InteractiveFly: GeneBrief

Ionotropic receptor 76b: Biological Overview | References

Gene name - Ionotropic receptor 76b

Synonyms -

Cytological map position - 76F1-76F1

Function - ionotropic glutamate receptor

Keywords - chemosensory detection of various amines and salt - a probable co-receptor subunit

Symbol - Ir76b

FlyBase ID: FBgn0036937

Genetic map position - chr3L:20108484-20111534

NCBI classification - Type 2 periplasmic binding fold superfamily

FlyBase gene group - Antennal Ionotropic Receptors

Cellular location - surface transmembrane

NCBI link: EntrezGene
Ir76b orthologs: Biolitmine
Recent literature
Lee, Y., Poudel, S., Kim, Y., Thakur, D. and Montell, C. (2018). Calcium taste avoidance in Drosophila. Neuron 97(1): 67-74.e64. PubMed ID: 29276056
Many animals, ranging from vinegar flies to humans, discriminate a wide range of tastants, including sugars, bitter compounds, NaCl, and sour. However, the taste of Ca(2+) is poorly understood, and it is unclear whether animals such as Drosophila melanogaster are endowed with this sense. This study examined Ca(2+) taste in Drosophila and showed that high levels of Ca(2+) are aversive. The repulsion was mediated by two mechanisms-activation of a specific class of gustatory receptor neurons (GRNs), which suppresses feeding and inhibition of sugar-activated GRNs, which normally stimulates feeding. The distaste for Ca(2+), and Ca(2+)-activated action potentials required several members of the variant ionotropic receptor (IR) family (IR25a, IR62a, and IR76b). Consistent with the Ca(2+) rejection, it was found that high concentrations of Ca(2+) decreased survival. It is concluded that gustatory detection of Ca(2+) represents an additional sense of taste in Drosophila and is required for avoiding toxic levels of this mineral.
Chen, H. L., Stern, U. and Yang, C. H. (2019). Molecular control limiting sensitivity of sweet taste neurons in Drosophila. Proc Natl Acad Sci U S A 116(40): 20158-20168. PubMed ID: 31527261
To assess the biological value of environmental stimuli, animals' sensory systems must accurately decode both the identities and the intensities of these stimuli. While much is known about the mechanism by which sensory neurons detect the identities of stimuli, less is known about the mechanism that controls how sensory neurons respond appropriately to different intensities of stimuli. The ionotropic receptor IR76b has been shown to be expressed in different Drosophila chemosensory neurons for sensing a variety of chemicals. This study show that IR76b plays an unexpected role in lowering the sensitivity of Drosophila sweet taste neurons. First, IR76b mutants exhibited clear behavioral responses to sucrose and acetic acid (AA) at concentrations that were too low to trigger observable behavioral responses from WT animals. Second, IR76b is expressed in many sweet neurons on the labellum, and these neurons responded to both sucrose and AA. Removing IR76b from the sweet neurons increased their neuronal responses as well as animals' behavioral responses to sucrose and AA. Conversely, overexpressing IR76b in the sweet neurons decreased their neuronal as well as animals' behavioral responses to sucrose and AA. Last, IR76b's response-lowering ability has specificity: IR76b mutants and WT showed comparable responses to capsaicin when the mammalian capsaicin receptor VR1 was ectopically expressed in their sweet neurons. These findings suggest that sensitivity of Drosophila sweet neurons to their endogenous ligands is actively limited by IR76b and uncover a potential molecular target by which contexts can modulate sensitivity of sweet neurons.
Dhakal, S., Sang, J., Aryal, B. and Lee, Y. (2021). Ionotropic receptors mediate nitrogenous waste avoidance in Drosophila melanogaster. Commun Biol 4(1): 1281. PubMed ID: 34773080
Ammonia and its amine-containing derivatives are widely found in natural decomposition byproducts. A biased chemoreceptor screening was conducted to investigate the mechanisms by which different concentrations of ammonium salt, urea, and putrescine in rotten fruits affect feeding and oviposition behavior. Three ionotropic receptors, including the two broadly required IR25a and IR76b receptors, were detected and the narrowly tuned IR51b receptor. These three IRs were fundamental in eliciting avoidance against nitrogenous waste products, which is mediated by bitter-sensing gustatory receptor neurons (GRNs). The aversion of nitrogenous wastes was evaluated by the cellular requirement by expressing Kir2.1 and behavioral recoveries of the mutants in bitter-sensing GRNs. Furthermore, by conducting electrophysiology assays, it was confirmed that ammonia compounds are aversive in taste as they directly activated bitter-sensing GRNs. Therefore, our findings provide insights into the ecological roles of IRs as a means to detect and avoid toxic nitrogenous waste products in nature.
Shrestha, B. and Lee, Y. (2021). Mechanisms of Carboxylic Acid Attraction in Drosophila melanogaster. Mol Cells. PubMed ID: 34711686
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. It was 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, these findings demonstrate that at least two different kinds of receptors are required to discriminate attractive carboxylic acids from other acids.
Vulpe, A. and Menuz, K. (2021). Ir76b is a Co-receptor for Amine Responses in Drosophila Olfactory Neurons. Front Cell Neurosci 15: 759238. PubMed ID: 34867202
Two large families of olfactory receptors, the Odorant Receptors (ORs) and Ionotropic Receptors (IRs), mediate responses to most odors in the insect olfactory system. Individual odorant binding "tuning" OrX receptors are expressed by olfactory neurons in basiconic and trichoid sensilla and require the co-receptor Orco. The situation for IRs is more complex. Different tuning IrX receptors are expressed by olfactory neurons in coeloconic sensilla and rely on either the Ir25a or Ir8a co-receptors; some evidence suggests that Ir76b may also act as a co-receptor, but its function has not been systematically examined. Surprisingly, recent data indicate that nearly all coeloconic olfactory neurons co-express Ir25a, Ir8a, and Ir76b. This study demonstrates that Ir76b and Ir25a function together in all amine-sensing olfactory receptor neurons. In most neurons, loss of either co-receptor abolishes amine responses. In contrast, amine responses persist in the absence of Ir76b or Ir25a in ac1 sensilla but are lost in a double mutant. Responses mediated by acid-sensing neurons do not require Ir76b, despite their expression of this co-receptor. This study also demonstrates that one population of coeloconic olfactory neurons exhibits Ir76b/Ir25a-dependent and Orco-dependent responses to distinct odorants. Together, the data establish the role of Ir76b as a bona fide co-receptor, which acts in partnership with Ir25a. Given that these co-receptors are among the most highly conserved olfactory receptors and are often co-expressed in chemosensory neurons, the data suggest Ir76b and Ir25a also work in tandem in other insects.
Aryal, B., Dhakal, S., Shrestha, B. and Lee, Y. (2022). Molecular and neuronal mechanisms for amino acid taste perception in the Drosophila labellum. Curr Biol 32(6): 1376-1386. PubMed ID: 35176225
Amino acids are essential nutrients that act as building blocks for protein synthesis. Recent studies in Drosophila have demonstrated that glycine, phenylalanine, and threonine elicit attraction, whereas tryptophan elicits aversion at ecologically relevant concentrations. This study demonstrated that eight amino acids, including arginine, glycine, alanine, serine, phenylalanine, threonine, cysteine, and proline, differentially stimulate feeding behavior by activating sweet-sensing gustatory receptor neurons (GRNs) in L-type and S-type sensilla. In turn, this process is mediated by three GRs (GR5a, GR61a, and GR64f), as well as two broadly required ionotropic receptors (IRs), IR25a and IR76b. However, GR5a, GR61a, and GR64f are only required for sensing amino acids in the sweet-sensing GRNs of L-type sensilla. This suggests that amino acid sensing in different type sensilla occurs through dual mechanisms. Furthermore, the findings indicated that ecologically relevant high concentrations of arginine, lysine, proline, valine, tryptophan, isoleucine, and leucine elicit aversive responses via bitter-sensing GRNs, which are mediated by three IRs (IR25a, IR51b, and IR76b). More importantly, these results demonstrate that arginine, lysine, and proline induce biphasic responses in a concentration-dependent manner. Therefore, amino acid detection in Drosophila occurs through two classes of receptors that activate two sets of sensory neurons in physiologically distinct pathways, which ultimately mediates attraction or aversion behaviors.
Dweck, H. K. M., Talross, G. J. S., Luo, Y., Ebrahim, S. A. M. and Carlson, J. R. (2022). Ir56b is an atypical ionotropic receptor that underlies appetitive salt response in Drosophila. Curr Biol 32(8): 1776-1787. PubMed ID: 35294865
Salt taste is one of the most ancient of all sensory modalities. However, the molecular basis of salt taste remains unclear in invertebrates. This study shows that the response to low, appetitive salt concentrations in Drosophila depends on Ir56b, an atypical member of the ionotropic receptor (Ir) family. Ir56b acts in concert with two coreceptors, Ir25a and Ir76b. Mutation of Ir56b virtually eliminates an appetitive behavioral response to salt. Ir56b is expressed in neurons that also sense sugars via members of the Gr (gustatory receptor) family. Misexpression of Ir56b in bitter-sensing neurons confers physiological responses to appetitive doses of salt. Ir56b is unique among tuning Irs in containing virtually no N-terminal region, a feature that is evolutionarily conserved. Moreover, Ir56b is a "pseudo-pseudogene": its coding sequence contains a premature stop codon that can be replaced with a sense codon without loss of function. This stop codon is conserved among many Drosophila species but is absent in a number of species associated with cactus in arid regions. Thus, Ir56b serves the evolutionarily ancient function of salt detection in neurons that underlie both salt and sweet taste modalities.
Aryal, B. and Lee, Y. (2022). Histamine avoidance through three gustatory receptors in Drosophila melanogaster. Insect Biochem Mol Biol 144: 103760. PubMed ID: 35346814
Histamine is a fermented food product that exerts adverse health effects on animals when consumed in high amounts. This biogenic amine is fermented by microorganisms from histidine through the activity of histidine decarboxylase. Drosophila melanogaster can discriminate histidine and histamine using GR22e and IR76b in bitter-sensing gustatory receptor neurons (GRNs). In this study, RNA interference screens were conducted to examine 28 uncharacterized gustatory receptor genes using electrophysiology and behavioral experiments, including the binary food choice and proboscis extension response assays. GR9a and GR98a were first identified as specific histamine receptors by evaluating newly generated null mutants and recovery experiments by expressing their wild-type cDNA in the bitter-sensing GRNs. It was further determined that histamine sensation was mainly mediated by the labellum but not by the legs, as demonstrated by the proboscis extension response assay. These findings indicated that toxic histamine directly activates bitter-sensing GRNs in S-type sensilla, and this response is mediated by the GR9a, GR22e, and GR98a gustatory receptors.

