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• W2064273702 abstract "•Gr43a is the main receptor for larval sugar preference•Drosophila larvae do not require classic sugar receptors for sugar sensing•Immediate fructose preference is mediated by pharyngeal Gr43a sensory neurons•Late sugars preference is mediated by Gr43a-expressing brain neurons 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 [1Wang Z. Singhvi A. Kong P. Scott K. Taste representations in the Drosophila brain.Cell. 2004; 117: 981-991Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 2Thorne N. Chromey C. Bray S. Amrein H. Taste perception and coding in Drosophila.Curr. Biol. 2004; 14: 1065-1079Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar]. 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 [3Dahanukar A. Foster K. van der Goes van Naters W.M. Carlson J.R. A Gr receptor is required for response to the sugar trehalose in taste neurons of Drosophila.Nat. Neurosci. 2001; 4: 1182-1186Crossref PubMed Scopus (207) Google Scholar, 4Miyamoto T. Chen Y. Slone J. Amrein H. Identification of a Drosophila glucose receptor using Ca2+ imaging of single chemosensory neurons.PLoS ONE. 2013; 8: e56304Crossref PubMed Scopus (52) Google Scholar, 5Ueno K. Ohta M. Morita H. Mikuni Y. Nakajima S. Yamamoto K. Isono K. Trehalose sensitivity in Drosophila correlates with mutations in and expression of the gustatory receptor gene Gr5a.Curr. Biol. 2001; 11: 1451-1455Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 6Slone J. Daniels J. Amrein H. Sugar receptors in Drosophila.Curr. Biol. 2007; 17: 1809-1816Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 7Jiao Y. Moon S.J. Montell C. A Drosophila gustatory receptor required for the responses to sucrose, glucose, and maltose identified by mRNA tagging.Proc. Natl. Acad. Sci. USA. 2007; 104: 14110-14115Crossref PubMed Scopus (156) Google Scholar, 8Dahanukar A. Lei Y.T. Kwon J.Y. Carlson J.R. Two Gr genes underlie sugar reception in Drosophila.Neuron. 2007; 56: 503-516Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar], while the majority of the remaining Gr genes are likely to encode bitter taste receptors [9Lee Y. Moon S.J. Montell C. Multiple gustatory receptors required for the caffeine response in Drosophila.Proc. Natl. Acad. Sci. USA. 2009; 106: 4495-4500Crossref PubMed Scopus (156) Google Scholar, 10Moon S.J. Köttgen M. Jiao Y. Xu H. Montell C. A taste receptor required for the caffeine response in vivo.Curr. Biol. 2006; 16: 1812-1817Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 11Moon S.J. Lee Y. Jiao Y. Montell C. A Drosophila gustatory receptor essential for aversive taste and inhibiting male-to-male courtship.Curr. Biol. 2009; 19: 1623-1627Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar], albeit some function as pheromone [12Wang L. Han X. Mehren J. Hiroi M. Billeter J.C. Miyamoto T. Amrein H. Levine J.D. Anderson D.J. Hierarchical chemosensory regulation of male-male social interactions in Drosophila.Nat. Neurosci. 2011; 14: 757-762Crossref PubMed Scopus (150) Google Scholar, 13Miyamoto T. Amrein H. Suppression of male courtship by a Drosophila pheromone receptor.Nat. Neurosci. 2008; 11: 874-876Crossref PubMed Scopus (142) Google Scholar, 14Bray S. Amrein H. A putative Drosophila pheromone receptor expressed in male-specific taste neurons is required for efficient courtship.Neuron. 2003; 39: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar] and carbon dioxide [15Kwon J.Y. Dahanukar A. Weiss L.A. Carlson J.R. The molecular basis of CO2 reception in Drosophila.Proc. Natl. Acad. Sci. USA. 2007; 104: 3574-3578Crossref PubMed Scopus (349) Google Scholar, 16Jones W.D. Cayirlioglu P. Kadow I.G. Vosshall L.B. Two chemosensory receptors together mediate carbon dioxide detection in Drosophila.Nature. 2007; 445: 86-90Crossref PubMed Scopus (471) Google Scholar] 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 [17Miyamoto T. Slone J. Song X. Amrein H. A fructose receptor functions as a nutrient sensor in the Drosophila brain.Cell. 2012; 151: 1113-1125Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar], 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. 