The Interactive Fly

Zygotically transcribed genes

Ionic Receptors: Divergent ligand-gated ion channels


  • Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila
  • The Drosophila IR20a clade of ionotropic receptors are candidate taste and pheromone receptors
  • Drosophila Ionotropic Receptor 25a mediates circadian clock resetting by temperature
  • Distinct signaling of Drosophila chemoreceptors in olfactory sensory neurons
  • Candidate ionotropic taste receptors in the Drosophila larva
  • Ionotropic chemosensory receptors mediate the taste and smell of polyamines: Neuropeptides modulate female chemosensory processing upon mating in Drosophila
  • Olfactory receptor pseudo-pseudogenes
  • Evolution of acid-sensing olfactory circuits in Drosophilids
  • A molecular and neuronal basis for amino acid sensing in the Drosophila larva
  • A molecular and cellular context-dependent role for Ir76b in detection of amino acid taste
  • Ionotropic receptors mediate Drosophila oviposition preference through sour gustatory receptor neurons

    Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila

    Ionotropic glutamate receptors (iGluRs; see Ionotropic receptor 8a) mediate neuronal communication at synapses throughout vertebrate and invertebrate nervous systems. This paper characterizes a family of iGluR-related genes in Drosophila, which have been named ionotropic receptors (IRs). These receptors do not belong to the well-described kainate, AMPA, or NMDA classes of iGluRs, and they have divergent ligand-binding domains that lack their characteristic glutamate-interacting residues. IRs are expressed in a combinatorial fashion in sensory neurons that respond to many distinct odors but do not express either insect odorant receptors (ORs) or gustatory receptors (GRs). IR proteins accumulate in sensory dendrites and not at synapses. Misexpression of IRs in different olfactory neurons is sufficient to confer ectopic odor responsiveness. Together, these results lead to the proposal that the IRs comprise a novel family of chemosensory receptors. Conservation of IR/iGluR-related proteins in bacteria, plants, and animals suggests that this receptor family represents an evolutionarily ancient mechanism for sensing both internal and external chemical cues (Benton, 2009).

    Species as diverse as bacteria, plants, and humans have the capacity to sense small molecules in the environment. Chemical cues can transmit the presence of food, alarm signals, and messages from conspecifics that signify mating compatibility. Peripheral chemical recognition largely relies on membrane receptor proteins that interact with external ligands and convert this binding into intracellular responses. The vast majority of identified chemosensory receptors in multicellular organisms belong to the seven transmembrane domain G protein-coupled receptor (GPCR) superfamily, including odorant, gustatory and pheromone receptors in mammals, birds, reptiles, amphibians, fish, and nematodes. Unicellular organisms also use GPCRs for chemoreception, such as the pheromone receptors in budding yeast (Benton, 2009).

    Insects can detect a wide range of environmental chemicals: bitter, sweet, and salty tastants, odors, pheromones, humidity, carbon dioxide, and carbonated water. Most of these chemosensory stimuli are recognized by members of two evolutionarily related insect-specific chemosensory receptor families, the Odorant Receptors (ORs) and Gustatory Receptors (GRs). These proteins contain seven predicted transmembrane domains but are evolutionarily unrelated to GPCRs and adopt a distinct membrane topology. Recent analysis has indicated that insect ORs function as odor-gated ion channels, setting them mechanistically apart from metabotropic vertebrate ORs (Benton, 2009).

    Comprehensive analysis of the expression of these receptors in Drosophila, has hinted at the existence of other types of insect chemosensory receptors. This is particularly apparent in the major olfactory organ, the third segment of the antenna, which bears three types of olfactory sensory hairs (sensilla): basiconic, trichoid, and coeloconic. All olfactory sensory neurons (OSNs) innervating basiconic and trichoid sensilla generally express one OR, along with the OR83b co-receptor. However, with the exception of OR35a/OR83b-expressing neurons, OSNs housed in coeloconic sensilla do not express OR83b or members of the OR or GR gene families. Nevertheless, electrophysiological analysis has revealed the existence of multiple types of coeloconic OSNs tuned to acids, ammonia and humidity, suggesting that other types of insect chemosensory receptors exist (Benton, 2009).

