The Interactive Fly

Zygotically transcribed genes

Ionotropic 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

    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

    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).

  • Ionotropic receptors mediate Drosophila oviposition preference through sour gustatory receptor neurons
  • Ionotropic Receptor 76b is required for gustatory aversion to excessive Na+ in Drosophila
  • Molecular and cellular organization of taste neurons in adult Drosophila pharynx
  • In vivo assembly and trafficking of olfactory ionotropic receptors
  • Mosquito heat seeking is driven by an ancestral cooling receptor
  • Olfactory receptor-dependent receptor repression in Drosophila
  • Ir56d-dependent fatty acid responses in Drosophila uncovers taste discrimination between different classes of fatty acids
  • Mechanisms of Carboxylic Acid Attraction in Drosophila melanogaster
  • Ir76b is a Co-receptor for Amine Responses in Drosophila Olfactory Neurons
    • Ionotropic Channels
      • Ionotropic receptor 8a: a member of a novel family of chemosensory receptor ion channels - expressed on antennal neurons
      • Ionotropic receptor 21a: mediator of heat seeking in the malaria vector Anopheles gambiae - Ir21a mediates heat avoidance in Drosophila
      • Ionotropic receptor 25a: part of an input pathway to the circadian clock that detects small temperature differences - required for humidity preference - co-receptor subunit in chemosensation and thermosensation
      • Ionotropic receptor 64a: chemosensory receptor ion channel, detection of acid
      • Ionotropic receptor 76b: chemosensory detection of various amines and salt - a probable co-receptor subunit
      • Ionotropic receptor 84a: chemosensory glutamate receptor family protein, regulation of male courtship behavior, response to aromatic odors

    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).

    Ionotropic Receptor 76b is required for gustatory aversion to excessive Na+ in Drosophila

    Avoiding ingestion of excessively salty food is essential for cation homeostasis that underlies various physiological processes in organisms. The molecular and cellular basis of the aversive salt taste, however, remains elusive. Through a behavioral reverse genetic screening, feeding suppression by Na(+)-rich food was found to require Ionotropic Receptor 76b (Ir76b) in Drosophila labellar gustatory receptor neurons (GRNs). Concentrated sodium solutions with various anions caused feeding suppression dependent on Ir76b. Feeding aversion to caffeine and high concentrations of divalent cations and sorbitol was unimpaired in Ir76b-deficient animals, indicating sensory specificity of Ir76b-dependent Na(+) detection and the irrelevance of hyperosmolarity-driven mechanosensation to Ir76b-mediated feeding aversion. Ir76b-dependent Na(+)-sensing GRNs in both L- and s-bristles are required for repulsion as opposed to the previous report where the L-bristle GRNs direct only low-Na(+) attraction. This work extends the physiological implications of Ir76b from low-Na(+) attraction to high-Na(+) aversion, prompting further investigation of the physiological mechanisms that modulate two competing components of Na(+)-evoked gustation coded in heterogeneous Ir76b-positive GRNs (Lee, 2017).

    Molecular and cellular organization of taste neurons in adult Drosophila pharynx

    The Drosophila pharyngeal taste organs are poorly characterized despite their location at important sites for monitoring food quality. Functional analysis of pharyngeal neurons has been hindered by the paucity of molecular tools to manipulate them, as well as their relative inaccessibility for neurophysiological investigations. This study generated receptor-to-neuron maps of all three pharyngeal taste organs by performing a comprehensive chemoreceptor-GAL4/LexA expression analysis. The organization of pharyngeal neurons reveals similarities and distinctions in receptor repertoires and neuronal groupings compared to external taste neurons. The mapping results were validated by pinpointing a single pharyngeal neuron required for feeding avoidance of L-canavanine. Inducible activation of pharyngeal taste neurons reveals functional differences between external and internal taste neurons and functional subdivision within pharyngeal sweet neurons. These results provide roadmaps of pharyngeal taste organs in an insect model system for probing the role of these understudied neurons in controlling feeding behaviors (Chen, 2017).

    In Drosophila, taste neurons located in sensilla in several body regions sense and distinguish nutritive substances such as sugars, amino acids, and low salt, and potentially harmful ones such as high salt, acids, and a diverse variety of bitter compounds. Hair-like sensilla on the labellum, distal segments of the legs (tarsi), anterior wing margins, and ovipositor have access to chemicals in external substrates. Pit-like sensilla (taste pegs) on the oral surface have access only once the fly extends its proboscis and opens the labellar palps; similar sensilla in the pharynx have access only when food intake is initiated. Based on its anatomical position, the pharynx is considered to act as a gatekeeper to control ingestion, promoting the intake of appetitive foods and blocking that of toxins (Chen, 2017).