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 [1Wang Z. Singhvi A. Kong P. Scott K. Taste representations in the Drosophila brain.Cell. 2004; 117: 981-991Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 2Thorne N. Chromey C. Bray S. Amrein H. Taste perception and coding in Drosophila.Curr. Biol. 2004; 14: 1065-1079Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar]. 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 [3Dahanukar A. Foster K. van der Goes van Naters W.M. Carlson J.R. A Gr receptor is required for response to the sugar trehalose in taste neurons of Drosophila.Nat. Neurosci. 2001; 4: 1182-1186Crossref PubMed Scopus (207) Google Scholar, 4Miyamoto T. Chen Y. Slone J. Amrein H. Identification of a Drosophila glucose receptor using Ca2+ imaging of single chemosensory neurons.PLoS ONE. 2013; 8: e56304Crossref PubMed Scopus (52) Google Scholar, 5Ueno K. Ohta M. Morita H. Mikuni Y. Nakajima S. Yamamoto K. Isono K. Trehalose sensitivity in Drosophila correlates with mutations in and expression of the gustatory receptor gene Gr5a.Curr. Biol. 2001; 11: 1451-1455Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 6Slone J. Daniels J. Amrein H. Sugar receptors in Drosophila.Curr. Biol. 2007; 17: 1809-1816Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 7Jiao Y. Moon S.J. Montell C. A Drosophila gustatory receptor required for the responses to sucrose, glucose, and maltose identified by mRNA tagging.Proc. Natl. Acad. Sci. USA. 2007; 104: 14110-14115Crossref PubMed Scopus (156) Google Scholar, 8Dahanukar A. Lei Y.T. Kwon J.Y. Carlson J.R. Two Gr genes underlie sugar reception in Drosophila.Neuron. 2007; 56: 503-516Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar], while the majority of the remaining Gr genes are likely to encode bitter taste receptors [9Lee Y. Moon S.J. Montell C. Multiple gustatory receptors required for the caffeine response in Drosophila.Proc. Natl. Acad. Sci. USA. 2009; 106: 4495-4500Crossref PubMed Scopus (156) Google Scholar, 10Moon S.J. Köttgen M. Jiao Y. Xu H. Montell C. A taste receptor required for the caffeine response in vivo.Curr. Biol. 2006; 16: 1812-1817Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 11Moon S.J. Lee Y. Jiao Y. Montell C. A Drosophila gustatory receptor essential for aversive taste and inhibiting male-to-male courtship.Curr. Biol. 2009; 19: 1623-1627Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar], albeit some function as pheromone [12Wang L. Han X. Mehren J. Hiroi M. Billeter J.C. Miyamoto T. Amrein H. Levine J.D. Anderson D.J. Hierarchical chemosensory regulation of male-male social interactions in Drosophila.Nat. Neurosci. 2011; 14: 757-762Crossref PubMed Scopus (150) Google Scholar, 13Miyamoto T. Amrein H. Suppression of male courtship by a Drosophila pheromone receptor.Nat. Neurosci. 2008; 11: 874-876Crossref PubMed Scopus (142) Google Scholar, 14Bray S. Amrein H. A putative Drosophila pheromone receptor expressed in male-specific taste neurons is required for efficient courtship.Neuron. 2003; 39: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar] and carbon dioxide [15Kwon J.Y. Dahanukar A. Weiss L.A. Carlson J.R. The molecular basis of CO2 reception in Drosophila.Proc. Natl. Acad. Sci. USA. 2007; 104: 3574-3578Crossref PubMed Scopus (349) Google Scholar, 16Jones W.D. Cayirlioglu P. Kadow I.G. Vosshall L.B. Two chemosensory receptors together mediate carbon dioxide detection in Drosophila.Nature. 2007; 445: 86-90Crossref PubMed Scopus (471) Google Scholar] 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 [17Miyamoto T. Slone J. Song X. Amrein H. A fructose receptor functions as a nutrient sensor in the Drosophila brain.Cell. 2012; 151: 1113-1125Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar], 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. The larval taste system is compartmentalized, and taste neurons are found in five major anatomical sites (Figure 1A) [18Stocker R.F. Design of the larval chemosensory system.Adv. Exp. Med. Biol. 2008; 628: 69-81Crossref PubMed Scopus (34) Google Scholar, 19Kwon J.Y. Dahanukar A. Weiss L.A. Carlson J.R. Molecular and cellular organization of the taste system in the Drosophila larva.J. Neurosci. 