    A bioinformatic screen has been carried out for insect-specific genes enriched in OSNs (Benton, 2007). Among these, a large expansion was found of the ionotropic glutamate receptor (iGluR) gene family of unknown biological function (Littleton, 2000). This study provides evidence that these variant iGluRs represent a novel class of chemosensory receptor (Benton, 2009).

    The screen identified 6 antennal-expressed genes encoding proteins annotated as ionotropic glutamate receptors (iGluRs) (Littleton, 2000). Using these novel receptor sequences as queries, exhaustive BLAST searches of the Drosophila genome identified a family of 61 predicted genes and 1 pseudogene. These genes are distributed throughout the genome, both as individual sequences and in tandem arrays of up to four genes. This family was named the Ionotropic Receptors (IRs), and individual gene names were assigned to the IRs using nomenclature conventions of Drosophila ORs (Benton, 2009).

    Phylogenetic analysis of predicted IR protein sequences revealed that they are not closely related to members of the canonical families of iGluRs (AMPA, kainate, NMDA, or delta). However, they appear to have a similar modular organization to iGluRs, comprising an extracellular N-terminus, a bipartite ligand-binding domain, whose two lobes (S1 and S2) are separated by an ion channel domain, and a short cytoplasmic C-terminus. It is noted that the gene structure and protein sequence of most receptors are presently only computational predictions. Nevertheless, the family is extremely divergent, exhibiting overall amino acid sequence identity of 10-70%. The most conserved region between IRs and iGluRs spans the ion channel pore, suggesting that IRs retain ion-conducting properties (Benton, 2009).

    The ligand-binding domains are considerably more variable, although alignment of small regions of the S1 and S2 lobes of IRs and iGluRs allowed examination of conservation in amino acid positions that make direct contact with glutamate or artificial agonists in iGluRs. While all iGluRs have an arginine (R) residue in S1 that binds the glutamate α-carboxyl group, only 19/61 (31%) IRs retain this residue. In the first half of the S2 domain, 9/61 (15%) of IRs retain a threonine (T), which contacts the glutamate γ-carboxyl group in all AMPA and kainate receptors. Interestingly, the iGluRs that lack this T residue (NR1, NR3A, delta) have glycine or serine and not glutamate as a preferred ligand. Finally, in the second half of the S2 domain, 100% of the iGluRs have a conserved aspartate (D) or glutamate (E) that interacts with the α-amino group of the glutamate ligand, compared with 10/61 (16%) IRs. Of 61 IRs, only three (IR8a, IR75a, IR75c) retain the R, D/E, and T residues characteristic of iGluRs, although these residues lie within a divergent structural backbone. Other IRs have a diversity of different amino acids at one or more of these positions. Thus, the ligand-binding specificity of most or all IRs is likely to be both distinct from that of iGluRs and varied within the IR family (Benton, 2009).

    The expression of the IR family was determined by both tissue-specific RT-PCR and RNA in situ hybridization. Fifteen IR genes are expressed in the antenna. Transcripts of these genes were not detected elsewhere in the adult head, body or appendages, except for IR25a and IR76b, which are also expressed in the proboscis. Expression of the remaining 46 IR genes was not reproducibly detected in any adult tissue. It is unclear whether these genes are not expressed, expressed at different life stages, or expressed in at levels below the detection threshold of these assays (Benton, 2009).