    Three distinct internal taste organs are present in the adult fly pharynx: the labral sense organ (LSO), the ventral cibarial sense organ (VCSO), and dorsal cibarial sense organ (DCSO). The VCSO and DCSO are paired on opposite sides of the rostrum, whereas the LSO is located in the haustellum. The organization and neuronal composition of all three organs, based on both light and electron microscopy data, have been described in detail. Nine separate sensilla are present in the LSO, of which 1-6 are innervated by a single mechanosensory neuron each. The remaining three, named 7-9, are uniporous sensilla, a feature that ascribes chemosensory function to them. Sensillum 7 is the largest one, with eight chemosensory neurons. Sensilla 8 and 9 have two neurons each (one mechanosensory and one chemosensory). Although one study reported two sensilla in the VCSO, this and other studies have observed three sensilla in the VCSO, innervated by a total of eight chemosensory neurons. The DCSO has two sensilla, each containing three chemosensory neurons. Notwithstanding the availability of detailed anatomical descriptions of pharyngeal taste organs, little is known about their function. The internal location of these organs poses challenges for electrophysiological analysis of taste neurons located within them. Additionally, few molecular tools are currently described to manipulate the function of selected pharyngeal taste neurons (Chen, 2017).

    The expression and function of members of several chemosensory receptor gene families such as gustatory receptors (Grs), ionotropic receptors (Irs), Pickpocket (Ppk) channels, and transient receptor potential channels (Trps) have been found in external gustatory receptor neurons (GRNs) of the labellum and the tarsal segments. A number of Gr- and Ir-GAL4 drivers are also shown to label pharyngeal organs, but only a few, including Gr43a and members of sweet Gr clade, Gr2a, Ir60b, and TrpA1, have been mapped to specific taste neurons (Chen, 2017).

    This study generated receptor-to-neuron maps for three pharyngeal taste organs by a systematic expression analysis of chemoreceptor reporter lines that represent Gr, Ir, and Ppk receptor families. The maps reveal a large and diverse chemoreceptor repertoire in the pharynx. Some receptors are expressed in combinations that are predictive of neuronal sweet or bitter taste function based on analysis of external GRNs. By contrast, some pharyngeal taste neurons express receptor combinations that are distinct from any that have been reported in other organs, leaving open questions about their functional roles. This study validated he receptor-to-neuron maps derived from reporter gene expression by assessing roles of pharyngeal GRNs predicted to detect L-canavanine, a bitter tastant for which a complete receptor repertoire has been reported. Interestingly, a systematic activation analysis of different classes of pharyngeal taste neurons reveals functional differences between external and internal taste neurons for bitter avoidance and functional subdivision within pharyngeal sweet neurons for sweet acceptance. Together, this study provides a molecular map of pharyngeal taste organs, which will serve as a resource for future studies of the roles of pharyngeal taste neurons in food evaluation (Chen, 2017).

    Internal pharyngeal taste organs are the least explored taste organs, despite their obvious importance in insect feeding behaviors, which are crucial drivers for damaging crops and vectoring disease. The receptor-to-neuron maps of pharyngeal taste organs suggest a high degree of molecular complexity, with co-expression of different chemoreceptor family members in many pharyngeal GRNs. In particular, none of the pharyngeal GRNs were found to express Gr genes alone; rather, one or more Ir genes were always expressed in the same neurons. Gr and Ir genes are also co-expressed in some external sweet and bitter-sensing GRNs. Thus, both classes of receptors are likely to contribute to responses of Gr/Ir-expressing neurons in the LSO and VCSO, but whether they interact functionally or act independently remains to be determined. In the LSO, expression of sweet Grs and Ir76b overlaps in pharyngeal sweet GRNs, as observed in tarsi as well. In the pharynx, this study also found co-expression of ppk28 with Ir genes, which has not been described for external GRNs. These observations invite explorations of possible crosstalk, and its functional significance, between the two classes of receptors (Chen, 2017).

