Behavioral responses to odorants in drosophila require nervous system expression of the beta integrin gene myospheroid

Integrins are cell adhesion molecules that mediate numerous developmental processes in addition to a variety of acute physiological events. Two reports implicate a Drosophila beta integrin, betaPS, in olfactory behavior. To further investigate the role of integrins in Drosophila olfaction, we used Gal4-driven expression of RNA interference (RNAi) transgenes to knock down expression of myospheroid (mys), the gene that encodes betaPS. Expression of mys-RNAi transgenes in the wing reduced betaPS immunostaining and produced morphological defects associated with loss-of-function mutations in mys, demonstrating that this strategy knocked down mys function. Expression of mys-RNAi transgenes in the antennae, antennal lobes, and mushroom bodies via two Gal4 lines, H24 and MT14, disrupted olfactory behavior but did not alter locomotor abilities or central nervous system structure. Olfactory behavior was normal in flies that expressed mys-RNAi transgenes via other Gal4 lines that specifically targeted the antennae, the projection neurons, the mushroom bodies, bitter and sweet gustatory neurons, or Pox neuro neurons. Our studies confirm that mys is important for the development or function of the Drosophila olfactory system. Additionally, our studies demonstrate that mys is required for normal behavioral responses to both aversive and attractive odorants. Our results are consistent with a model in which betaPS mediates events within the antennal lobes that influence odorant sensitivity.     

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.     

Exploring the neural mechanisms of aversion to bitter gourd phytochemicals in insects using Drosophila

Bitter gourd (Momordica charantia L.) has compounds that repel insect pests. Unlike conventional pesticides, these compounds are eco-friendly and beneficial for human health. However the mechanisms by which these compounds repel insects and affect their physiology remains poorly known. Here we used Drosophila melanogaster (Meigen) to address these issues. We tested a wild strain, and a laboratory bred Canton S strain. Bitter gourd extract reduced the viability of developing flies, but did not affect survival in adults. Flies avoided bitter gourd extract in a food choice assay, and consumed a significantly low amount of food mixed with bitter gourd – indicating that it acts as an antifeedant. Transgenic flies with impaired aversive taste sensitive neurons showed a reduced aversion towards bitter gourd extract showing that these compounds act through the bitter sensitive gustatory neurons. Finally, flies also retained the memory of consuming bitter gourd extract for at least 24 hours, suggesting an additional cognitive mechanism for long term aversion. Our study provides the first evidence of bitter gourd compounds acting as antifeedants and also as potent reinforcers of aversive memory in drosophilids. We suggest that flies can be used to understand the physiological and neural mechanisms underlying the mode of action of other such phyto-extracts with the goal of developing potent but less harmful pest control formulations.     

How social network structure affects decision-making in Drosophila melanogaster

Animals use a number of different mechanisms to acquire crucial information. During social encounters, animals can pass information from one to another but, ideally, they would only use information that benefits survival and reproduction. Therefore, individuals need to be able to determine the value of the information they receive. One cue can come from the behaviour of other individuals that are already using the information. Using a previous extended dataset, we studied how individual decision-making is influenced by the behaviour of conspecifics in Drosophila melanogaster. We analysed how uninformed flies acquire and later use information about oviposition site choice they learn from informed flies. Our results suggest that uninformed flies adjust their future choices based on how coordinated the behaviours of the informed individuals they encounter are. Following social interaction, uninformed flies tended either to collectively follow the choice of the informed flies or to avoid it. Using social network analysis, we show that this selective information use seems to be based on the level of homogeneity of the social network. In particular, we found that the variance of individual centrality parameters among informed flies was lower in the case of a ‘follow’ outcome compared with the case of an ‘avoid’ outcome.     

Learned odor discrimination in Drosophila without combinatorial odor maps in the antennal lobe

A unifying feature of mammalian and insect olfactory systems is that olfactory sensory neurons (OSNs) expressing the same unique odorant-receptor gene converge onto the same glomeruli in the brain [1-7]. Most odorants activate a combination of receptors and thus distinct patterns of glomeruli, forming a proposed combinatorial spatial code that could support discrimination between a large number of odorants [8-11]. OSNs also exhibit odor-evoked responses with complex temporal dynamics [11], but the contribution of this activity to behavioral odor discrimination has received little attention [12]. Here, we investigated the importance of spatial encoding in the relatively simple Drosophila antennal lobe. We show that Drosophila can learn to discriminate between two odorants with one functional class of Or83b-expressing OSNs. Furthermore, these flies encode one odorant from a mixture and cross-adapt to odorants that activate the relevant OSN class, demonstrating that they discriminate odorants by using the same OSNs. Lastly, flies with a single class of Or83b-expressing OSNs recognize a specific odorant across a range of concentration, indicating that they encode odorant identity. Therefore, flies can distinguish odorants without discrete spatial codes in the antennal lobe, implying an important role for odorant-evoked temporal dynamics in behavioral odorant discrimination.     