2011; 31: 15300-15309Crossref PubMed Scopus (73) Google Scholar]. Two of these sites, the terminal and ventral organ (TO and VO; Figure 1A), sample soluble chemicals externally. Three internal taste organs, the dorsal, ventral, and posterior pharyngeal sense organs (DPS, VPS, and PPS) monitor chemicals as food passes through the pharynx (Figure 1A). About 40 Gr genes are expressed in neurons of both the external taste organs, as well as in internal pharyngeal sense organs; however, none of the eight sugar Gr genes are expressed in larval chemosensory neurons [19Kwon J.Y. Dahanukar A. Weiss L.A. Carlson J.R. Molecular and cellular organization of the taste system in the Drosophila larva.J. Neurosci. 2011; 31: 15300-15309Crossref PubMed Scopus (73) Google Scholar], even though larvae can sense most sugars [20Schipanski A. Yarali A. Niewalda T. Gerber B. Behavioral analyses of sugar processing in choice, feeding, and learning in larval Drosophila.Chem. Senses. 2008; 33: 563-573Crossref PubMed Scopus (56) Google Scholar]. To identify larval sugar receptor(s), we adapted the two-choice taste assay developed by Schipanski and colleagues [20Schipanski A. Yarali A. Niewalda T. Gerber B. Behavioral analyses of sugar processing in choice, feeding, and learning in larval Drosophila.Chem. Senses. 2008; 33: 563-573Crossref PubMed Scopus (56) Google Scholar]. w1118 (i.e., Gr43a+ control) larvae show strong preference for fructose and sucrose across a wide concentration range (500 mM to 0.8 mM; Figure S1 available online). Larvae exhibit an immediate attraction (i.e., within 2 min) to both sugars at concentrations as low as 20 mM, while preference at even lower concentrations is delayed and not noticeable until 4 min or more. An immediate preference for glucose and trehalose is observed only at very high concentrations (1 M and 500 mM), but a preference for both sugars is also noticeable at 100 mM concentrations after 16 min (Figure S1). These observations confirm previous findings [20Schipanski A. Yarali A. Niewalda T. Gerber B. Behavioral analyses of sugar processing in choice, feeding, and learning in larval Drosophila.Chem. Senses. 2008; 33: 563-573Crossref PubMed Scopus (56) Google Scholar] and reveal that larvae exhibit a distinct attraction for sugar containing agar in a time- and concentration-dependent fashion. While the sugar Gr genes appear not to be expressed in larvae, Gr43a was found in two internal taste neurons of larvae [19Kwon J.Y. Dahanukar A. Weiss L.A. Carlson J.R. Molecular and cellular organization of the taste system in the Drosophila larva.J. Neurosci. 2011; 31: 15300-15309Crossref PubMed Scopus (73) Google Scholar]. To investigate Gr43a expression in more detail, we employed Gr43aGAL4, an allele in which the coding sequence of Gr43a was replaced by that of Gal4 using homologous recombination [17Miyamoto T. Slone J. Song X. Amrein H. A fructose receptor functions as a nutrient sensor in the Drosophila brain.Cell. 2012; 151: 1113-1125Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar]. Gr43aGAL4 is expressed in approximately four neurons in the pharyngeal taste clusters (DPS, VPS, and PPS), while no expression is observed in the external taste organs (TO and VO) [19Kwon J.Y. Dahanukar A. Weiss L.A. Carlson J.R. Molecular and cellular organization of the taste system in the Drosophila larva.J. Neurosci. 2011; 31: 15300-15309Crossref PubMed Scopus (73) Google Scholar] (Figure 1B). Combining Gr43aGAL4 and Gr66a-GFP-IRES-GFP-IRES-GFP, a marker for bitter-sensing neurons, revealed no overlap in expression between these two genes (Figures 1C and 1D), which is consistent with a possible function of Gr43a in sugar sensing. We note that Gr43aGAL4 is also expressed in neurons associated with the proventriculus (Figure 1B; Figure S2). Furthermore, immunostaining identified Gr43aGAL4 expression in the brain, including neurons in the protocerebrum, the mushroom body and the ventral nerve cord (VNC; Figure 1E; Figure S2). We next carried out behavioral two-choice preference assays for fructose and glucose using w1118 control larvae, larvae lacking all eight sugar Gr genes, Gr43aGAL4 mutant larvae, and larvae in which the Gr43aGAL4 null mutation is rescued by a UAS-Gr43a transgene (Figure 2A). The preference for fructose was abolished in Gr43a mutants