    Analysis of where in the antenna IR genes are expressed compared to ORs was performed by double RNA in situ hybridization with probes for the OR co-receptor OR83b and one of several IR genes, including IR64a, IR76b, IR31a, and IR40a. IRs are not expressed in basiconic and trichoid sensilla, as they are not co-expressed with OR83b, and IR expression persists in mutants for the proneural gene absent md neurons and olfactory sensilla (amos), which completely lack these sensilla types. However, expression of these IRs is dependent upon the proneural gene atonal, which specifies the coeloconic sensilla as well as a feather-like projection called the arista, and a three-chambered pocket called the sacculus. Thus, ORs and IRs are expressed in developmentally distinct sensory lineages in the antenna. One exception is the subpopulation of coeloconic OSNs that expresses both IR76b and OR35a and OR83b. It was confirmed that IR-expressing cells in the antenna are neurons by demonstrating that they co-express the neuronal marker elav (Benton, 2009).

    A comprehensive map was generated of IR expression. Each IR was observed to have a topologically-defined expression pattern that is conserved across individuals of both sexes. IR8a and IR25a, which encode closely related receptors, are broadly expressed, detected in overlapping populations of neurons around the sacculus and in the main portion of the antenna. IR25a but not IR8a is also detected in the arista. IR21a is expressed in approximately 6 neurons in the arista, as well as 5-10 neurons near the third chamber of the sacculus. Three IRs display specific expression in neurons surrounding the sacculus: IR40a and IR93a are co-expressed in 10-15 neurons adjacent to the first and second sacculus chambers, while IR64a is found in 10-15 neurons surrounding the third chamber (Benton, 2009).

    The remaining 9 IRs are expressed in coeloconic OSNs distributed across the antenna. Double and triple RNA in situ hybridization revealed that individual neurons express between 1 and 3 different IR genes and are organized into specific clusters of two or three neurons. Four distinct clusters (cluster A-cluster D), containing two (cluster C) or three (cluster A, B, and D) neurons, could be defined by their expression of stereotyped combinations of IR genes. Cluster C includes a coeloconic neuron that expresses OR35a and OR83b in addition to IR76b. Although each cluster is distinct, there is overlap between the IRs they express. IR76b is expressed in one neuron in all four clusters, IR75d in three clusters and IR75a in two clusters. In additional to these selectively-expressed receptors, individual neurons are likely to express one or both of the broadly-expressed IR8a and IR25a. The combinatorial expression patterns of the IRs raise the possibility that these genes define specific functional properties of these neurons (Benton, 2009).

    Definition of four distinct clusters of IR-expressing neurons in the antenna is consistent with the identification of four types of coeloconic sensilla, named ac1-ac4, which have distinct yet partially overlapping sensory specificities (Yao, 2005). To examine whether IR expression correlates with the chemosensory properties of these OSNs, the spatial organization of IR-expressing neurons was compared using probes for unique IR markers for each cluster type to these functionally distinct sensilla types. As a unique molecular marker for Cluster B is lacking, this cluster was defined as those containing IR75a-expressing OSNs (present in Cluster B and Cluster C) that are not paired with OR35a-expressing cluster C neurons. It was found that each cluster has a different, though overlapping, spatial distribution in the antenna. For example, Cluster A neurons (marked by IR31a) are restricted to a zone at the anterior of the antenna, just below the arista, while cluster C neurons (marked by IR75b) are found exclusively in the posterior of the antenna. These stereotyped IR neuron distributions were observed in antennae from over 20 animals (Benton, 2009).

    The initial description of the coeloconic sensilla classes did not describe their spatial distribution (Yao, 2005). This study therefore recorded odor-evoked responses in >100 coeloconic sensilla in several dozen animals across most of the accessible antennal surface, using a panel of odorants that allowed identification unambiguously of each sensilla type (ammonia for ac1, 1,4-diaminobutane for ac2, propanal and hexanol for ac3, and phenylacetaldehyde for ac4) (Yao, 2005). After electrophysiological identification, the location of the sensilla on the antennal surface was noted (Benton, 2009).