Re-patterning sleep architecture in Drosophila through gustatory perception and nutritional quality

Organisms perceive changes in their dietary environment and enact a suite of behavioral and metabolic adaptations that can impact motivational behavior, disease resistance, and longevity. However, the precise nature and mechanism of these dietary responses is not known. We have uncovered a novel link between dietary factors and sleep behavior in Drosophila melanogaster. Dietary sugar rapidly altered sleep behavior by modulating the number of sleep episodes during both the light and dark phase of the circadian period, independent of an intact circadian rhythm and without affecting total sleep, latency to sleep, or waking activity. The effect of sugar on sleep episode number was consistent with a change in arousal threshold for waking. Dietary protein had no significant effect on sleep or wakefulness. Gustatory perception of sugar was necessary and sufficient to increase the number of sleep episodes, and this effect was blocked by activation of bitter-sensing neurons. Further addition of sugar to the diet blocked the effects of sweet gustatory perception through a gustatory-independent mechanism. However, gustatory perception was not required for diet-induced fat accumulation, indicating that sleep and energy storage are mechanistically separable. We propose a two-component model where gustatory and metabolic cues interact to regulate sleep architecture in response to the quantity of sugar available from dietary sources. Reduced arousal threshold in response to low dietary availability may have evolved to provide increased responsiveness to cues associated with alternative nutrient-dense feeding sites. These results provide evidence that gustatory perception can alter arousal thresholds for sleep behavior in response to dietary cues and provide a mechanism by which organisms tune their behavior and physiology to environmental cues.     

A Drosophila protein specific to pheromone-sensing gustatory hairs delays males' copulation attempts

In insects, increasing evidence suggests that small secreted pheromone binding proteins (PBPs) and odorant binding proteins (OBPs) are important for normal olfactory detection of airborne pheromones and odorants far from their source. In contrast, it is unknown whether extracellular ligand binding proteins participate in perception of less volatile chemicals, including many pheromones, that are detected by direct contact with chemosensory organs. CheB42a, a small Drosophila melanogaster protein unrelated to known PBPs or OBPs, is expressed and likely secreted in only a small subset of gustatory sensilla on males' front legs, the site of gustatory perception of contact pheromones. Here we show that CheB42a is expressed specifically in the sheath cells surrounding the taste neurons expressing Gr68a, a putative gustatory pheromone receptor for female cuticular hydrocarbons that stimulate male courtship. Surprisingly, however, CheB42a mutant males attempt to copulate with females earlier and more frequently than control males. Furthermore, CheB42a mutant males also attempt to copulate more frequently with other males that secrete female-specific cuticular hydrocarbon pheromones, but not with females lacking cuticular hydrocarbons. Together, these data indicate that CheB42a is required for a normal gustatory response to female cuticular hydrocarbon pheromones that modulate male courtship.     

A microarray analysis of genes involved in relating egg production to nutritional intake in Drosophila melanogaster

Egg chambers of Drosophila are reabsorbed under conditions of nutritional shortage by inducing apoptosis at stages 8 and 9, midway through oogenesis. Nutritional shortage leads to an increase in ecdysone concentration in flies. Apoptosis at stage 8/9 is also induced by 20-hydroxyecdysone injection into the females maintained with adequate nutrition. The expression pattern in the ovary of some ecdysone response genes, E75A, BR-C, is different according to the nutritional environment and the overexpression of these genes induces apoptosis. Apoptosis is suppressed by Juvenile hormone analog treatment of females under nutritional shortage. We predict nutritional and stress response genes control hormone levels and the increase in ecdysone concentration in the flies following starvation induces the ovarian apoptosis. We therefore used a microarray approach to identify the genes involved in receiving the nutritional signal from the environment and translating it in the ovary, thus initiating and executing apoptosis.     

Mechanisms of Carboxylic Acid Attraction in Drosophila melanogaster

Sour is one of the fundamental taste modalities that enable taste perception in animals. Chemoreceptors embedded in taste organs are pivotal to discriminate between different chemicals to ensure survival. Animals generally prefer slightly acidic food and avoid highly acidic alternatives. We recently proposed that all acids are aversive at high concentrations, a response that is mediated by low pH as well as specific anions in Drosophila melanogaster. Particularly, some carboxylic acids such as glycolic acid, citric acid, and lactic acid are highly attractive to Drosophila compared with acetic acid. The present study determined that attractive carboxylic acids were mediated by broadly expressed Ir25a and Ir76b, as demonstrated by a candidate mutant library screen. The mutant deficits were completely recovered via wild-type cDNA expression in sweet-sensing gustatory receptor neurons. Furthermore, sweet gustatory receptors such as Gr5a, Gr61a, and Gr64a-f modulate attractive responses. These genetic defects were confirmed using binary food choice assays as well as electrophysiology in the labellum. Taken together, our findings demonstrate that at least two different kinds of receptors are required to discriminate attractive carboxylic acids from other acids.     

The course of habituation of the proboscis extension reflex can be predicted by sucrose responsiveness in Drosophila

The proboscis extension reflex (PER) is triggered when insects' gustatory receptors contact appetitive stimuli, so it provides a behavioral readout for perceptual encoding of tastants. Research on the experience dependent modulation of PER in Drosophila has been hindered by the difficulty of obtaining reliable measures of memory-driven change in PER probability in the background of larger changes induced by physiological state. In this study, we showed that the course of PER habituation can be predicted by the degree of sucrose responsiveness in Drosophila. We assessed early response parameters, including the number of proboscis extensions and labellar movements in the first five trials, the trial to start responding, and the trial to make the first stop to quantify responsiveness, which predicted the upcoming pattern of both the short-term and 1 hour memory of PER habituation for individual flies. The cAMP signaling pathway mutant rutabaga displayed deficits in attunement of perceptual salience of sucrose to physiological demands and stimulus-driven sensitization.