but not in larvae lacking the sugar Gr genes. Importantly, Gr43a mutant larvae expressing a UAS-Gr43a transgene regained their preference for fructose (Figure 2A; Figure S3A). When larvae were tested for glucose preference, all genotypes but the Gr43a mutants exhibited a pronounced attraction to this sugar (Figure 2A). However, establishing this preference required prolonged assay time (16 min). Regardless, these observations indicate that the preference for glucose and fructose is dependent on a single Gr protein, Gr43a, which is surprising since this receptor is narrowly tuned to fructose and does not respond to glucose [17Miyamoto T. Slone J. Song X. Amrein H. A fructose receptor functions as a nutrient sensor in the Drosophila brain.Cell. 2012; 151: 1113-1125Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 21Sato K. Tanaka K. Touhara K. Sugar-regulated cation channel formed by an insect gustatory receptor.Proc. Natl. Acad. Sci. USA. 2011; 108: 11680-11685Crossref PubMed Scopus (175) Google Scholar]. We therefore tested larvae with two additional sugars, melezitose and sorbitol (Figure 2B). Melezitose is a complex nutritious sugar containing a fructose moiety and indeed was sensed immediately, just like fructose. Sorbitol, a tasteless sugar alcohol, is sensed in a moderately delayed fashion (but faster than glucose) by wild-type larvae but not sensed at all in Gr43a mutants. Importantly, the phenotype of Gr43a mutants to both melezitose and sorbitol is rescued with the expected temporally displayed preference in the presence of a UAS-Gr43a transgene (Figure 2B). Lastly, lack of preference is specific to sugars because Gr43a mutant and control larvae showed similar preference for agar containing casein peptone (an amino acid/peptide mixture), a mixture of three amino acids or low concentration of sodium chloride (Figure 2C; Figure S3B). Taken together, our analysis shows that in Drosophila larvae, (1) fructose is the most stimulating sugar substrate, (2) Gr43a is the main sugar receptor and acts independently of the sugar Gr proteins, (3) the sugar Gr proteins play no significant role in sugar sensing, and (4) nonfructose-containing sugars, such as glucose and trehalose, are probably sensed by a postprandial mechanism that relies on conversion of (a fraction of) these sugars into fructose ([22Harvey R.A. Ferrier D.R. Biochemistry: Lippincott’s Illustrated Reviews. 5th Edition. Lippincott, Williams and Wilkins, Philadelphia2011Google Scholar], see below). The two-choice feeding assay reveals a time-dependent component for establishing a food preference. This is evident when comparing the response for six different sugars versus agar alone at 100 mM concentration over a 16 min assay period. Larvae show an immediate preference (i.e., within the first 2 min) for fructose and sugars containing a fructose moiety (sucrose, melezitose), while they fail to establish such a preference for glucose, trehalose, or sorbitol (Figures 2A and 2B; Figures S1 and S3A). Notably, after 16 min, wild-type larvae, but not Gr43a mutant larvae, exhibit a robust, late preference for all these sugars as well (Figures 2A and 2B). We propose that the late preference for glucose, trehalose, and sorbitol is mediated by internal Gr43a-expressing neurons, probably those in the brain. In adult flies, a fraction of ingested nutritious carbohydrates are converted into fructose, leading to transient fructose increase in the hemolymph, which in turn mediates satiation-dependent feeding activity [17Miyamoto T. Slone J. Song X. Amrein H. A fructose receptor functions as a nutrient sensor in the Drosophila brain.Cell. 2012; 151: 1113-1125Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar]. To address the role of the brain neurons in fructose sensing, we investigated the temporal preference dynamics of Gr43aGAL4 mutant larvae expressing a UAS-Gr43a transgene with and without Cha7.4kb-GAL80 (Figure 3). GAL80 binds to and suppresses transcriptional activity of GAL4 [23Suster M.L. Seugnet L. Bate M. Sokolowski M.B. Refining GAL4-driven transgene expression in Drosophila with a GAL80 enhancer-trap.Genesis. 