    This mapping process allowed a correlation of the electrophysiological and molecular properties of the coeloconic sensilla. For example, ac1 sensilla were detected only in a region on the anterior antennal surface just ventral to the arista, and therefore are most likely correspond to cluster A, containing IR31a-IR75d-IR76b/IR92a-expressing neurons. The data fit well with the previous assignment of the OR35a-expressing neuron to the ac3 sensillum (Yao, 2005), which is found on the posterior of the antenna and is the only coeloconic sensillum class that unambiguously houses two neurons (Yao, 2005). While these results allow initial assignment of IRs to different coeloconic sensilla classes, it is noted that assignment of specific odor responses to individual IR-expressing OSNs is not possible from these data alone (Benton, 2009).

    All neurons expressing a given OR extend axons that converge upon a single antennal lobe glomerulus, resulting in the representation of a cognate odor ligand as a spatially-defined pattern of neural activity within the brain. To ask whether IR-expressing neurons have the same wiring logic, the targeting of OSNs expressing IR76a was investigated by constructing an IR76a-promoter GAL4 driver that recapitulates the endogenous expression pattern. Labeling of these neurons with mCD8:GFP revealed convergence of their axons on to a single glomerulus, ventral medial 4 (VM4), in the antennal lobe. This glomerulus is one of approximately eight that was previously unaccounted for by maps of axonal projections of OR-expressing OSNs (Benton, 2009).

    To determine where IRs localize in sensory neurons, antibodies were generated against IR25a. Broad expression of IR25a protein was detected in sensory neurons of the arista, sacculus, and coeloconic sensilla. All anti-IR25a immunoreactivity was abolished in an IR25a null mutant. Low levels of IR25a could be detected in the axon segment adjacent to the cell body in some neurons but no staining was observed along the axons as they entered the brain, or at synapses within antennal lobe glomeruli. In coeloconic neurons, prominent anti-IR25a staining was detected both in the cell body and in the distal tip of the dendrite, which corresponds to the ciliated outer dendritic segment innervating the sensory hair. Relatively low levels were detected in the inner dendrites, suggesting the existence of a transport mechanism to concentrate receptor protein in cilia. A similar subcellular localization was observed in sacculus and aristal sensory neurons. The specific targeting of an IR to sensory cilia suggests a role for these proteins in sensory detection (Benton, 2009).

    To test the hypothesis that IR genes encode chemosensory receptors, whether ectopic IR expression could induce novel olfactory specificities was investigated. Three IRs expressed in ac4 sensilla (IR84a, IR76a and IR75d) were individually mis-expressed in ac3 sensilla using the OR35a-GAL4 driver. Single sensillum recordings were used to examine which, if any, of these three IRs, could confer sensitivity to phenylacetaldehyde, the only known robust ligand for ac4 but not ac3 sensilla (Yao, 2005). Mis-expression of IR84a conferred a strong response to phenylacetaldehyde that was not observed in control strains or in animals mis-expressing either IR76a or IR75d. Ectopically-expressed IR84a did not confer sensitivity to the structurally related odor, phenylacetonitrile, which does not activate either ac3 or ac4 neurons (Yao, 2005). This indicates that mis-expressed IR84a does not simply generate non-specific ligand sensitivity in these neurons (Benton, 2009).

    Next the novel odor responses conferred by IR84a mis-expression were compared to the endogenous phenylacetaldehyde responses of ac4 sensilla by generating dose-response curves. Stimulus evoked spike frequencies of ac3 sensilla ectopically expressing IR84a are quantitatively very similar to those in ac4 sensilla, even exceeding the endogenous ac4 responses at higher odor concentrations. These elevated responses are likely to be due to the contribution of weak endogenous phenylacetaldehyde responses that were observed in ac3 sensilla at high stimulus concentrations, as subtraction of these values produces an IR84a-dependent phenylacetaldehyde dose-response curve that is statistically the same as that of ac4 sensilla. Thus, ectopic expression of a single IR in ac3 is sufficient to confer a novel ligand- and receptor-specific odor sensitivity that is physiologically indistinguishable from endogenous responses (Benton, 2009).