Amino acid taste is expected to be a universal property among animals. Although sweet, bitter, salt, and water tastes have been well characterized in insects, the mechanisms underlying amino acid taste remain elusive. From a Drosophila RNAi screen, this study identified an ionotropic receptor, Ir76b, as necessary for yeast preference. Using calcium imaging, the Ir76b+ amino acid taste neurons in legs were identified and were found to be overlapping partially with sweet neurons but not those that sense other tastants. Ir76b mutants have reduced responses to amino acids, which are rescued by transgenic expression of Ir76b and a mosquito ortholog AgIr76b. Co-expression of Ir20a with Ir76b is sufficient for conferring amino acid responses in sweet-taste neurons. Notably, Ir20a also serves to block salt response of Ir76b. Overall, this study establishes the role of a highly conserved receptor in amino acid taste and suggests a mechanism for mutually exclusive roles of Ir76b in salt- and amino-acid-sensing neurons (Ganguly, 2017).

The importance of dietary protein and amino acids has been investigated for several insects including Drosophila and reveals that, like mammals, insects must acquire some essential amino acids via foods. Females, in particular, require large supplies of amino acids for synthesizing egg yolk. Restriction of amino acids thus has a direct impact on female fecundity. Amino acid deprivation also significantly affects larval growth and development, as well as adult lifespan (Ganguly, 2017).

Given the importance of amino acids in food sources, it is perhaps not surprising that insects demonstrate taste sensitivity to amino acids. Behavioral analyses in various insects, including honeybees, ants, and the dengue fever vector, Aedes aegypti, show that mixtures of some amino acids and sugar are preferred over sugar alone. Moreover, electrophysiological recordings show that selected amino acids evoke action potentials in taste hairs of some insects. For instance, in blowflies and fleshflies some individual amino acids were found to activate either sweet- or salt-sensing neurons; others were found to have inhibitory effects on these taste neurons. Studies in blood-feeding tsetse flies identified neurons in tarsal taste hairs that are exquisitely sensitive to several individual amino acids, as well as to a mixture of amino acids found in human sweat. Amino-acid-sensing neurons have also been described in cabbage butterflies and Helicoverpa moths (Ganguly, 2017).

Drosophila exhibit strong feeding preference for yeasts and yeast extract, which serve as a major source of protein. Mated females, as well as adult flies fed on a protein deficient diet, can identify and select yeast over sucrose in binary choice assays. A recent study reports behavioral taste sensitivity to free amino acids, albeit only in flies raised on a diet lacking in protein. In these experiments, flies extended their proboscis upon stimulation of taste hairs with amino acid solutions, indicating a role for taste hairs as amino acid sensors. However, little is known about the molecular and cellular basis of amino acid taste (Ganguly, 2017).

Many amino acids taste savory or sweet to humans. Mammals detect amino acids using a heteromeric receptor comprised of two subunits, T1R1 and T1R3, expressed in fungiform taste buds. The T1R1/T1R3 receptor has broad specificity for L-amino acids and does not respond to the D isomers. T1Rs, which are G-protein-coupled receptors related to metabotropic glutamate receptors, have no counterparts in insect genomes (Ganguly, 2017).

This study investigated behavioral and cellular responses in the fly to amino acids, identifying them as critical cues for feeding preference to yeast extract. Mated females exhibit feeding preference for individual amino acids, which are preferred to different extents in binary choice experiments with sucrose. From an RNAi screen, this study identifed a requirement for a highly conserved chemosensory ionotropic receptor, Ir76b, in mediating feeding preference for yeast extract. Using genetic silencing and calcium imaging experiments, the role of Ir76b+ neurons in behavioral and cellular responses to amino acids in mated females was characterized. Responses to all tested amino acids were found to be abolished in Ir76b mutants, and rescued by transgenic expression of Ir76b. Moreover, Ir76b function is conserved across millions of years of evolution -- expression of the Ir76b ortholog from Anopheles gambiae also rescues the behavioral deficits in Ir76b mutant flies. Ir76b has been recently described as a salt-taste receptor (Zhang, 2013); however, this study found that amino-acid-sensing neurons do not respond to salt. Analysis of additional candidates from the initial RNAi screen reveals additional Irs involved in amino acid taste. Co-expression of one of these, Ir20a, with Ir76b, is sufficient to confer amino acid sensitivity to sweet-taste neurons. Moreover, the presence of Ir20a blocks Ir76b-mediated salt response as measured in cellular and behavioral assays. Taken together, these results demonstrate a highly conserved gustatory role for Ir76b in detection of amino acids, in addition to its function as a salt-taste receptor. These studies also identify a potential role for Ir20a in facilitating mutually exclusive functions of Ir76b in salt and amino acid taste neurons (Ganguly, 2017).

Analysis of Ir76b expression and function is consistent with a model in which this receptor marks two functionally exclusive populations of cells, one that responds to salt and another that responds to amino acids. In the latter, Ir76b combines with Ir20a, and possibly other Irs, which gate its activity to amino acid ligands (Ganguly, 2017).

Ir genes encode proteins related to ionotropic glutamate receptors and represent an ancient family of chemoreceptors, based on their occurrence in genomes of all protostomes. Their expression and function has been extensively characterized in the fly olfactory system, in which they are expressed in combinations of up to four receptors in olfactory receptors neurons (Abuin, 2011). In keeping with their ancient origin, Irs have been associated with detection of broadly appealing or noxious stimuli, including acids, amines, and ammonia More recently, Ir gene expression has been analyzed in gustatory neurons of both adult and larval stages and accords possible roles in taste recognition to several members of the family (Koh, 2014, Stewart, 2015). However, with the exception of Ir76b, taste functions of Ir proteins remain to be characterized. Given that many Ir genes are co-expressed with either Gr5a or Gr66a in sweet or bitter-taste neurons (Koh, 2014), another open question is whether, and if so how, Ir proteins coordinate with other classes of receptors (Ganguly, 2017).

Ir76b has been proposed to function as a Na+ leak channel that is fixed in a permeable state (Zhang, 2013). In this model, Ir76b-mediated sodium conductance remains low until contact with salt-laced foods, because the sensillar lymph is rich in potassium but contains low sodium. Ectopic expression of Ir76b yields the predicted outcome: sensitivity to sodium chloride in a concentration-dependent manner (Zhang, 2013). This was surprising because Ir76b is expressed in a variety of neurons that do not respond to salt, including amino-acid-sensing neurons in tarsi. The identification of Ir20a as one co-receptor that promotes amino acid response and blocks salt response is consistent with the idea that Ir76b conductance is regulated differently in salt and amino acid taste neurons by other members of the Ir family. Notably, although expression of Ir20a blocked salt response of Ir76b+ neurons in L-type sensilla, it was not sufficient to confer sensitivity to amino acids. Moreover, Ir candidates may have been missed within the limited scope of the initial RNAi screen using yeast extract, which could have several redundant attractive cues. Thus, in all likelihood additional Irs operate in combination with Ir76b and Ir20a to form amino acid receptors. The presence of Ir47a and Ir56d in tarsal neurons as well as labellar sweet-taste neurons makes them appealing candidates for such roles. It is also possible that different Irs fulfill the role of Ir20a in other amino-acid-sensing neurons. A few observations support this idea. First, Ir20a mutants do not phenocopy Ir76b mutants. Second, Ir20a displays a restricted pattern of expression in two to three neurons in the fifth segment, representing only a small fraction of Ir76b+ neurons. Third, there appears to be some diversity in amino acid responses across taste neurons, invoking differences in receptor repertoires. Notably, there is precedent for participation of Ir76b in functional heteromeric receptors with two other Irs in olfactory neurons (Abuin, 2011, Benton, 2009, Silbering, 2011). An appealing hypothesis is that Ir76b might operate likewise in taste neurons, in complexes with combinations of Irs that may have distinct amino acid recognition properties. The occurrence of receptor combinations may also explain why different amino acids evoke responses of different strengths (Ganguly, 2017).