    Pharyngeal GRNs also exhibit distinctive functional groupings. All external bitter GRNs have always been found grouped with sweet GRNs in taste hairs. By contrast, canonical sweet and bitter GRNs appear to segregate in different sensilla in the LSO, which is most well characterized for this perspective. L8 and L9 may be functionally identical and house only one Gr66a-expressing bitter GRN each, whereas L7 contains two sweet GRNs (L7-1 and L7-2). Moreover, external hairs typically have two to four GRNs, each of which has a distinct functional profile. In the LSO duplications are found (L7-1 and L7-2 are identical, as are L7-4 and L7-5), although differences between these pairs of GRNs may emerge as additional chemoreceptors are mapped in the pharynx. Finally, it is difficult to ascribe putative functions to most pharyngeal GRNs based on existing knowledge of receptor function in external counterparts. The L7-3 Gr-expressing neuron, for example, does not express members of the sweet clade, but neither does it express any of the common bitter Grs (Gr32a, Gr66a, and Gr89a) that would corroborate its role as a bitter GRN. Similarly, with the exception of salt neurons that may express Ir76b alone, there are few known functions for GRNs that solely express Ir genes. One possibility is that some of these GRNs possess novel chemoreceptor family ligand interactions. For example, L7-7 is involved in sensing sucrose but limiting sugar ingestion, representing an Ir neuron that operates in a negative circuit module for sugar intake. In addition, another recent study suggests that TRPA1 expression in L8 and L9 of the LSO is involved in feeding avoidance to bacterial endotoxins lipopolysaccharides (LPS). Alternatively, some pharyngeal GRNs may evaluate characteristics other than palatability, such as temperature or viscosity. Ir25a, which is broadly expressed in all 24 pharyngeal GRNs, is required for cool sensing and thermosensing. It will be worth investigating whether one or more pharyngeal GRNs act to integrate information about temperature and chemical quality of food substrates (Chen, 2017).

    Expression analyses also hint at some functional subdivisions between pharyngeal taste organs. The LSO contains a smaller proportion of Gr-expressing neurons than the VCSO, which also expresses a larger number of Gr genes that are co-expressed with Gr66a. Thus, broader bitter taste function might be expected in the VCSO. By contrast, sweet taste function appears to be more dominant in the LSO; its sweet GRNs express more sweet Gr-GAL4 drivers than the ones in the VCSO, and their activation is sufficient to drive feeding preference. VCSO sweet GRNs fail to promote ingestion by themselves but may contribute to an increase in feeding preference when activated simultaneously with those in the LSO. Thus, there may be synergistic or hierarchical interactions between LSO and VCSO sweet taste circuits, with the latter coming into play only once the former is activated. The finding that Gr and Ir genes are expressed in the LSO and VCSO but only Ir genes in the DCSO is also striking and raises the possibility that the DCSO, which is present at the most internal location relative to the others, may serve a unique role in controlling ingestion (Chen, 2017).

    Based on its molecular signature, the V5 neuron was identified as an L-canavanine-sensing neuron in the pharynx. As predicted, feeding avoidance of L-canavanine is dependent on V5. It was thus unexpected that capsaicin-mediated activation of bitter pharyngeal GRNs, which include V5, did not induce strong feeding avoidance either in the absence or presence of sugar. Because the strength and pattern of pharyngeal neuronal activation by bitter tastants or capsaicin is unknown, it is possible that capsaicin response may be weaker than that of canonical bitter tastants. Alternatively, sweet and bitter inputs from internal and external neurons may be summed differently. It is known that activation of one or few external sweet neurons can lead to proboscis extension, for example, but a larger number of bitter neurons may need to be activated for avoidance (Chen, 2017).

    The afferents of pharyngeal GRNs target regions of the SEZ that are distinct from areas in which afferents from labellar and tarsal GRNs terminate. Interestingly, pharyngeal GRN projections between molecularly different classes of neurons, as well as between GRNs of the LSO and VCSO, are also distinct. Projections of sugar-sensing GRNs were found in separate ipsilateral regions, whereas those of neurons predicted to detect aversive tastants were found at the midline, suggesting the presence of contralateral termini. These observations may inform future functional studies of pharyngeal GRNs. L7-6 neurons, for example, would be predicted to sense aversive compounds based on the presence of their termini at the midline. Analysis of pharyngeal GRN projections also suggests distinct connectivity to higher order neuronal circuits. With the molecular tools described here, future investigations of pharyngeal GRNs and pharyngeal taste circuits will provide insight into how internal taste is integrated with external taste to control various aspects of feeding behavior (Chen, 2017).