    To extend this analysis to a second IR, whether mis-expression of one of the IR genes uniquely expressed in ammonia-sensitive ac1 neurons (IR31a and IR92a) was sufficient to confer ectopic responsiveness to this odor was examined. Because ac3 sensilla neurons display endogenous ammonia-evoked responses at modest stimulus concentrations, these experiments used the IR76a-promoter GAL4 transgene to mis-express these receptors in ammonia-insensitive ac4 sensilla (Yao, 2005). ac4 sensilla mis-expressing IR92a, but not IR31a, displayed responses to ammonia. 1,4-diaminobutane, a control stimulus that does not activate either ac1 or ac4 neurons (Yao, 2005), did not stimulate ac4 sensilla mis-expressing IR92a. It was noted that the magnitude of the ectopic IR92a ammonia response is lower than native ammonia-evoked responses of ac1 sensilla (Yao, 2005). This may be due to the lack of co-factors present in ac1 sensilla but not in ac4 sensilla. Nevertheless, these results suggest that IR92a comprises at least part of an ammonia-specific chemosensory receptor (Benton, 2009).

    The specific combinatorial expression patterns of IRs in sensory neurons and the diversity in their ligand-binding domains is difficult to rationalize with a general role in signal transduction, independent of ligand recognition. More importantly, the novel olfactory sensitivity induced by ectopic expression of IR84a and IR92a provides evidence that IR proteins function directly as ligand-specific, chemosensory receptors. While these experiments demonstrate a sufficiency of IRs for conferring odor-responsiveness, definitive proof of their necessity will require analysis of loss-of-function mutations (Benton, 2009).

    In animal nervous systems, iGluRs mediate neuronal communication by forming glutamate-gated ion channels, and it is speculated that IRs also form ion channels, gated by odors and other chemosensory stimuli. A growing number of ionotropic mechanisms in chemoreception are known. For example, members of the transient receptor potential (TRP) family of ion channels are the primary receptors for nociceptive compounds including capsaicin and menthol and have also been implicated in gustatory detection of acids. Insect ORs also display functional properties of ion channels. Proof that IRs function as ion channels will necessitate electrophysiological characterization of these receptors in heterologous expression systems, and evidence for direct binding of chemosensory ligands to IRs will require biochemical assays in vitro (Benton, 2009).

    iGluRs normally function as heterotetrameric assemblies of variable subunit composition that exhibit differing functional properties such as ligand sensitivity and ion permeability. The current analysis indicates that up to five different IRs may be co-expressed in a single sensory neuron, raising the possibility that these receptors also form multimeric protein assemblies with subunit-dependent characteristics. Of particular interest are the two broadly-expressed members of the family, IR8a and IR25a, which may represent common subunits in many different types of IR complexes. Their function is unclear, but it is possible that they have a co-receptor function with other IRs, analogous to that of OR83b. Preliminary analysis of IR25a mutants revealed no obvious defects in odor-evoked responses in coeloconic sensilla, but this may be due to redundancy of IR25a with IR8a or the existence of homomeric IR receptors without IR8a or IR25a. Other IRs, such as IR75a and IR76b, are expressed in two or more types of coeloconic sensory neurons. In these cases, the response properties may be defined by the combination of IRs expressed in these distinct neuronal populations. However, the present lack of knowledge of relevant ligands for several coeloconic OSNs makes it difficult to match specific ligands to individual IR neurons based on the expression map alone (Benton, 2009).