Sex-dependent variations in food choice have been described previously, but the extent to which they depend on variation in sensitivity of taste neurons remains to be examined. The results of calcium imaging experiments suggest that differences in tarsal sensitivity to amino acids may underlie sexual dimorphism in yeast and amino acid preference. Moreover, the observation that overexpression of Ir76b caused an increase in the preference for yeast extract implies that levels of Ir76b are limiting, particularly in male flies. Sexual dimorphism in expression levels of Ir76b is therefore expected. However, transcriptome analysis revealed otherwise. Moreover, neither Ir76b-GAL4 nor Ir20a-GAL4 showed any sexual dimorphism in expression in tarsal or pharyngeal neurons, where both are expressed. Thus, the mechanisms by which amino acid taste and yeast preference are enhanced in females as compared to males are likely to be dependent on as yet unknown sex-specific factors in Ir76b+ neurons. Interestingly, Ir76b-GAL4 is not expressed in fru+ neurons, suggesting that fru circuitry may not underlie the sex specificity of peripheral amino acid responses (Ganguly, 2017).

Amino acid and yeast preferences are also upregulated in females upon mating. Virgin females behave much like males in binary choice assays. Interestingly, preliminary explorations with calcium imaging showed that amino acid responses are present in tarsi of virgin females, indicating that the low preference for yeast in virgins does not arise from an inability to sense amino acids. Previous studies have shown that the post-mating shift in food preference depends on sex peptide (Ribeiro, 2010), which is synthesized by male accessory glands and transferred to the female reproductive tract during copulation, although the manner in which sex peptide receptor (SPR) circuitry impinges on taste circuitry is not known. A recent study found that SPR function in Ir76b+ neurons plays a role in sexually dimorphic responses to polyamines (Hussain, 2016). However, this study found that RNAi-mediated knockdown of SPR in Gr5a+ or Ir76b+ neurons did not affect the behavioral shift to yeast extract in mated females. Thus, the functional overlap between SPR+ and amino-acid-sensing circuitry is likely to occur downstream of the sensory neuron. Consistent with this model, a role has been identified for fru+/dsx+/ppk+/SPR+ neurons in the reproductive tract that convey information either directly or indirectly to the subesophageal zone (Ganguly, 2017).

In mammals, amino acids are detected by a dedicated population of taste receptor cells. By contrast, this study found that amino-acid-sensing neurons overlap with sucrose-sensing neurons in fly tarsi. However, behavioral experiments suggest that the fly can differentiate between sucrose and amino acids, supporting the idea that the two have distinct percepts in the brain. The lack of amino acid sensitivity in labellar sweet taste neurons might provide one avenue with which to distinguish the two categories of tastants. Furthermore, previous studies in other insects suggest possible synergistic interactions between sugars and amino acids when presented in mixtures. Such interactions may be achieved, at least in part, via the co-expression of amino acid and sweet taste receptors in a subset of neurons. Indeed, this appears to be the case in fleshflies and blowflies that detect some amino acids via sweet-sensing neurons (Ganguly, 2017).

Ir76b is highly conserved in insect genomes (Croset, 2010), and the functional substitution of DmIr76b with AgIr76b suggests that its role in taste detection is conserved as well. Although this study highlights the importance of Ir76b and amino acid detection for selection of proteinaceous food sources by phytophagous insects like Drosophila, free amino acids are also found in human sweat and may serve as critical cues for blood-feeding disease vectors such as mosquitoes and tsetse flies. The identification of Ir76b as a receptor for amino acid taste invites further exploration of molecular mechanisms of amino acid taste in human disease vectors and may lead to targets for control of insect feeding behaviors (Ganguly, 2017).

An expression atlas of variant ionotropic glutamate receptors identifies a molecular basis of carbonation sensing

Through analysis of the Drosophila ionotropic receptors (IRs), a family of variant ionotropic glutamate receptors, it was revealed that most IRs are expressed in peripheral neuron populations in diverse gustatory organs in larvae and adults. This study characterize IR56d, which defines two anatomically-distinct neuron classes in the proboscis: one responds to carbonated solutions and fatty acids while the other represents a subset of sugar- and fatty acid-sensing cells. Mutational analysis indicates that IR56d, together with the broadly-expressed co-receptors IR25a and IR76b, is essential for physiological responses to carbonation and fatty acids, but not sugars. It was further demonstrated that carbonation and fatty acids both promote IR56d-dependent attraction of flies, but through different behavioural outputs. This work provides a toolkit for investigating taste functions of IRs, defines a subset of these receptors required for carbonation sensing, and illustrates how the gustatory system uses combinatorial expression of sensory molecules in distinct neurons to coordinate behaviour (Sanchez-Alcaniz, 2018).

IRs are best-characterised in Drosophila melanogaster, which possesses 60 intact Ir genes. Of these, the most thoroughly understood are the 17 receptors expressed in the adult antenna. Thirteen of these are expressed in discrete populations of sensory neurons, and function as olfactory receptors for volatile acids, aldehydes and amines or in humidity detection. The remaining four are expressed in multiple, distinct neuron populations and function, in various combinations, as co-receptors with the selectively-expressed tuning IRs (Sanchez-Alcaniz, 2018).

By contrast, little is known about the sensory functions of the remaining, large majority of non-antennal IRs. Previous analyses described the expression of transgenic reporters for subsets of these receptors in small groups of gustatory sensory neurons (GSNs) in several different contact chemosensory structures. While these observations strongly implicate these genes as having gustatory functions, the evidence linking specific taste ligands to particular receptors, neurons and behaviours remains sparse. For example, IR52c and IR52d are expressed in sexually-dimorphic populations of leg neurons and implicated in male courtship behaviours, although their ligands are unknown. Reporters for IR60b, IR94f and IR94h are co-expressed in pharyngeal GSNs that respond to sucrose, which may limit overfeeding or monitor the state of externally digested food. IR62a is essential for behavioural avoidance of high Ca2+ concentrations, but the precise neuronal expression of this receptor is unclear. As in the olfactory system, these selectively-expressed IRs are likely to function with the IR25a and/or IR76b co-receptors, which are broadly-expressed in contact chemosensory organs, and required for detection of multiple types of tastants, including polyamines, inorganic, carboxylic and amino acid and Ca2+ (Sanchez-Alcaniz, 2018).

This study describes a set of transgenic reporters for the entire Ir repertoire. These were used to survey the expression of this receptor family in both larval and adult stages. Using this molecular map, IR56d was identified as a selectively-expressed receptor that acts with IR25a and IR76b to mediate physiological and attractive behavioural responses to carbonation, a previously orphan taste class. Furthermore, this study extends recent studies to show that IR56d is also required in sugar-sensing GR neurons to mediate distinct behavioural responses to fatty acids (Sanchez-Alcaniz, 2018).

This work describes that non-antennal IRs function to detect a myriad of chemical stimuli to evoke a variety of behavioural responses. Such properties presumably apply to the vast, divergent IR repertoires of other insect species, for example, the 455 family members in the German cockroach Blatella germanica, or the 135 IRs in the mosquito Aedes aegypti. Within Drosophila no obvious relationships were detected between IR phylogeny and stage- or organ-specific expression patterns. Phylogenetic proximity may therefore be the most indicative of functional relationships between IRs, as is the case for those expressed in the antenna. If this hypothesis is correct, the expression data presented in this study suggest that functionally-related clades of receptors act in several types of chemosensory organ (Sanchez-Alcaniz, 2018).

An important caveat to the transgenic approach used to reveal expression is the faithfulness of these reporters to the endogenous expression pattern of Ir genes. Although this strategy has been widely (and successfully) used for antennal IRs and other chemosensory receptor families, it is impossible to determine reporter fidelity without a complementary tool (e.g. receptor-specific antibodies or tagging of the endogenous genomic locus). Discrepancies were noted between the expression of some of the Ir-Gal4 lines and those described previously; many of these probably reflect differences in the length of regulatory regions used to create these distinct transgenes. Precise comparison of independently-constructed transgenic constructs may in fact be useful in informing the location of enhancer elements directing particular temporal or spatial expression patterns. Moreover, transgenic reporters provide powerful genetic tools for visualisation and manipulation of specific neuronal populations. The reagents generated in this study should therefore provide a valuable resource for further exploration of the IRs in insect gustation (Sanchez-Alcaniz, 2018).

Using the atlas, IR56d-together with the broadly-expressed co-receptors IR25a and IR76b- were identified as essential for responses of labellar taste peg (Pit-like sensilla) neurons to carbonation. Such observations implicate IR56d as the previously unknown tuning receptor for this stimulus. However, these IRs do not appear to be sufficient for carbonation detection, as their misexpression in other neurons failed to confer sensitivity to carbonated stimuli. This observation suggests that additional molecules or cellular specialisations are required. Such a factor may be rather specific to taste pegs, given the minimal/absent responses of Ir56d-expressing taste bristle/leg neurons to carbonation, but does not appear to be another IR, as no other IR reporters were detected that expressed in this population of cells (Sanchez-Alcaniz, 2018).

While precise mechanistic insights into carbonation sensing will require the ability to reconstitute IR56d-dependent carbonation responses in heterologous systems, it is interesting to compare how insects and mammals detect this stimulus. The main mammalian gustatory carbonation sensor, the carbonic anhydrase Car4 is an enzyme tethered to the extracellular surface of sour (acid) taste receptor cells in lingual taste buds, where it is thought to catalyse the conversion of aqueous CO2 into hydrogencarbonate (bicarbonate) ions (HCO3-) and protons (H+). The resulting free protons, but not hydrogencarbonate ions, provide a relevant signal for the sour-sensing cells. By contrast, IR56d neurons are not responsive to low pH, suggesting a different chemical mechanism of carbonation detection. The observation that IR56d is also essential for sensitivity to hexanoic acid suggests that IR56d could recognise the common carboxyl group of hydrogencarbonate and fatty acid ligands. However, IR56d neurons are not responsive to all organic acids, indicating that this cannot be the only determinant of ligand recognition (Sanchez-Alcaniz, 2018).