2004; 39: 240-245Crossref PubMed Scopus (83) Google Scholar]. Cha7.4kb-GAL80 suppresses GAL4 expression almost completely in the two internal taste clusters and the neurons associated with the proventriculus but not the neurons in the brain (Figure 3A). As expected, “Gr43a brain-only larvae” showed the same preference dynamics for glucose and sorbitol as control larvae; however, their immediate (2 min) preference to fructose was severely reduced, albeit not completely abolished (Figure 3B). This may be explained by residual expression of Gr43a in proventricular neurons of some larvae (Figure 3A) or by rapid uptake of fructose into the hemolymph. We then tested sugar preference in larvae in which Gr43a function was restricted to peripheral neurons by expressing tetanus toxin (TNT) in the brain (Gr43aGAL4/+; UAS-TNT/ Cha7.4kb-GAL80; TNT inhibits neural transmission [24Martin J.R. Keller A. Sweeney S.T. Targeted expression of tetanus toxin: a new tool to study the neurobiology of behavior.Adv. Genet. 2002; 47: 1-47Crossref PubMed Google Scholar]). Indeed, these larvae exhibited an immediate preference for fructose but failed to generate a late preference for glucose and sorbitol (Figure 3C). In adults, feeding of all nutritious sugars, such as fructose, sorbitol, or glucose, leads to a significant increase of hemolymph fructose [17Miyamoto T. Slone J. Song X. Amrein H. A fructose receptor functions as a nutrient sensor in the Drosophila brain.Cell. 2012; 151: 1113-1125Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar]. To examine the effects of dietary sugars on larval hemolymph fructose, we let larvae feed on agarose only (control) or agarose containing 100 mM sugars (fructose, sorbitol, or glucose) before their hemolymph was collected and examined for the three major hemolymph sugars. As expected, hemolymph fructose levels increased significantly when fed fructose or sorbitol, while hemolymph glucose/trehalose levels did not change (Figure 4). When larvae were fed glucose, hemolymph glucose and trehalose levels increased, the latter significantly, while no change in hemolymph fructose was observed. The sweet taste of sugars is crucial for providing animals a hedonic stimulus for feeding on nutritious carbohydrates. Here, we have shown that the sugar Gr genes mediating sweet taste in adult flies are dispensable for sugar sensing during the larval stage. Instead, Gr43a is the major sweet taste receptor in larvae. Gr43a is narrowly tuned to fructose and the fructose-containing disaccharide sucrose [17Miyamoto T. Slone J. Song X. Amrein H. A fructose receptor functions as a nutrient sensor in the Drosophila brain.Cell. 2012; 151: 1113-1125Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 21Sato K. Tanaka K. Touhara K. Sugar-regulated cation channel formed by an insect gustatory receptor.Proc. Natl. Acad. Sci. USA. 2011; 108: 11680-11685Crossref PubMed Scopus (175) Google Scholar] found in most fruit. In contrast to adult flies, in which Gr43a has only an auxiliary role in tasting dietary sugars and in which its main role is to sense fructose in the brain hemolymph [17Miyamoto T. Slone J. Song X. Amrein H. A fructose receptor functions as a nutrient sensor in the Drosophila brain.Cell. 2012; 151: 1113-1125Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar], larvae appear to rely almost exclusively on this receptor for carbohydrate sensing. Why does Drosophila employ distinct sugar receptors during the different life stages? Larvae and adults face different sets of challenges to satisfy similar dietary needs of carbohydrates and proteins. Larvae are highly restricted in their choice of carbohydrates, which is determined by where they have been deposited as eggs. Thus, they consume a relatively homogenous source of sugar. The major gustatory challenge, therefore, is not to locate and identify sugars but to verify its presence and assure that it is void of hazardous chemicals that may be produced by microorganisms colonizing ripening and decaying fruit. Indeed, at least 40 putative bitter receptors are expressed in the larval taste sensory neurons [19Kwon J.Y. Dahanukar A. Weiss L.A. Carlson J.R. Molecular and cellular organization of the taste system in the Drosophila larva.J. Neurosci. 