    The IR repertoire is remarkably similar in size, overall genomic organization and sequence divergence to Drosophila ORs. Like the ORs, individual IRs are specifically expressed in small subpopulations of chemosensory neurons, and this expression is regulated by relatively short (< 1-2 kb) upstream regulatory regions. Furthermore, at least one population of IR-expressing neurons converges on to a single glomerulus in the antennal lobe, similar to the wiring logic established for OR-expressing neurons both in invertebrate and vertebrate olfactory systems. Some differences are observed, however, in the organizational logic of IR and OR expression. Most OR-expressing neurons express a single OR gene, along with OR83b, in distinct clusters that innervate specific olfactory hairs. In contrast, many IR-expressing neurons identified in the antenna express 2 or 3 IR genes, in addition to one or both of the broadly-expressed IR8a and IR25a genes. Moreover, overlap is observed both between the molecular composition of different IR neurons and the combination of neurons that innervate a given sensillum. For example, IR76b is co-expressed with at least two other different IR genes in at least two different sensilla - with IR92a in ac1 and with IR76a in ac4 - as well as being co-expressed with OR35a and OR83b in ac3. While the precise biological logic of IR co-expression awaits the matching of specific chemosensory ligands to IR-expressing neurons, combinatorial expression of IRs may contribute more significantly to their role in sensory detection than for ORs (Benton, 2009).

    Why does Drosophila possess two types of antennal chemosensory receptors? Although both may be ionotropic, IRs and ORs are not simply slight evolutionary variants. The receptor families are molecularly unrelated, are under the control of distinct developmental programs, and housed within sensory structures of radically different morphology. Thus, it seems likely that these chemosensory receptors fulfill distinct functions in chemosensation. Analysis of the chemosensory behaviors mediated by IR sensory circuits (now possible with the identification of specific molecular markers for these pathways) may provide insights into the contributions of these different olfactory subsystems. IRs may also have functions in other chemosensory modalities, as two antennal IRs are also detected in the proboscis, and the expression of 46 members of the repertoire remains unknown (Benton, 2009).

    Chemosensation is an ancient sensory modality that predates the evolution of the eukaryotes. Are there traces of conservation in the molecular mechanism by which prokaryotes and eukaryotes sense external chemicals? iGluRs have long been recognized to have prokaryotic origins. Their ion channel domain is homologous to bacterial potassium channels, and the ligand binding domain is structurally related to bacterial periplasmic binding proteins (PBPs), extracellular proteins that scavenge or sense amino acids, carbohydrates and metal ions by coupling to transporters or chemotaxis receptors. Evolutionary connections between iGluRs and PBP function have not often been considered, perhaps in part due to their very weak primary sequence similarity, the widespread occurrence of the PBP fold -also present, for example, in bacterial transcription regulators - and the dedicated role for iGluRs in mediating or regulating synaptic transmission, a process seemingly distant from bacterial solute uptake and chemotaxis (Benton, 2009).

    This discovery of a family of divergent iGluR-like proteins that may act as peripheral chemosensors provides a link between the disparate functions of these protein modules. While a role for IRs in detecting diverse external ligands is analogous to the function of bacterial PBPs, the primary sequence and neuronal expression of IRs is clearly closer to the properties of iGluRs. Intriguingly, a large family of iGluR-related proteins, the GLRs has also been identified in the plant Arabidopsis thaliana (Lam, 1998; Chiu, 1999). Almost nothing is known about their physiological functions, but bioinformatic analysis of GLRs suggests that glutamate is unlikely to be their natural ligand (Dubos, 2003; Qi, 2006). It is possible that GLRs may have roles as chemosensors, for example in detection of soil nutrients or airborne volatiles. Thus, while iGluRs have been intensely studied for their roles in synaptic communication, this characterization of the IRs leads to the suggestion that the ancestral function of this protein family may have been in detecting diverse chemical ligands to mediate both intercellular communication and environmental chemical sensing (Benton, 2009).

    The Drosophila IR20a clade of ionotropic receptors are candidate taste and pheromone receptors

    Insects use taste to evaluate food, hosts, and mates. Drosophila has many "orphan" taste neurons that express no known taste receptors. The Ionotropic Receptor (IR) superfamily is best known for its role in olfaction, but virtually nothing is known about a clade of approximately 35 members, the IR20a clade. Here, a comprehensive analysis of this clade reveals expression in all taste organs of the fly. Some members are expressed in orphan taste neurons, whereas others are coexpressed with bitter- or sugar-sensing Gustatory receptor (Gr) genes. Analysis of the closely related IR52c and IR52d genes reveals signatures of adaptive evolution, roles in male mating behavior, and sexually dimorphic expression in neurons of the male foreleg, which contacts females during courtship. These neurons are activated by conspecific females and contact a neural circuit for sexual behavior. Together, these results greatly expand the repertoire of candidate taste and pheromone receptors in the fly (Koh, 2014).