    In vivo assembly and trafficking of olfactory ionotropic receptors

    Ionotropic receptors (IRs) are a large, divergent subfamily of ionotropic glutamate receptors (iGluRs) that are expressed in diverse peripheral sensory neurons and function in olfaction, taste, hygrosensation and thermosensation. Analogous to the cell biological properties of their synaptic iGluR ancestors, IRs are thought to form heteromeric complexes that localise to the ciliated dendrites of sensory neurons. IR complexes are composed of selectively expressed 'tuning' receptors and one of two broadly expressed co-receptors (IR8a or IR25a). This study identified a sequence in the co-receptor LBD, the 'co-receptor extra loop' (CREL), which is conserved across IR8a and IR25a orthologues but not present in either tuning IRs or iGluRs. The CREL contains a single predicted N-glycosylation site, which bears a sugar modification in recombinantly expressed IR8a. Using the Drosophila olfactory system as an in vivo model, a transgenically encoded IR8a mutant was found in which the CREL that cannot be N-glycosylated is impaired in localisation to cilia in some, though not all, populations of sensory neurons expressing different tuning IRs. This defect can be complemented by the presence of endogenous wild-type IR8a, indicating that IR complexes contain at least two IR8a subunits and that this post-translational modification is dispensable for protein folding or complex assembly. Analysis of the subcellular distribution of the mutant protein suggests that its absence from sensory cilia is due to a failure in exit from the endoplasmic reticulum. CREL N-glycosylation site is likely to be located on the external face of a heterotetrameric IR complex. These data reveal an important role for the IR co-receptor LBD in control of intracellular transport, provide novel insights into the stoichiometry and assembly of IR complexes and uncover an unexpected heterogeneity in the trafficking regulation of this sensory receptor family (Abuin, 2019).

    Mosquito heat seeking is driven by an ancestral cooling receptor

    Mosquitoes transmit pathogens that kill >700,000 people annually. These insects use body heat to locate and feed on warm-blooded hosts, but the molecular basis of such behavior is unknown. This study identified Ionotropic receptor IR21a, a receptor conserved throughout insects, as a key mediator of heat seeking in the malaria vector Anopheles gambiae. Although Ir21a mediates heat avoidance in Drosophila, it drives heat seeking and heat-stimulated blood feeding in Anopheles. At a cellular level, Ir21a is essential for the detection of cooling, suggesting that during evolution mosquito heat seeking relied on cooling-mediated repulsion. These data indicate that the evolution of blood feeding in Anopheles involves repurposing an ancestral thermoreceptor from non-blood-feeding Diptera (Greppi, 2020).

    Insect-borne diseases kill over 700,000 people annually, with >400,000 deaths resulting from malaria, a disease caused by protozoan Plasmodium spp. parasites that are transmitted by blood-feeding anopheline mosquitoes. Host seeking by mosquitoes and other pathogen-spreading insects relies on the detection of host-associated cues, including carbon dioxide (CO2), odors, and body heat. Receptors for CO2 and host odors have been characterized in mosquitoes, but receptors that promote heat seeking and heat-induced blood feeding have remained elusive. As vector mosquitoes are descendants of non-blood-feeding ancestors, it remains unknown whether the emergence of heat seeking and warming-induced blood feeding in mosquitoes involved the generation of novel thermoreceptors or the repurposing of existing thermoreceptors (Greppi, 2020).

    To date, mosquito orthologs of two Drosophila warmth receptors, TRPA1 and GR28b, have been tested as candidate heat-seeking receptors in the yellow fever mosquito Aedes aegypti. However, neither is required for heat seeking in Aedes. Rather, TRPA1 promotes heat avoidance in both Aedes and Drosophila. Although efforts have focused on warmth receptors, insects also possess cooling-activated receptors, which should be equally capable of supporting heat seeking through cooling-mediated repulsion. In Drosophila, cooling detection is mediated by IR21a, IR25a, and IR93a, three members of the ionotropic receptor (IR) family, a group of invertebrate-specific sensory receptors related to ionotropic glutamate receptors. IR21a is specifically required for cooling detection in the fly and can confer cooling sensitivity when ectopically expressed, while IR25a and IR93a are more broadly acting co-receptors that support cooling detection and other IR-dependent sensory modalities. At the behavioral level in Drosophila, IR21a, IR25a, and IR93a help the fly achieve optimal body temperatures by supporting avoidance of excessively cool and warm temperatures. Beyond Drosophila, IR21a, Ir25a, and IR93a are each widely conserved from Diptera (flies and mosquitoes) to Isoptera (termites), raising the possibility that their thermosensory functions may also be conserved. Using Anopheles gambiae, a major vector of malaria in sub-Saharan Africa, tests were performed to see whether IR21a is required for detecting cooling in mosquitoes and subsequently whether it can drive heat attraction and heat-stimulated blood feeding (Greppi, 2020).