Characterisation of IR56d neurons extends previous reports to reveal an unexpected complexity in the molecular and neuronal basis by which attractive taste stimuli are encoded. The taste bristle population of IR56d neurons represents a subset of sugar-sensing cells that are also responsive to fatty acids, glycerol and, minimally, to carbonation. Although activation of these neurons promotes PER, it was found that carbonation-evoked stimulation is insufficient to trigger this behaviour, which suggests that taste bristles are not a relevant sensory channel for this stimulus. While members of a specific clade of GRs are well-established to mediate responses to sugars and glycerol, the detection mechanisms of fatty acids appear to be more complex. Earlier work demonstrated an important role of a phospholipase C homologue (encoded by norpA) in labellar fatty acid responses. More recently, GR64e was implicated as a key transducer of fatty acid-dependent signals, but suggested to act downstream of NorpA, rather than as a direct fatty acid receptor. By contrast, an independent study of the legs showed that all sugar-sensing Gr genes (including Gr64e) were dispensable for fatty acid detection and provided evidence instead for an important role of IR25a and IR76b in these responses. Analysis of Ir56d mutants indicates an IR-dependent fatty acid-detection mechanism also exists in the labellum; future work will be needed to relate this to the roles of GR64e and NorpA (Sanchez-Alcaniz, 2018).

The IR56d taste peg population is, by contrast, sensitive to carbonation and fatty acids (but not sugars or glycerol), and these responses can be ascribed to IR56d (a Gr64eLexA reporter is not expressed in taste peg neurons). Although these neurons mediate taste-acceptance behaviour, they do not appear to promote proboscis extension or food ingestion. Recent work using optogenetic neuronal silencing experiments provided evidence that taste peg neuron activity is important for sustaining, rather than initiating, feeding on yeast, by controlling the number of sips an animal makes after proboscis extension. These observations are concordant with the internal location of taste pegs on the labellum, as they will not come into contact with food until the proboscis has been extended, and could explain the positional preference for carbonated substrates that were observed. This study has attempted to determine whether carbonation can influence sipping behaviour using flyPAD. Although these experiments did not reveal a statistically-significant effect, interpretation is complicated by the difficulty of providing and maintaining carbonation stimuli in the solid medium used in flyPAD assays. Future development of other approaches to provide this stimulus in feeding assays will be necessary. Nevertheless, the data strengthen the view that carbonation, a non-nutritious microbial fermentation product, regulates-via activation of IR56d taste peg neurons-a distinct motor programme to PER as part of a multicomponent behavioural response (Sanchez-Alcaniz, 2018).

A molecular and neuronal basis for amino acid sensing in the Drosophila larva

Amino acids are important nutrients for animals, reflected in conserved internal pathways in vertebrates and invertebrates for monitoring cellular levels of these compounds. In mammals, sensory cells and metabotropic glutamate receptor-related taste receptors that detect environmental sources of amino acids in food are also well-characterised. By contrast, it is unclear how insects perceive this class of molecules through peripheral chemosensory mechanisms. This study investigated amino acid sensing in Drosophila melanogaster larvae, which feed ravenously to support their rapid growth. Larvae were shown to display diverse behaviours (attraction, aversion, neutral) towards different amino acids, which depend upon stimulus concentration. Some of these behaviours require IR76b, a member of the variant ionotropic glutamate receptor repertoire of invertebrate chemoreceptors. IR76b is broadly expressed in larval taste neurons, suggesting a role as a co-receptor. A subpopulation of these neurons were identified that displays physiological activation by some, but not all, amino acids, and which mediate suppression of feeding by high concentrations of at least a subset of these compounds. These data reveal the first elements of a sophisticated neuronal and molecular substrate by which these animals detect and behave towards external sources of amino acids (Croset, 2016).

Amino acids are vital for all organisms, both as constituents of proteins and as signalling molecules1. In animals, many of the twenty canonical L-amino acids that serve as building blocks for protein synthesis can be produced endogenously, but a subset must be obtained through their diet. Of these 'essential' amino acids, nine are common to mammals and insects (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine); insects also require a nutritional source of arginine2. In addition to a minimum basal intake, the precise ratio of dietary amino acids is crucial (Croset, 2016).

Consistent with the fundamental importance of amino acids, the cellular mechanisms that sense these molecules are widely conserved. A central component of this pathway is the Target of Rapamycin (TOR) kinase, which integrates information from the levels of amino acids (and other environmental signals) to control cell growth and metabolism. Signalling elements upstream of TOR that mediate uptake and/or direct detection of amino acids are, however, only starting to be identified; these include both substrate-specific cytosolic amino acid-binding proteins and transmembrane transporters (Croset, 2016).

Because of the dietary requirement for many amino acids, animals have also evolved peripheral chemosensory pathways that can detect environmental sources of these nutrients and induce adaptive behaviours. In humans, some, but not all, amino acids elicit an appetitive, savoury “umami” taste, and rodents display diverse behaviours towards different amino acids. The best-characterised amino acid sensory receptor in mammals is a heteromeric complex of the T1R1 and T1R3 G protein-coupled receptors, which are distantly-related to metabotropic glutamate receptors. T1R1/T1R3 proteins are expressed in specific cells in lingual taste buds, and are required for amino acid-evoked electrophysiological responses in the lingual nerve. Although this heteromeric receptor is a key mechanism for environmental amino acid sensing, the observed heterogeneity in perception suggests that these stimuli act through additional pathways (Croset, 2016).

Dietary amino acids are also critical for insects, notably to support a high rate of egg production in females. Although there has been some investigation of behavioural responses of insects towards individual amino acids, the sensory mechanisms allowing them to perceive these chemicals in the environment are largely unknown. Electrophysiological responses of chemosensory neurons in adult feeding organs to individual (or mixes of) amino acids have been described in several species, but the behavioural roles of these neurons are unexplored. In the genetic model Drosophila melanogaster, where substantial progress has been made in revealing the molecular and neuronal basis of sweet and bitter tastants, amino acid sensing mechanisms are surprisingly poorly understood. This might reflect, in part, the observation that adult Drosophila display robust behavioural responses towards amino acids only when previously deprived of these nutrients, similar to locusts. It remains unclear whether such plasticity in behaviour reflects the function of specific peripheral chemoreceptive pathways or internal metabolic amino acid sensors (Croset, 2016).

Larval Drosophila represent an appealing system for investigating sensory detection of amino acids, because these animals display a remarkably persistent appetite to support a 250-fold increase in mass from hatching to pupation. This study has investigated the behavioural responses of larvae to amino acids and delineated some of the relevant chemosensory receptors and neurons (Croset, 2016).

This is the first study to investigate the molecular and neuronal mechanisms underlying amino acid detection in the Drosophila larva. Despite the numerical simplicity of this animal's chemosensory system, these data reveal an unexpected complexity in how it responds to this class of compounds. Different amino acids can produce behavioural responses of opposing valence. Moreover an amino acid stimulus that produces appetitive behaviour at low concentration can evoke aversion at higher concentrations. Behavioural valence does not correlate with the essential/non-essential dietary requirements for amino acids, nor with their physico-chemical properties. These observations suggest that the assignment of value to amino acids is a complex process, potentially related to the diverse properties and biological functions of these chemicals. It is important to note that the presumed 'innate' behaviours measured in third instar larvae are not necessarily fixed across life stages and strains of different genetic background, as previously noted in adult Drosophila (Croset, 2016).

This study has identified a single class of broadly (but not universally) tuned amino acid sensing neurons, marked by IR60c-Gal4, in the main gustatory organ of the larva. Unexpectedly, the physiological profile of these neurons does not show concordance with the observed behavioural sensitivity or valence for these compounds. This observation implies the existence of additional sensory pathways for amino acids not revealed in the present study, or which exhibit responses below the detection threshold of the experimental assays (Croset, 2016).

The demonstration that at least some of the behavioural responses to amino acids require the IR76b co-receptor provides the first evidence for the molecular mechanism of this taste modality. It seems very unlikely, however, that IR76b functions as an amino acid sensor itself. This receptor is broadly expressed in multiple populations of chemosensory neurons, and, in adults, has been implicated in sensory transduction of diverse odours in the olfactory system, and in polyamine and salt sensing in distinct subsets of neurons in the gustatory system. IR76b has been suggested to function independently as a salt sensor (potentially by acting as sodium leak channel), but in other contexts it appears to function as a co-receptor with other more selectively-expressed IRs that determine ligand specificity. It is proposed that IR76b must also act with one, or more, different tuning IRs to mediate responses to amino acids. Although the IR60c-Gal4 expressing neurons have been identified as amino acid sensors, IR60c does not itself appear to be required for physiological responses of these cells, suggesting that other receptors act with IR76b in these neurons. Regardless, the implication of a variant iGluR in peripheral amino acid sensing in insects provides an interesting molecular analogy to the role of the mGluR-related T1R1/T1R3 umami sensors in mammals, and might be reflective of a broader role of the iGluR superfamily in amino acid sensing (Croset, 2016).