    Candidate ionotropic taste receptors in the Drosophila larva

    This paper examines in Drosophila a group of approximately 35 ionotropic receptors (IRs), the IR20a clade, about which remarkably little is known. Of 28 genes analyzed, GAL4 drivers representing 11 showed expression in the larva. Eight drivers labeled neurons of the pharynx, a taste organ, and three labeled neurons of the body wall that may be chemosensory. Expression was not observed in neurons of one taste organ, the terminal organ, although these neurons express many drivers of the Gr (Gustatory receptor) family. For most drivers of the IR20a clade, expression was observed in a single pair of cells in the animal, with limited coexpression, and only a fraction of pharyngeal neurons are labeled. The organization of IR20a clade expression thus appears different from the organization of the Gr family or the Odor receptor (Or) family in the larva. A remarkable feature of the larval pharynx is that some of its organs are incorporated into the adult pharynx, and several drivers of this clade are expressed in the pharynx of both larvae and adults. Different IR drivers show different developmental dynamics across the larval stages, either increasing or decreasing. Among neurons expressing drivers in the pharynx, two projection patterns can be distinguished in the CNS. Neurons exhibiting these two kinds of projection patterns may activate different circuits, possibly signaling the presence of cues with different valence. Taken together, the simplest interpretation of these results is that the IR20a clade encodes a class of larval taste receptors (Steward, 2015).

    Drosophila Ionotropic Receptor 25a mediates circadian clock resetting by temperature

    Circadian clocks are endogenous timers adjusting behaviour and physiology with the solar day. Visual and non-visual photoreceptors are responsible for synchronizing circadian clocks to light, but clock-resetting is also achieved by alternating day and night temperatures with only 2-4 degrees C difference. This study shows that Drosophila Ionotropic Receptor 25a (IR25a) is required for behavioural synchronization to low-amplitude temperature cycles. This channel is expressed in sensory neurons of internal stretch receptors previously implicated in temperature synchronization of the circadian clock. IR25a is required for temperature-synchronized clock protein oscillations in subsets of central clock neurons. Extracellular leg nerve recordings reveal temperature- and IR25a-dependent sensory responses, and IR25a misexpression confers temperature-dependent firing of heterologous neurons. It is proposed that IR25a is part of an input pathway to the circadian clock that detects small temperature differences. This pathway operates in the absence of known 'hot' and 'cold' sensors in the Drosophila antenna, revealing the existence of novel periphery-to-brain temperature signalling channels (Chen, 2015).

    Distinct signaling of Drosophila chemoreceptors in olfactory sensory neurons

    In Drosophila, olfactory sensory neurons (OSNs) rely primarily on two types of chemoreceptors, odorant receptors (Ors) and ionotropic receptors (Irs), to convert odor stimuli into neural activity. The cellular signaling of these receptors in their native OSNs remains unclear because of the difficulty of obtaining intracellular recordings from Drosophila OSNs. This study developed an antennal preparation that enabled the first recordings from targeted Drosophila OSNs through a patch-clamp technique. Brief odor pulses triggered graded inward receptor currents with distinct response kinetics and current-voltage relationships between Or- and Ir-driven responses. When stimulated with long-step odors, the receptor current of Ir-expressing OSNs did not adapt. In contrast, Or-expressing OSNs showed a strong Ca2+-dependent adaptation. The adaptation-induced changes in odor sensitivity obeyed the Weber-Fechner relation; however, surprisingly, the incremental sensitivity was reduced at low odor backgrounds but increased at high odor backgrounds. This model for odor adaptation revealed two opposing effects of adaptation, desensitization and prevention of saturation, in dynamically adjusting odor sensitivity and extending the sensory operating range (Cao, 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).