    Two mutant alleles of A. gambiae Ir21a were generated using CRISPR-Cas9. Ir21a+7bp contains a 7-base pair (bp) insertion, introducing a frameshift positioned to disrupt IR21a's translation within the second of IR21a's three transmembrane domains; this lesion is predicted to generate a nonfunctional receptor. In Ir21aEYFP, a disruption cassette containing an enhanced yellow fluorescent protein (EYFP) marker, was inserted into IR21a's fourth exon, a lesion also predicted to create a nonfunctional receptor. Both mutants lacked detectable IR21a protein expression, consistent with their acting as Ir21a null mutations (Greppi, 2020).

    Genome-wide analyses of A. gambiae sensory tissues suggest that Ir21a RNA is specifically expressed in the antenna. To visualize IR21a protein expression and localization with cellular resolution, anti-IR21a antisera were generated. The antenna's most distal segment (flagellomere 13) contains three coeloconic sensilla that house sensitive thermoreceptors. In females, IR21a expression was detected in three sensory neurons in flagellomere 13, one innervating each of the coeloconic sensilla. Consistent with a role in thermosensory transduction, IR21a strongly localized to the sensory ending of each of these neurons. IR21a immunostaining was absent in Ir21a mutants, confirming antisera specificity. The male antennal tip also contains thermoreceptors, and IR21a expression was detected in sensory endings there as well (Greppi, 2020).

    Extracellular recordings were performed from the IR21a-positive coeloconic sensilla at the antennal tip. In wild-type mosquitoes, the activity of the Cooling Cell, a thermosensory neuron stimulated by cooling and inhibited by warming, was readily detected. On rare occasions of exceptional signal to noise, a smaller-amplitude spike was also detected, corresponding to a Heating Cell activated by warming and inhibited by cooling. Cooling Cell responses were highly thermosensitive: an ~0.5°C drop from ~30°C increased spiking by ~40%, and an ~0.5°C drop from ~37°C increased spiking by ~80%. Response adaptation initiated rapidly, followed by a slower decline to baseline. Heating inhibited spiking, in a similarly transient manner. Importantly, Cooling Cells remained highly active at warm temperatures (e.g., 37°C) and were neither more active nor more thermosensitive at colder temperatures. Thus, while often referred to as Cold Cells in the classical literature, cooling and not cold is their activating stimulus. In addition, while often referred to as 'phasic-tonic' receptors, their responses to temperature shifts adapted fully, albeit slowly, requiring sustained observation (>20 s) to fully appreciate. Therefore, although they fire robustly at constant temperature, Cooling Cells are phasic thermoreceptors. Their rate of baseline firing was relatively temperature insensitive [with a fold change upon 10°C increase (Q10) of ~1.6, reflecting a slight increase with warmth], enabling the cell to respond to small temperature fluctuations over a wide range of absolute temperatures. Taken together, these data indicate that Cooling Cells are phasic thermoreceptors that respond to temperature change rather than absolute temperature and that they are capable of responding to abrupt changes in temperature over the wide range of absolute temperatures relevant for host seeking (Greppi, 2020).

    Cooling Cell thermosensitivity was eliminated in A. gambiae Ir21a mutants. The large-amplitude spike detected in Ir21a mutants was neither activated by cooling nor inhibited by warming. Rather, its activity increased slightly upon warming, with a Q10 under 2, which is average for a biological process. Thus, Ir21a is essential for thermosensing by Cooling Cells in the mosquito, demonstrating that A. gambiae IR21a's molecular function is conserved with its Drosophila ortholog (Greppi, 2020).