Neuropeptides modulate female chemosensory processing upon mating in Drosophila: Ionotropic chemosensory receptors mediate the taste and smell of polyamines

A female's reproductive state influences her perception of odors and tastes along with her changed behavioral state and physiological needs. The mechanism that modulates chemosensory processing, however, remains largely elusive. Using Drosophila, this study has identified a behavioral, neuronal, and genetic mechanism that adapts the senses of smell and taste, the major modalities for food quality perception, to the physiological needs of a gravid female. Pungent smelling polyamines, such as putrescine and spermidine, are essential for cell proliferation, reproduction, and embryonic development in all animals. A polyamine-rich diet increases reproductive success in many species, including flies. Using a combination of behavioral analysis and in vivo physiology, this study shows that polyamine attraction is modulated in gravid females through a G-protein coupled receptor, the sex peptide receptor (SPR), and its neuropeptide ligands, MIPs (myoinhibitory peptides), which act directly in the polyamine-detecting olfactory and taste neurons. This modulation is triggered by an increase of SPR expression in chemosensory neurons, which is sufficient to convert virgin to mated female olfactory choice behavior. Together, these data show that neuropeptide-mediated modulation of peripheral chemosensory neurons increases a gravid female's preference for important nutrients, thereby ensuring optimal conditions for her growing progeny (Hussain, 2016b).

The behavior of females in most animal species changes significantly as a consequence of mating. Those changes are interpreted from an evolutionary standpoint as the female's preparation to maximize the fitness of her offspring. In general, they entail a qualitative and quantitative change in her diet, as well as the search for an optimal site where her progeny will develop. In humans, the eating behavior and perception of tastes and odors of a pregnant woman are modulated in concert with altered physiology and the specific needs of the embryo. While several neuromodulatory molecules such as noradrenaline are found in the vertebrate olfactory and gustatory systems, little is known about how reproductive state and pregnancy shape a female's odor and taste preferences. Very recent work in the mouse showed that olfactory sensory neurons (OSNs) are modulated during the estrus cycle. Progesterone receptor expressed in OSNs decreases the sensitivity of pheromone-detecting OSNs and thereby reduces the non-sexually receptive female's interest in male pheromones. The mechanisms of how mating, pregnancy, and lactation shape the response of the female olfactory and gustatory systems remain poorly understood (Hussain, 2016b).

The neuronal underpinnings of mating and its consequences on female behaviors have arguably been best characterized in Drosophila. Shortly after copulation, female flies engage in a series of post-mating behaviors contrasting with those of virgins: their sexual receptivity decreases, and they feed to accumulate essential resources needed for the production of eggs; finally, they lay their eggs. This suite of behaviors results from a post-mating trigger located in the female's reproductive tract. Sensory neurons extending their dendrites directly into the oviduct are activated by a component of the male's ejaculate, the sex peptide (SP). Sex peptide receptor (SPR) expressed by these sensory neurons triggers the post-mating switch. Mated females mutant for SPR produce and lay fewer eggs while maintaining a high sexual receptivity. In addition to SP, male ejaculate contains more than 200 proteins, which are transferred along with SP into the female. These have been implicated in conformational changes of the uterus, induction of ovulation, and sperm storage (Hussain, 2016b).

Additional SPR ligands have been identified that are not required for the canonical post-mating switch, opening the possibility that this receptor is involved in the neuromodulation of other processes. These alternative ligands, the myoinhibitory peptides (MIPs)/allatostatin-Bs, unlike SP, have been found outside of drosophilids, in many other insect species such as the silkmoth (Bombyx mori), several mosquito species, and the red flour beetle (Tribolium castaneum). They are expressed in the brain of flies and mosquitoes, including in the centers of olfactory and gustatory sensory neuron projections, the antennal lobe (AL), and the subesophageal zone (SEZ), respectively. Although these high-affinity SPR ligands have recently been implicated in the control of sleep in Drosophila males and females, nothing thus far suggests a function in reproductive behaviors (Hussain, 2016b).

To identify optimal food and oviposition sites, female flies rely strongly on their sense of smell and taste. Drosophila females prefer to oviposit in decaying fruit and use byproducts of fermentation such as ethanol and acetic acid to choose oviposition sites. Their receptivity to these byproducts is enhanced by their internal state. It was shown, for instance, that the presence of an egg about to be laid results in increased attraction to acetic acid. Yet the mechanisms linking reproductive state to the modulation of chemosensory processing remain unknown (Hussain, 2016b).

This study has examined the causative mechanisms that integrate reproductive state into preference behavior and chemosensory processing. Focus was placed on the perception of another class of byproducts of fermenting fruits, polyamines. Polyamines such as putrescine, spermine, and spermidine are important nutrients that are associated with reproductive success across animal species. A diet high in polyamines indeed increases the number of offspring of a fly couple, and female flies prefer to lay their eggs on polyamine-rich food (Hussain, 2016a). Importantly, previous studies have characterized the chemosensory mechanisms flies use to find and evaluate polyamine-rich food sources and oviposition sites. In brief, volatile polyamines are detected by OSNs on the fly's antenna, co-expressing two ionotropic receptors (IRs), IR41a and IR76b. Interestingly, the taste of polyamines is also detected by IR76b in labellar gustatory receptor neurons (GRNs) (Hussain 2016a; Hussain, 2016b).

This beneficial role of polyamines has a well-characterized biological basis: polyamines are essential for basic cellular processes such as cell growth and proliferation, and are of specific importance during reproduction. They enhance the quality of sperm and egg and are critical during embryogenesis and postnatal development. While the organism can generate polyamines, a significant part is taken in with the diet. Moreover, endogenous synthesis of polyamines declines with ageing and can be compensated for through a polyamine-rich diet. Therefore, these compounds represent a sensory cue as well as an essential component of the diet of a gravid female fly (Hussain, 2016b and references therein).

This study shows that the olfactory and gustatory perception of polyamines is modulated by the female's reproductive state and guides her choice behavior accordingly. This sensory and behavioral modulation depends on SPR and its conserved ligands, the MIPs that act directly on the chemosensory neurons themselves. Together, these results suggest that mating-state-dependent neuropeptidergic modulation of chemosensory neurons matches the female fly's decision-making to her physiological needs (Hussain, 2016b).

Mechanistically, this study shows that virgin females, or mated females lacking the G-protein coupled receptor SPR, display reduced preference for polyamine-rich food and oviposition sites. Using targeted gene knockdown, mutant rescue, overexpression, and in vivo calcium imaging, a new role was uncovered for SPR and its conserved ligands, MIPs, in directly regulating the sensitivity of chemosensory neurons and modulating taste and odor preferences according to reproductive state. Together with recent work in the mouse, these results emphasize that chemosensory neurons are potent targets for tuning choice behavior to reproductive state (Hussain, 2016b).

Reproductive behaviors such as male courtship and female egg-laying strongly depend on the mating state. While previous work has suggested that mating modulates odor- or taste-driven choice behavior of Drosophila females, how mating changes the processing of odors and tastes remained elusive. This study shows that a female-specific neuropeptidergic mechanism acts in peripheral chemosensory neurons to enhance female preference for essential nutrients. The data suggests that this modulation is autocrine and involves the GPCR SPR and its conserved MIP ligands. Notably, MIPs are expressed in chemosensory cells in the apical organs of a distant organism, the annelid (Platynereis) larvae, in which they trigger settlement behavior via an SPR-dependent signaling cascade. Importantly, as SP and not MIP induces the SPR-dependent canonical post-mating switch, the current findings report the first gender and mating-state-dependent role of these peptides. Whether this regulation is also responsible for previously reported changes in preference behavior upon mating remains to be seen, but it is anticipated that this type of regulation is not only specific to polyamines. On the other hand, mating-dependent changes for salt preference-salt preference is also dependent on IR76b receptor but in another GRN type-might undergo a different type of regulation, as RNAi-mediated knockdown of SPR in salt receptor neurons had no effect on salt feeding. Instead, the change in salt preference is mediated by the canonical SP/SPR pathway and primarily reflects the fact that mating has taken place. The mechanism of how salt detection and/or processing are modulated is not known. In contrast to salt preference and polyamine preference, acetic acid preference is strongly modulated by egg-laying activity and not just mating. The extent to which changes in salt or acetic acid preference are similar to the modulation of behavior to polyamine that this study has described can currently not be tested, because the olfactory neurons that mediate acetic acid preference have not been determined (Hussain, 2016b).

While SPR regulates the neuronal output of both olfactory and gustatory neurons, the behavioral and physiological data surprisingly revealed that it does so through two opposite neuronal mechanisms. SPR signaling increases the presynaptic response of GRNs and decreases it in OSNs. Behaviorally, these two types of modulation produce the same effect: they enhance the female's attraction to polyamine and tune it to levels typical for decaying or fermenting fruit. How these two effects are regulated by the same receptor and ligand pair remains open. GPCRs can recruit and activate different G-proteins. SPR was previously shown to recruit the inhibitory Gαi/o-type, thereby down-regulating cAMP levels in the cell. In the female reproductive tract, SP inhibits SPR-expressing internal sensory neurons and thereby promotes several post-mating behaviors. This type of inhibitory G-protein signaling could also explain the data in the olfactory system. Here, mating decreases the presynaptic activity of polyamine-detecting OSNs, and conversely, RNAi knockdown of SPR increases their responses strongly. This decrease in neuronal output also shifts the behavioral preference from low to high polyamine levels. While the relationship between behavior and GRN activity is much more straightforward in the gustatory system (increased neuronal response, increased preference behavior), it implies that another G-protein might be activated downstream of SPR. G-protein Gαi/s increases cAMP levels and Gαq enhances phospholipase C (PLC) and calcium signaling. In addition, Gβγ subunits regulate ion channels and other signaling effectors, including PLC. Future work will address the exact mechanisms of this bi-directional modulation through SPR signaling. Nonetheless, it is interesting to speculate that different cells, including sensory neurons, could be modulated differentially by the same molecules depending on cell-specific states and the availability of signaling partners (Hussain, 2016b).