    Olfactory receptor pseudo-pseudogenes

    Pseudogenes are generally considered to be non-functional DNA sequences that arise through nonsense or frame-shift mutations of protein-coding genes. Although certain pseudogene-derived RNAs have regulatory roles, and some pseudogene fragments are translated, no clear functions for pseudogene-derived proteins are known. Olfactory receptor families contain many pseudogenes, which reflect low selection pressures on loci no longer relevant to the fitness of a species. This study reports the characterization of a pseudogene in the chemosensory variant ionotropic glutamate receptor repertoire of Drosophila sechellia, an insect endemic to the Seychelles that feeds almost exclusively on the ripe fruit of Morinda citrifolia. This locus, D. sechellia Ir75a (see Drosophila Ir75a), bears a premature termination codon (PTC) that appears to be fixed in the population. However, D. sechellia Ir75a encodes a functional receptor, owing to efficient translational read-through of the PTC. Read-through is detected only in neurons and is independent of the type of termination codon, but depends on the sequence downstream of the PTC. Furthermore, although the intact Drosophila melanogaster Ir75a orthologue detects acetic acid-a chemical cue important for locating fermenting food found only at trace levels in Morinda fruit-D. sechellia Ir75a has evolved distinct odour-tuning properties through amino-acid changes in its ligand-binding domain. Functional PTC-containing loci were identified within different olfactory receptor repertoires and species, suggesting that such 'pseudo-pseudogenes' could represent a widespread phenomenon (Prieto-Godino, 2016).

    Evolution of acid-sensing olfactory circuits in Drosophilids

    Animals adapt their behaviors to specific ecological niches, but the genetic and cellular basis of nervous system evolution is poorly understood. This study compared the olfactory circuits of the specialist Drosophila sechellia-which feeds exclusively on Morinda citrifolia fruit-with its generalist cousins D. melanogaster and D. simulans. D. sechellia was shown to exhibit derived odor-evoked attraction and physiological sensitivity to the abundant Morinda volatile hexanoic acid, and how the responsible sensory receptor (the variant ionotropic glutamate receptor IR75b) and attraction-mediating circuit have evolved were characterized. A single amino acid change in IR75b is sufficient to recode it as a hexanoic acid detector. Expanded representation of this sensory pathway in the brain relies on additional changes in the IR75b promoter and trans-acting loci. By contrast, higher-order circuit adaptations are not apparent, suggesting conserved central processing. This work links olfactory ecology to structural and regulatory genetic changes influencing nervous system anatomy and function (Prieto-Godino, 2017).

    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).

    A molecular and cellular context-dependent role for Ir76b in detection of amino acid taste

    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, the 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).

    Ionotropic receptors mediate Drosophila oviposition preference through sour gustatory receptor neurons

    Carboxylic acids are present in many foods, being especially abundant in fruits. Yet, relatively little is known about how acids are detected by gustatory systems and whether they have a potential role in nutrition or provide other health benefits. This study identified sour gustatory receptor neurons (GRNs) in tarsal taste sensilla of Drosophila melanogaster. Most tarsal sensilla were found to harbor a sour GRN that is specifically activated by carboxylic and mineral acids but does not respond to sweet- and bitter-tasting chemicals or salt. One pair of taste sensilla features two GRNs that respond only to a subset of carboxylic acids and high concentrations of salt. All sour GRNs prominently express two Ionotropic Receptor (IR) genes, IR76b and IR25a, and this study shows that both these genes are necessary for the detection of acids. Furthermore, IR25a and IR76b were shown to be essential in sour GRNs of females for oviposition preference on acid-containing food. These investigations reveal that acids activate a unique set of taste cells largely dedicated to sour taste, and they indicate that both pH/proton concentration and the structure of carboxylic acids contribute to sour GRN activation. Together, these studies provide new insights into the cellular and molecular basis of sour taste (Chen, 2017).


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    Zygotically transcribed genes

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