    In female mosquitoes, heat seeking is part of a multimodal host-seeking program activated upon exposure to CO2, with body heat serving as an important cue close to the host (within ~10 to 15 cm). To assess heat seeking, female mosquitoes were provided a 20-s puff of 4% CO2 and exposed to two targets, a control target at ambient temperature (~26°C) and a heated target at ~37°C. Wild-type mosquitoes exhibited robust heat seeking, with 43 ± 3% of CO2-activated mosquitoes landing on the 37°C target (average ± SEM). The loss of Ir21a greatly reduced this behavior, with only 15 ± 4% of Ir21aEYFP mutants and 14 ± 4% of Ir21a+7bp mutants landing on the 37°C target. In all cases, the control target was largely ignored, confirming temperature's importance in the assay. While heat seeking was greatly reduced in Ir21a mutants, it was not entirely eliminated. This residual activity likely reflects signaling from other as-yet-uncharacterized thermosensors. However, the strong reduction of heat seeking in Ir21a mutants identifies this receptor as a major driver of mosquito attraction to warmth (Greppi, 2020).

    To test the specificity of the Ir21a mutant behavioral deficit for heat seeking, their ability to perform an activation-dependent behavior less reliant on thermosensation was tested. While body heat is a powerful short-range cue, a multimodal combination of longer-range chemosensory and visual cues mediates initial approach, suggesting that such behavior should be largely unaffected by a specific thermosensory deficit. To assess approach behavior, mosquitoes were activated by five human breaths and presented a human hand, positioned on a platform to prevent physical contact with the mosquitoes but otherwise providing host-associated cues. Hand approach was strong in the wild type, with 57 ± 2% of mosquitoes landing on the surface beneath the hand. Hand-associated cues were critical, as hand withdrawal prompted rapid dispersal. Ir21aEYFP mutants remained robustly responsive, exhibiting maximum levels of approach (55 ± 2%) similar to wild-type levels. Careful examination of response kinetics revealed that, compared to wild type, their initial accumulation rate decreased by ~25%, and their dispersal rate upon hand removal increased by ~40%, potentially reflecting subtle contributions of the warmth gradient created by the presence of the hand to the avidity of host approach. Similar results were obtained for Ir21a+7bp. Overall, these data demonstrate that the loss of Ir21a does not broadly disrupt orientation toward sensory cues and argue against the presence of global behavioral deficits in the mutants. These results are consistent with prior work indicating that the disruption of single sensory modalities is insufficient to completely eliminate host approach (Greppi, 2020).

    Heat strongly stimulates mosquito blood feeding. To assess the effect of warmth on blood feeding, artificial membrane feeders were used to present human blood meals at different temperatures. One meal was held at room temperature (RT, ~23°C) and the other warmed to ~31°C, a temperature similar to the surface temperatures (~29°C to 33°C) of human torsos and extremities in a 23°C to 24°C room (26). In each trial, green food coloring was added to one meal so that the consumption of warm versus RT food could be distinguished. Each class of trial was assessed independently, and each yielded similar results. In wild type, elevated temperature robustly promoted feeding, as reflected in the greater percentages of mosquitoes consuming warm versus RT meals. For both Ir21aEYFP and Ir21+7bp mosquitoes, this preference for warm blood was significantly reduced. Thus, similar to heat seeking, warmth-promoted blood feeding was reduced in the absence of Ir21a (Greppi, 2020).

    These data identify IR21a as a key mediator of heat-seeking behavior in A. gambiae mosquitoes. Although a cooling-activated receptor driving heat seeking is superficially counterintuitive, repulsion from cooling would yield a similar behavioral outcome as attraction to warming. Furthermore, Cooling Cells are bidirectional and are not only activated by cooling but also inhibited by heating; each phase of the response could modulate downstream circuits to control behavior. Ultimately, the detection of temperature change by the Cooling Cells is critical, but is just one step in heat seeking, a response that involves the processing of multiple sensory inputs to generate a coherent response. Identification of a key molecular receptor for heat seeking provides a starting point for a deeper understanding of this complex behavior and its contribution to the multimodal process that culminates in mosquito blood feeding (Greppi, 2020).

    The conservation of IR21a's thermosensory function between Drosophila and Anopheles, whose last common ancestor lived ~250 million years ago, suggests thermosensing is an ancestral function of IR21a. As this ancestor predates the evolution of blood feeding, its IR21a would have regulated other behaviors, such as thermoregulation. Thus, the current findings indicate that the evolution of blood feeding in A. gambiae mosquitoes involved repurposing an ancestral thermoreceptor to facilitate host seeking. Alterations in the connectivity or function of downstream circuits would likely have been crucial in this behavioral shift. Given the conservation of IR21a as well as IR25a and IR93a (IR21a's coreceptors in Drosophila) across insects, these IRs may be used in heat seeking not only by other mosquitoes but also across a range of hematophagous insect taxa (Greppi, 2020).