While the data provides a neuronal and molecular mechanism of how chemosensory processing itself is affected by mating, it remains unclear how mating regulates MIP/SPR signaling in chemosensory neurons. The data indicates that SPR levels strongly increase in chemosensory organs upon mating. In addition, MIP levels appear to be mildly increased by mating. This suggests that mating regulates primarily the expression of the GPCR resembling the modulation of sNPFR expression during hunger states. On the other hand, MIP overexpression also induced mated-like preference behavior in virgin flies, suggesting a somewhat more complex situation. For instance, it is possible that overexpression of MIP induces the expression of SPR. Alternatively, active MIP levels might also be regulated at the level of secretion or posttranslational processing, and overexpression might override this form of regulation. In the case of hunger, sNPFR levels are increased through a reduction of insulin signaling. SP could be viewed as the possible equivalent of insulin for mating state. Females mated to SP mutant males, however, do not show a significant change in olfactory perception of polyamines. It is yet important to note that male sperm contains roughly 200 different proteins, some of which might be involved in mediating the change in MIPs/SPR signaling upon mating. In the mosquito, which does not possess SP, the steroid hormone 20E serves as the post-mating switch. Interestingly, mating or treatment with 20E induces in particular the expression of the enzymes required for the synthesis of polyamines in the female spermatheca, a tissue involved in sperm storage and egg-laying. Whether such a mechanism also exists in Drosophila is not known (Hussain, 2016b).

In addition to mating and signals transferred by mating, it is possible that egg-laying activity contributes to the regulation of MIPs/SPR signaling in chemosensory neurons through a mechanism that involves previously identified mechanosensory neurons of the female's reproductive tract; such neurons may sense the presence of an egg to be laid. Indeed, females that cease to lay eggs return to polyamine preferences as found before mating. On the other hand, SP mutant male-mated females and ovoD1 sterile females still show enhanced attraction to polyamine odor, although they lay very few or no eggs. Conversely, knockdown of SPR in IR41a neurons reduced polyamine odor attraction but had a marginal effect on the number of eggs laid. Nevertheless, somewhat reduced numbers of eggs laid were observed upon inactivation of IR76b neurons. At this point, possible reasons can only be speculated. Although IR76b receptor is not expressed in ppk-positive internal SPR neurons, no expression of IR76b-Gal4 is found in neurons innervating the rectum and possibly gut. Hence, there might be an IR76b-mediated interplay between metabolism and nutrient uptake that influences egg-laying. However, females mated to SP-mutant males do not display an increase in feeding, indicating that preference for polyamines does not depend on the metabolic cost of egg-laying. This conclusion is strengthened by the data obtained with mated ovoD1 sterile females, who show similar attraction to polyamines as compared to mated controls. Due to very few or no eggs laid by SP mutant male-mated females and ovoD1 females, respectively, it is not possible to fully exclude a contribution of egg-laying activity to taste-dependent oviposition choice behavior (Hussain, 2016b).

A further argument against an important role of egg-laying activity in the current paradigm comes from the observation that the sensory modulation of OSNs and GRNs occurs rapidly after mating and is maintained only for a few hours. Similarly, SPR expression increases within the same time window shortly after mating. Egg-laying, however, continues for several days after this. In addition, overexpression of SPR was sufficient to switch virgin OSN calcium responses and female behavioral preferences to that of mated females without increasing the number of eggs laid. All in all, these data are more consistent with the hypothesis that mating and not egg-laying activity per se is the primary inducer of sensory modulation leading to the behavioral changes of females (Hussain, 2016b).

It remains that the exact signal triggered by mating that regulates odor and taste preference for polyamines, through the mechanism identified in this study, needs to still be determined. Furthermore, the role of metabolic need and polyamine metabolism is not completely clear. This is similar to the situation found for increased salt preference after mating. While more salt is beneficial for egg-laying, sterile females still increase their preference for salt upon mating. Regardless, in the case of polyamines, it is tempting to speculate that exogenous (by feeding) and endogenous (by enzymatic activity or expression) polyamine sources are regulated by reproductive state and together contribute to reach optimal levels for reproduction in the organism. (Hussain, 2016b).

The results bear some similarities to recent work on the modulation of OSN sensitivity in hunger states (Root, 2011). sNPF/sNPFR signaling modulates the activity of OSNs in the hungry fly. MIPs/SPR might play a very similar role in the mated female. Overexpression of sNPFR in OSNs of fed flies was sufficient to trigger enhanced food search behavior. Likewise, an increase in SPR signaling in taste or smell neurons converts virgin to mated female preference behavior. Therefore, different internal states appear to recruit similar mechanisms to tune fly behavior to internal state. Furthermore, such modulation of first order sensory neurons appears not only be conserved within a species, but also for regulation of reproductive state-dependent behavior across species. For instance, a recent study in female mice showed that progesterone-receptor signaling in OSNs modulates sensitivity and behavior to male pheromones according to the estrus cycle. Also in this case, sensory modulation accounts in full for the switch in preference behavior. What is the biological significance of integrating internal state at the level of the sensory neuron? First, silencing of neurons in a state-dependent manner shields the brain from processing unnecessary information. As sensory information may not work as an on/off switch, it is possible that an early shift in neural pathway activation might reduce costly inhibitory activity to counteract activation once the sensory signal has been propagated. Second, another interesting possibility is that peripheral modulation might help to translate transient changes in internal state into longer-lasting behavioral changes that manifest in higher brain centers. This might be especially important in the case of female reproductive behaviors such as mate choice or caring for pups or babies. In contrast to hunger, which increases with time of starvation, the effect of mating decays slowly over time as the sperm stored in the female's spermatheca is used up. This study has shown that the effect of mating on chemosensory neurons mediated by MIPs/SPR signaling is strong within the first 6 h after mating and remains a trend at 1 wk post-mating. However, it triggers a long-lasting behavioral switch, which is observed for over a week. Therefore, this transient modulation and altered sensitivity to polyamines could be encoded more permanently in the brain when the animal encounters the stimulus, for instance, in the context of an excellent place to lay her eggs. Because polyamine preference continues to be high for as long as stored sperm can fertilize the eggs, it is speculated that this change in preference might be maintained by a memory mechanism in higher centers of chemosensory processing. Thus, short-term sensory enhancement not only increases perceived stimulus intensity, it may also help to associate a key sensation to a given reward or punishment. These chemosensory associations are of critical importance in parent-infant bonding in mammals, including humans, which form instantly after birth and last for months, years, or a lifetime (Hussain, 2016b).

Ahn, J. E., Chen, Y. and Amrein, H. (2017). Molecular basis of fatty acid taste in Drosophila. Elife 6. PubMed ID: 29231818

Molecular basis of fatty acid taste in Drosophila

Behavioral studies have established that Drosophila appetitive taste responses towards fatty acids are mediated by sweet sensing Gustatory Receptor Neurons (GRNs). This study shows that sweet GRN activation requires the function of the Ionotropic Receptor genes IR25a, IR76b and IR56d. The former two IR genes are expressed in several neurons per sensillum, while IR56d expression is restricted to sweet GRNs. Importantly, loss of appetitive behavioral responses to fatty acids in IR25a and IR76b mutant flies can be completely rescued by expression of respective transgenes in sweet GRNs. Interestingly, appetitive behavioral responses of wild type flies to hexanoic acid reach a plateau at ~1%, but decrease with higher concentration, a property mediated through IR25a/IR76b independent activation of bitter GRNs. With previous report on sour taste, these studies suggest that IR-based receptors mediate different taste qualities through cell-type specific IR subunits (Ahn, 2017).

IR genes have emerged as a second large gene family encoding chemoreceptors in insects. In the Drosophila olfactory system, IRs function as multimeric receptors in coeloconic olfactory sensory neurons (OSN) and are thought to sense volatile carboxylic acids, amines and aldehydes. Expression analyses have shown that each coeloconic OSN expresses up to four IR genes, including high levels of either IR8a or IR25a. IR25a and IR8a are distinct from other IRs in that they are more conserved to each other and iGluRs, and they share a long amino terminal domain absent in all other IRs. These observations, along with functional analyses of basiconic olfactory neurons that express combinations of IR genes, led to a model in which IR based olfactory receptors are tetrameric complexes thought to consist of up to three different subunits that contain at least one core unit (IR8a or IR25a) and two additional IRs that determine ligand binding specificity. The findings presented in this paper expand this concept to taste receptors that sense fatty acids through the sweet GRNs found in tarsal taste sensilla (Ahn, 2017).