2011; 31: 15300-15309Crossref PubMed Scopus (73) Google Scholar], some of which may sense such harmful chemicals. In contrast, adults explore diverse habitats to locate appropriate food sources, find mates, escape predators, and lay eggs. Thus, a set of Gr genes encoding different sugar receptors capable of detecting a variety of sugars is better suited to identify appropriate food sources in the complex environment of adult Drosophila. The two-choice feeding assay reveals two temporally distinct phases while acquiring a sugar preference, both of which are dependent on Gr43a. We propose that if a sugar can be sensed by taste neurons (such as fructose-containing sugars), a preference is established within 2 min (Figures 2 and 3C; Figure S1). The second phase in larval sugar perception is characterized by the establishment of a delayed preference, is dependent on Gr43a function in the brain (Figure 3), and most likely is mediated by fructose in the hemolymph, converted from ingested sugar (Figure 4) [17Miyamoto T. Slone J. Song X. Amrein H. A fructose receptor functions as a nutrient sensor in the Drosophila brain.Cell. 2012; 151: 1113-1125Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar]. The observation of a slow developing sugar preference is surprising, given the elapsed time from food intake and actual activation of brain neurons by fructose. Preliminary experiments suggest that crawl speed of larvae is dependent on the nutritious content of the agar, but more extensive behavioral analyses that consider turn frequency, digging activity, etc. will be necessary to uncover the rationale for the observed late sugar preference. In adult flies, only about six brain neurons express Gr43a; they were shown to sense circulating fructose and regulate consumption of nutritious carbohydrate [17Miyamoto T. Slone J. Song X. Amrein H. A fructose receptor functions as a nutrient sensor in the Drosophila brain.Cell. 2012; 151: 1113-1125Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar]. Larvae have several groups of Gr43aGAL4-expressing neurons, one probably corresponding to the neurons in the posterior superior lateral protocerebrum of adult flies, a group of mushroom body Kenyon cells, and about 20 to 30 neurons dispersed along the VNC. The Kenyon cells are unlikely to be involved in this process, as they are not necessary to mediate the late sugar preference (Figure S4), but they may play a role in associative learning during feeding. Regardless, our analysis suggests that some of the brain neurons in the larvae function as nutrient sensors by detecting hemolymph fructose, derived from dietary fructose or fructose converted from other nutritious carbohydrates. Measurements of hemolymph fructose in flies show significant increases after glucose-, sorbitol-, or fructose-based meals (3-, 5-, and 10-fold, respectively) [17Miyamoto T. Slone J. Song X. Amrein H. A fructose receptor functions as a nutrient sensor in the Drosophila brain.Cell. 2012; 151: 1113-1125Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar]. These values are higher than those observed in larvae, in which only feeding on fructose and sorbitol, but not on glucose, raises hemolymph fructose levels (Figure 4). Two scenarios may account for this difference, both implying a role of the blood-brain barrier (BBB): first, conversion of fructose may be restricted to the brain or, second, the conversion into fructose may be ubiquitous, but accumulation in the brain may be regulated by selective transporters for fructose across the BBB [25Burant C.F. Takeda J. Brot-Laroche E. Bell G.I. Davidson N.O. Fructose transporter in human spermatozoa and small intestine is GLUT5.J. Biol. Chem. 1992; 267: 14523-14526Abstract Full Text PDF PubMed Google Scholar]. The specificity of Gr43a for fructose and the location of this sensor in the brain, therefore, invoke a postingestive mechanism requiring conversion of dietary sugars into fructose. Whatever the mechanism, the brain neurons are both necessary and sufficient to mediate the late sugar preference in larvae. Gr43aGAL4 is also expressed in the larval proventriculus, reiterating the adult expression profile in which this structure separates the foregut from the midgut [17Miyamoto T. Slone J. Song X. Amrein H. A fructose receptor functions as a nutrient