    In addition to Ir21a's role in heat seeking, IR21a expression in the antennae of A. gambiae males suggests it continues to serve additional thermosensory functions. It will be interesting to assess whether IR21a mediates thermal preference in male and possibly female mosquitoes and the extent to which thermal preference and heat-seeking circuits overlap. Not all thermoreceptors appear to have been repurposed, as the TRPA1 warmth receptor has a similar role in flies and mosquitoes, mediating heat avoidance in both. At a practical level, exploiting and manipulating the sensory systems of vector insects offer an avenue for disease control strategies (Greppi, 2020).

    Olfactory receptor-dependent receptor repression in Drosophila

    In olfactory systems across phyla, most sensory neurons express a single olfactory receptor gene selected from a large genomic repertoire. This study describes previously unknown receptor gene-dependent mechanisms that ensure singular expression of receptors encoded by a tandem gene array [Ionotropic receptor 75c (Ir75c), Ir75b, and Ir75a, organized 5' to 3'] in Drosophila melanogaster. Transcription from upstream genes in the cluster runs through the coding region of downstream loci and inhibits their expression in cis, most likely via transcriptional interference. Moreover, Ir75c blocks accumulation of other receptor proteins in trans through a protein-dependent, posttranscriptional mechanism. These repression mechanisms operate in endogenous neurons, in conjunction with cell type-specific gene regulatory networks, to ensure unique receptor expression. These data provide evidence for inter-olfactory receptor regulation in invertebrates and highlight unprecedented, but potentially widespread, mechanisms for ensuring exclusive expression of chemosensory receptors, and other protein families, encoded by tandemly arranged genes (Mika, 2021).

    Ir56d-dependent fatty acid responses in Drosophila uncovers taste discrimination between different classes of fatty acids

    Chemosensory systems are critical for evaluating the caloric value and potential toxicity of food prior to ingestion. While animals can discriminate between 1000's of odors, much less is known about the discriminative capabilities of taste systems. Fats and sugars represent calorically potent and innately attractive food sources that contribute to hedonic feeding. Despite the differences in nutritional value between fats and sugars, the ability of the taste system to discriminate between different rewarding tastants is thought to be limited. In Drosophila, sweet taste neurons expressing the Ionotropic Receptor 56d (IR56d) are required for reflexive behavioral responses to the medium-chain fatty acid, hexanoic acid. Further, it was found that flies can discriminate between a fatty acid and a sugar in aversive memory assays, establishing a foundation to investigate the capacity of the Drosophila gustatory system to differentiate between various appetitive tastants. This study tested whether flies can discriminate between different classes of fatty acids using an aversive memory assay. The results indicate that flies are able to discriminate medium-chain fatty acids from both short- and long-chain fatty acids, but not from other medium-chain fatty acids. While IR56d neurons are broadly responsive to short-, medium-, and long-chain fatty acids, genetic deletion of IR56d selectively disrupts response to medium-chain fatty acids. Further, IR56d+GR64f+ neurons are necessary for proboscis extension response (PER) to medium-chain fatty acids, but both IR56d and GR64f neurons are dispensable for PER to short- and long-chain fatty acids, indicating the involvement of one or more other classes of neurons. Together, these findings reveal that IR56d is selectively required for medium-chain fatty acid taste, and discrimination of fatty acids occurs through differential receptor activation in shared populations of neurons. This study uncovers a capacity for the taste system to encode tastant identity within a taste category (Brown, 2021).

    Previous studies identified IR56d as a receptor for hexanoic acid and carbonation. The current findings suggest that IR56d is selectively involved in responses to medium-chain fatty acids, including 6C, 7C, and 8C fatty acids, and dispensable for responses to shorter and longer-chain fatty acids. Such receptor specificity for different classes of fatty acids based on chain length has not been documented in other systems. In flies, both sugars and fatty acids evoke activity in neurons that co-express the receptors GR64f and IR56d. The finding that short- and long-chain fatty acids also evoke activity in IR56d-expressing neurons posits that additional fatty acid receptors are present in these neurons. Previously, it has been found that deletion of Phospholipase C (PLC) signaling selectively impairs fatty acid response while leaving sweet taste intact, raising the possibility that activation of distinct intracellular signaling pathways could serve as a mechanism for discrimination between sucrose and fatty acid, while another suggests TRPA1 and GR64e are targets of PLC and are generally required for fatty acid sensing. Determining whether or not short- and long-chain fatty acids also signal through PLC may provide insight into whether signaling mechanisms are shared between different fatty acid receptors expressed in IR56d-expressing neurons (Brown, 2021).