This analysis extends the multimodal role of IR25a and IR76b to the taste systems. Consistent with gene expression arrays, this paper shows that up to three GRNs, including many sweet and bitter GRNs, co-express IR25a and IR76b. Functional studies have established a novel role for these two IR proteins in fatty acid taste, which revealed that these two subunits are not only critically important to elicit Proboscis Extension Reflex (PER) responses in flies when challenged with fatty acids, but are also necessary for fatty acid induced Ca2+ increases in tarsal sweet GRNs. Based on these findings and with consideration of their established role in other sensory systems, it is proposed that IR25a and IR76b play central roles in sweet GRNs in a multimeric receptor complex for initiating appetitive taste behavior to these chemicals. Intriguingly, both genes are also co-expressed in two other GRNs of most tarsal taste sensilla, strongly arguing for additional taste functions. While the subset of tarsal bitter GRNs activated by hexanoic acid does not require either gene, the third GRN (the sour GRN) is narrowly tuned to acids in an IR25a/IR76b dependent manner. These observations suggest that modality specific IRs are likely expressed in a cell-type specific fashion whereby they complement IR25a/IR76b to function as either a fatty acid or a sour taste receptor. Indeed, the screen identified IR56d, a gene that is expressed in sweet GRNs of tarsal taste sensilla, as a likely candidate encoding an IR subunit specific for a fatty acid taste receptor. It remains to be seen whether IR25a, IR76b and IR56d comprise all subunits that constitute this receptor or whether yet additional IRs are necessary to mediate responses to these chemicals (Ahn, 2017).

The fact that different food chemicals can activate a single class of neurons raises the question how flies discriminate between sugars and fatty acids. First, the difference is noted in sensitivity of appetitive GRNs to sugars and fatty acids, respectively: The most responsive GRN for sugars is the one associated with the 5v sensilla, followed by that with the 5s and finally the 5b sensilla, while the responsiveness for fatty acids is the reverse (5b > 5s > 5v). Second, fatty acids induces weaker PER responses from stimulation of the labial palps as opposed to tarsi, while sugars induce equally strong PER responses from stimulation of either taste organ. Third, at least some fatty acids activate bitter GRNs, and hence, generate more complex activation patterns in the brain than sugars, which are not known to activate neurons other than sweet GRNs. These properties may provide a rationale for differential coding of these two classes of chemicals in the brain. Finally, sugars but not fatty acids are soluble in water, and hence, the specific solvents in which these chemicals are presented provides different textural quality, which was recently shown to play a role in taste perception (Ahn, 2017).

NorpA, which encodes a phospholipase C (PLC), plays a critical role in sweet GRNs for appetitive feeding responses to fatty acids, but it is dispensable for behavioral responses to sugars. This study found that its absence also selectively abolishes Ca2+ responses to fatty acids, but not sugars, in sweet cells. NorpA is known for its role as downstream effector of G-protein coupled receptors in the fly's visual system, but interestingly it is also required for olfactory responses in neurons of the maxillary palps, which express ORs that are thought to function as ligand-gated ion channels. It is noted that fatty acid taste in mammals is in part mediated by two G-protein coupled receptors, GPR40 and GPR120, and that one of these (GPR120) was found to signal through a phospholipase C. Thus, future studies will be necessary to gain insights for how PLC mediates chemosensory responses through ORs and the phylogenetically unrelated IRs (Ahn, 2017).

Multimeric IR based receptors were recently shown to be required in non-chemosensory processes. Specifically, Dorsal Organ Cool Cells (DOCCs) located in the larval brain, express and require the function of three IRs (IR21a IR25a and IR93a), thereby allowing larvae to avoid temperatures below ~20°C. Similarly, two sets of cells in the antennal sacculus of adult flies, requiring the functions of IR25a and IR93a and either IR40a or IR68a, were shown to mediate a fly's preferred humidity environment, which is generally in the dry range, but is also dependent on the fly's hydration state. Intriguingly, these non-chemosensory IR complexes share a common theme with the fatty acid and carboxylic acid taste receptors in that they all require a core unit (IR25a) and two additional IRs that mediate specificity for a particular stimulus type (i.e., temperature, humidity, fat, acid) (Ahn, 2017).

An IR76b based sodium channel and an IR76b based amino acid receptor appear to lack an obligate core unit (IR25a or IR8a) found in olfactory receptors or fatty acid and carboxylic acid taste receptors. The IR76b sodium channel mediates salt responses in a heterologous systems independently of any other IRs, while a proposed multimeric IR76b containing receptor mediates amino acids taste in wild type and IR25a mutant flies (IR8a is not expressed in taste neurons). It will be interesting to elucidate the compositions of complete IR based amino acid and sour taste receptors, and (with regard of amino acid receptors) to identify the neurons that mediate this taste modality (Ahn, 2017).

The molecular basis for attractive salt-taste coding in Drosophila

Below a certain level, table salt (NaCl) is beneficial for animals, whereas excessive salt is harmful. However, it remains unclear how low- and high-salt taste perceptions are differentially encoded. This study identified a salt-taste coding mechanism in Drosophila melanogaster. Flies use distinct types of gustatory receptor neurons (GRNs) to respond to different concentrations of salt. A member of the newly discovered ionotropic glutamate receptor (IR) family, IR76b, functions in the detection of low salt and is a Na(+) channel. The loss of IR76b selectively impairs the attractive pathway, leaving salt-aversive GRNs unaffected. Consequently, low salt becomes aversive. This work demonstrates that the opposing behavioral responses to low and high salt were determined largely by an elegant bimodal switch system operating in GRNs (Zhang, 2013).

To address the fundamental question as to how low- and high-salt taste perceptions are differentially encoded in GRNs in insects the fruit fly was chosen as a model. The animal's behavioral responses were tested to different salt concentrations ranging from 1 mM to 1000 mM using a robust, food-color based preference assay. Akin to mammals, flies preferred low-salt food (1 mM to 100 mM) with a maximal preference at 50 mM NaCl, whereas they rejected high-salt food (> 200 mM). This pattern differs from both sugar and bitter taste in that flies prefer sweet and dislike bitter compounds regardless of the concentration (Zhang, 2013).

In Drosophila, the primary taste sensory organ, the labellum contains 31 sensilla, which are further classified by size as small (S), intermediate (I), and large (L) sensilla. Sensilla contain multiple GRNs, which respond to distinct stimuli, including bitter, sweet and salty tastants. The physiological responses of sensilla were tested to low salt (50 mM) and high salt (500 mM) were tested by performing tip recordings. The GRNs housed by two L-type sensilla (L4 and L6) produced the most robust firings in response to low salt, while the GRNs in three S-type sensilla (S4, S6 and S8) displayed the strongest responses to high salt. These three S-type sensilla respond differentially to bitter tastants, suggesting that each gustatory sensillum has a unique taste tuning profile. GRNs in I-type sensilla responded to salt, but none as robustly as the most sensitive L- or S-type sensilla. With the exception of S4 and S8, the S-type sensilla show robust responses to the broadest array of aversive tastants, while L-type sensilla produce the strongest physiological responses to attractive tastants, such as sugars. Thus, it was deduced that the responses to low and high salt were likely to be controlled by a balance between the GRNs housed in L- and S-type sensilla (Zhang, 2013).

Focus was placed on L4 and S6 sensilla, using NaCl ranging from 1 mM to 1000 mM. The firing of salt GRNs in the L4 sensilla increased progressively at low concentrations and peaked at 100 mM. In contrast, the salt GRNs in S6 sensilla were much less active than the salt GRNs in L4 sensilla, suggesting that these latter sensilla played a predominant role in low salt response. As the salt concentration increased above 100 mM, the firing of salt GRNs in L4 sensilla gradually declined. In contrast, the action potentials produced by S6 salt GRNs exhibited a remarkable increase (>100 mM) with a maximal response at 500 mM. At high salt concentrations, the firing of S6 salt GRNs far exceeded L4 salt GRNs, indicating that the high salt response was controlled predominantly by salt GRNs in S-type sensilla (Zhang, 2013).

A model is therefore proposed in which competition between GRNs in the S- and L-type sensilla accounts for the bidirectional behavioral responses to salt. At low concentrations, the low-salt GRNs dominate over the high-salt GRNs, thereby causing the animals to prefer low salt. At high salt levels, the high-salt GRNs overwhelm the low-salt GRNs, resulting in salt rejection (Zhang, 2013).

Several candidate salt receptors and channels were tested, none of which affected salt taste. Ionotropic receptors (IRs) comprise a class of olfactory receptors in Drosophila, which are distantly related to mammalian ionotropic glutamate receptors (iGluRs). Several Ir genes, such as Ir25a and Ir76b, also appear to be expressed in gustatory sensilla. The Ir25a2 mutant had no obvious deficits in sensing either low salt or high salt. A genetic analysis of Ir76b was carried out, and two null alleles, Ir76b1 and Ir76b2 were generated by P-element-mediated imprecise excision. A revertant line was also generated that underwent a precise P-element excision (Ir76bR1). Loss of Ir76b did not impair the responses to KCl, sucrose, water or bitter tastants (Zhang, 2013).

The Ir76b deletions resulted in severe defects in the attraction to low-salt concentrations (1 - 100 mM;. In contrast, the Ir76b mutants showed the same aversion to high salt as the Ir76b+ control (w1118). Ir76bR1 behavior was indistinguishable from the 'wild-type' Ir76b+ control (w1118) at all salt concentrations (Zhang, 2013).