sensor in the Drosophila brain.Cell. 2012; 151: 1113-1125Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar]. Interestingly, the Gr43a orthologs of the cotton bollworm Helicoverpa armigera and the silkworm Bombyx mori are expressed in the gut, indicating an important role for this fructose sensor in the gastrointestinal tract [21Sato K. Tanaka K. Touhara K. Sugar-regulated cation channel formed by an insect gustatory receptor.Proc. Natl. Acad. Sci. USA. 2011; 108: 11680-11685Crossref PubMed Scopus (175) Google Scholar, 26Xu W. Zhang H.J. Anderson A. A sugar gustatory receptor identified from the foregut of cotton bollworm Helicoverpa armigera.J. Chem. Ecol. 2012; 38: 1513-1520Crossref PubMed Scopus (52) Google Scholar]. Sensing of dietary fructose may induce expression/secretion of carbohydrate-modifying enzymes and/or regulate peristaltic movements of the midgut to aid in digestion. Future studies in Drosophila and other insects will illuminate the function of this unique gustatory receptor both in the gastrointestinal system and the brain. For behavioral analysis, w1118 larvae were used as wild-type controls, Gr43aGal4/Gal4 larvae as null mutants for Gr43a, R1 Gr5aLexA/R1 Gr5aLexA; R2/R2; Gr61a−ΔGr64/Gr61a− ΔGr64 larvae as triple mutant lacking all eight sweet Gr genes, and Gr43aGal4/Gr43aGal4;UAS-Gr43a/+ larvae as Gr43a rescue controls. For larvae with Gr43aGAL4 expression restricted to the brain, we used Gr43aGal4/Gr43aGal4;UAS-Gr43a/Cha7.4kb-Gal80 larvae, while Gr43aGal4/Gr43aGal4; UAS-Gr43a/+ and Gr43aGal4/Gr43aGal4; Cha7.4kb-Gal80/+ were used as controls. Expression analysis was carried out on Gr43aGal4/+; UAS-mCD8GFP/+ and Gr43aGAL4 UAS-mCD8RFP/Gr66a-GFP-IRES-GFP-IRES-GFP. R1 and R2 are rescue transgenes containing non-Gr genes deleted in ΔGr64 [6Slone J. Daniels J. Amrein H. Sugar receptors in Drosophila.Curr. Biol. 2007; 17: 1809-1816Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar]. Two-choice preference assay: flies were kept in mass culture under standard conditions. Third-instar, feeding-stage larvae (4 days after egg laying) of the indicated strains were used for all assays. Larvae were collected from culture tubes and briefly washed in water to remove food particles. We used 15 larvae for each preference assay and placed on the midline separating the two agar media (one containing sugar and the other lacking sugar) on a 55 mm plastic Petri dish. Images were taken after the indicated time points (2, 4, 8, and 16 min) to establish larval distribution and to calculate preference indices. Larvae that dug in the agarose or crawled onto the lid were excluded. A preference index (PREF) was then calculated as PREF = [number of larvae on sugar/agar − number of larvae on agar only] / total number. A positive PREF score indicates a preference for the sugar-containing agar, while a negative PREF score would indicate a preference for plain agar. Preparation of media is as follows: 55 mm Petri dishes are filled with a solution of 1% agarose. After the agarose has solidified, the agar is split into two identical halves using a razor blade, and one half is discarded. The emptied half of the dish is then replaced with agar containing the sugar at the indicated concentration. Plates were used within an hour after preparation. Third-instar larvae were kept on agar overnight, collected, rinsed in water, briefly dried, and weighted in batches of five. A batch of larvae was then transferred on plates for 16 min of either plain agarose or agarose plates with 100 mM glucose, sorbitol, or fructose. Each batch was rinsed to remove food particles, briefly dried off, and transferred to a clean dissection dish, filled with ∼50 μl of distilled water. Larvae were carefully torn apart using forceps to drain hemolymph into the water, and 40 μl of the hemolymph-containing solution was collected. Protein and cellular debris were removed by treatment with Barium Hydroxide and Zinc sulfate (0.3 N). This work was supported by grants from the National Institute of Health 1RO1GMDC05606 and 1RO1DC009014 to H.A. Download .pdf (1.03 MB) Help with pdf files Document S1. Supplemental Experimental Procedures and Figures S1–S4" @default.
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