Tip recordings were performed to monitor physiological abnormalities in the GRNs. The Ir76b mutations caused a decease in the number of action potentials by salt GRNs in L4 sensilla in response to 50 mM salt, demonstrating a functional defect in the GRNs. There were no significant changes in the firing frequencies of S6 GRNs in the Ir76b mutants, compared with wild-type. Using the Ir76b-Gal4 and UAS-Ir76b transgenes, normal attractive responses to low salt were restored in the Ir76b1 mutant (Zhang, 2013).

The physiological responses were tested to different salt concentrations (1 - 1000 mM NaCl). Loss of Ir76b caused significant reductions in the firing frequencies of the salt GRNs in L4 sensilla at all salt concentrations. Firing of salt GRNs in L6 sensilla were also impaired. However, there were no effects on the physiological responses of high-salt GRNs in S4 or S6 sensilla (Zhang, 2013).

Taken together, these studies indicated that removal of Ir76b selectively disrupted the attractive salt pathway, while leaving the aversive salt pathway intact. Consequently, Ir76b mutant animals avoided rather than preferred low salt, while they retained aversion to high salt (Zhang, 2013).

To examine the cellular distribution pattern of IR76b, antibodies were raised against IR76b. The antibodies marked GRNs in the labellum, and the staining was virtually eliminated in Ir76b mutants. Flies were generated expressing an Ir76b reporter (Ir76b-Gal4). In combination with UAS-mCD8::GFP or UAS-dsRed, prominent GRN staining was detected in the proboscis. Ir76b-expressing GRNs were in all L-type sensilla, including L4 and L6. Ir76b reporter expression was also detected in GRNs in the leg tarsi and wing margins, which sent projections to the ventral nerve cord. Ir76b reporter expression largely overlapped with the anti-IR76b staining, suggesting that the reporter reflected the bona fide cellular distribution of Ir76b (Zhang, 2013).

To determine if Ir76b-positive GRNs overlapped with Gr66a expressing bitter- or Gr5a expressing sugar-responsive GRNs, an Ir76b reporter was generated using the Q system (Ir76b-QF). Double-labeling showed that Ir76b-positive GRNs were distinct from either Gr66a or Gr5a GRNs. Gr66a- and Gr5a-positive GRNs project their axons from the labellum to non-overlapping regions in the brain, the subesophageal ganglion (SOG). The projections of Ir76b GRNs in the SOG showed minimal overlap with regions innervated by the axons of Gr5a and Gr66a GRNs. Thus, Ir76b GRNs represented a class of GRNs distinct from sugar or bitter-responsive GRNs (Zhang, 2013).

In olfactory receptor neurons (ORNs), IRs function either alone or in conjugation with other IRs. Whether misexpression of IR76b alone conferred salt taste was tested when introduced in non-salt responsive GRNs. Because Ir76b and Gr5a are expressed in different GRN populations, Ir76b was introduced into Gr5a-sugar neurons in an Ir76b1 background. Recordings were made from L2 sensilla, which showed few responses to low salt even in wild type. In response to NaCl, there was a robust train of action potentials produced by these Gr5a GRNs in L2 sensilla. In contrast, these same GRNs did not induce a response to NMDGCl or potentiate the response to sucrose. Thus, the action potentials were due to Na+ and not Cl, and was not a consequence of nonspecific elevation of Gr5a GRN activity. The behavioral deficit in low salt preference in Ir76b mutants was rescued by misexpressing Ir76b in Gr5a GRNs (Zhang, 2013).

To test if IR76b was capable of functioning as a Na+ permeable channel, whole-cell recordings were performed after expressing IR76b in HEK293T cells. The IR76b expressing cells showed increased current (IIR76b) relative to control cells. The nearly linear current-voltage (I-V) relationship indicated that IIR76b was not strongly voltage-dependent. Replacement of the external Cl- with gluconate anions had little effect on IIR76b. Using bionic conditions, the relative ion selectivity of IR76b was PNa (1.0) = PCs (1.0) > PK (0.4). The Na+ conductance properties of IR76b were similar to NALCN - a mouse Na+ leak channel, suggested that IR76b was in a constitutively open state (Zhang, 2013).

The ion conductance of iGluRs is controlled by residues in the third transmembrane (TM3) region that includes YTANLAAFLT. In the absence of ligand, the channels are closed. A spontaneous A288T mutation in TM3 of mouse GluRδ2 (Lurcher mutation; GluRδ2Lc) disrupts the closed conformation, resulting in a constitutive Na+ conductance. Notably, IR76b harbored a threonine (T293), in nearly the same position as the Lurcher substitution (A288T). A threonine is absent in corresponding positions of other fly IRs and mammalian iGluRs. Therefore, it was postulated that T293 enabled IR76b to be fixed in an open Na+-permeable state. To test this idea, IR76b was replaced with IR76bT293A, and the effects of this substitution were determined in vivo and in vitro. When expressing UAS-Ir76bT293A using the Gr5a-Gal4, no a salt response was detected in L2 sensilla. Moreover, the T293A mutation greatly attenuated the constitutive current in HEK293T cells (Zhang, 2013).

To explain how the fly uses IR76b to detect salt, it is proposed that IR76b is a Na+ leak channel, and is effective due to the unusual extracellular cation composition bathing the GRNs. Different from the body hemolymph that contains high Na+, the endolymph that bathes insect chemosensory neurons appears to have a lower Na+ concentration that is similar to the intracellular levels. Under resting conditions, there may be little Na+ conductance. After consuming Na+-containing foods, the Na+ levels in the endolymph rises, driving Na+ influx through IR76b. Excitation of salt-attractive GRNs induces the animals to consume salt. Loss of IR76b selectively impaired the attractive pathway, making the otherwise attractive low salt become to become aversive (Zhang, 2013).

This work establishes that the salt attractive pathway relies on a type of Na+ permeable channel not previously known to function in taste, and this channel, IR76b, bears no relationship to ENaC channels that are required for sensing low salt in mice. Some ENaC channels may be constitutively active, and lead to depolarization of taste receptor cells following a rise in cation levels at the cell surface. Thus, despite the divergence between fly IRs and mammalian ENaC channels, they may mediate salt taste through similar mechanisms. The competition model for low and high salt taste detection may represent a widely used mechanism for salt taste coding in other animals, including mammals (Zhang, 2013).


Search PubMed for articles about Drosophila Ir76b

Abuin, L., Bargeton, B., Ulbrich, M. H., Isacoff, E. Y., Kellenberger, S. and Benton, R. (2011). Functional architecture of olfactory ionotropic glutamate receptors. Neuron 69(1): 44-60. PubMed ID: 21220098

Benton, R., Vannice, K. S., Gomez-Diaz, C. and Vosshall, L. B. (2009). Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell 136(1): 149-162. PubMed ID: 19135896

Croset, V., Schleyer, M., Arguello, J. R., Gerber, B. and Benton, R. (2016). A molecular and neuronal basis for amino acid sensing in the Drosophila larva. Sci Rep 6: 34871. PubMed ID: 27982028

Ganguly, A., Pang, L., Duong, V.K., Lee, A., Schoniger, H., Varady, E. and Dahanukar, A. (2017). A molecular and cellular context-dependent role for Ir76b in detection of amino acid taste. Cell Rep 18: 737-750. PubMed ID: 28099851

Hussain, A., Zhang, M., Ucpunar, H. K., Svensson, T., Quillery, E., Gompel, N., Ignell, R. and Grunwald Kadow, I. C. (2016a). Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines. PLoS Biol 14(5): e1002454. PubMed ID: 27145030

Hussain, A., Ucpunar, H. K., Zhang, M., Loschek, L. F. and Grunwald Kadow, I. C. (2016b). Neuropeptides modulate female chemosensory processing upon mating in Drosophila. PLoS Biol 14: e1002455. PubMed ID: 27145127

Koh, T. W., He, Z., Gorur-Shandilya, S., Menuz, K., Larter, N. K., Stewart, S. and Carlson, J. R. (2014). The Drosophila IR20a clade of ionotropic receptors are candidate taste and pheromone receptors. Neuron 83(4): 850-865. PubMed ID: 25123314

Ribeiro, C. and Dickson, B. J. (2010). Sex peptide receptor and neuronal TOR/S6K signaling modulate nutrient balancing in Drosophila. Curr Biol 20(11): 1000-1005. PubMed ID: 20471268

Sanchez-Alcaniz, J. A., Silbering, A. F., Croset, V., Zappia, G., Sivasubramaniam, A. K., Abuin, L., Sahai, S. Y., Munch, D., Steck, K., Auer, T. O., Cruchet, S., Neagu-Maier, G. L., Sprecher, S. G., Ribeiro, C., Yapici, N. and Benton, R. (2018). An expression atlas of variant ionotropic glutamate receptors identifies a molecular basis of carbonation sensing. Nat Commun 9(1): 4252. PubMed ID: 30315166

Silbering, A. F., Rytz, R., Grosjean, Y., Abuin, L., Ramdya, P., Jefferis, G. S. and Benton, R. (2011). Complementary function and integrated wiring of the evolutionarily distinct Drosophila olfactory subsystems. J Neurosci 31(38): 13357-13375. PubMed ID: 21940430

Stewart, S., Koh, T. W., Ghosh, A. C. and Carlson, J. R. (2015). Candidate ionotropic taste receptors in the Drosophila larva. Proc Natl Acad Sci U S A 112(14): 4195-4201. PubMed ID: 25825777

Zhang, Y. V., Ni, J. and Montell, C. (2013). The molecular basis for attractive salt-taste coding in Drosophila. Science 340(6138): 1334-1338. PubMed ID: 23766326

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date revised: 24 November 2022

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