    An aversive taste memory assay confirmed previous findings that flies can discriminate between sugars and fatty acids, and led to the surprising observation that flies can distinguish between different classes of fatty acids, even though the baseline responsiveness to short-, medium-, and long-chain fatty acids was similar in innate preference assays. Fatty acids are a natural by-product of yeast fermentation, and their abundance in peaches, for example, declines after ripening . Further, fatty acids have antifungal activity, which scales with chain length (i.e., the greater the chain length, the greater the antifungal efficiency). Thus, the ability to discriminate between different classes of fatty acids is likely to be important in determining the stage of fruit ripeness, degree of fermentation, and the general palatability of a potential food source/oviposition site (Brown, 2021).

    The finding that flies can distinguish between different classes of fatty acids contrasts with the results of a previous study that applied a similar assay and found that flies were unable to discriminate between different sugars or bitter compounds. One possibility is that this is due to differences in fatty acid detection, which is dependent on IRs, and sweet and bitter tastant detection, which relies on GRs.The findings of this study highlight the complexity of taste discrimination, which extends beyond simple proboscis extension response (PER) as a readout for taste. For example, all types of fatty acids tested increase GR64f neural responsiveness; however, only GR64f neurons are required for PER to medium-chain fatty acids, thereby raising the possibility that short-, medium-, and long-chain fatty acid taste discrimination occurs through different neural channels. These findings stress the need to define the fatty acid receptors and neural circuits that govern responses to short- and long-chain fatty acid taste. Furthermore, the ability of the Drosophila taste system to discriminate suggests it may be more like the olfactory system than previously appreciated. Flies are able to distinguish between many different odorants, likely due to the complexity of olfactory coding at the level of the receptor as well as in the antennal lobe. However, flies can also discriminate between odorants sensed by a single olfactory receptor, suggesting that temporal coding also plays a role in discrimination. It is possible that similar mechanisms underlie discrimination between different classes of fatty acid tastants (Brown, 2021).

    The Drosophila genome encodes 66 IRs, which comprise a recently identified family of receptors implicated in taste, olfaction, and temperature sensation. IRs are involved in the detection of many different tastants and function as heteromers that confer sensory specificity. While IR56d expression is restricted to a subset of sweet taste neurons, it likely functions in a complex with IR25a and IR76b, all three of which are required fatty acid taste. Other tastants whose responses are mediated by IRs are also likely to be detected by IR complexes. For example, roles for IR25a, IR62a, and IR76b have been described for Ca2+ taste. The broad degree of co-expression of IRs in the brain and periphery can provide candidates for those involved in detecting short- and long-chain fatty acids (Brown, 2021).

    The identification of taste discrimination between different classes of fatty acids provides the opportunity to identify how different tastants are encoded in the brain and how these circuits are modified with experience. Although projections of primary taste neurons to the SEZ have been mapped in some detail, little is known about connectivity with downstream neurons and whether sensory neurons responsive to different appetitive tastants can activate different downstream circuits. Recent studies have identified a number of interneurons that modify feeding, including IN1, a cholinergic interneuron responsive to sucrose, E564 neurons that inhibit feeding, and Fdg neurons that are required for sucrose-induced feeding. Future work can investigate whether these and other downstream neurons are shared for fatty acid taste. Previous studies have found that incoming sensory information is selectively modulated within the SEZ in accordance with feeding state. It will be interesting to determine if similar modulation promotes differentiation of sugars and fatty acids, which are sensed by shared GRNs. Large-scale brain imaging has now been applied in flies to measure responsiveness to different tastants, and a comparison of brain activity patterns elicited by different classes of fatty acids may provide insight into differences in their sensory input and processing (Brown, 2021).

    All experiments in this study tested flies under starved conditions, which is necessary to elicit the PER that is used as a behavioral readout of taste acceptance. However, responses to many tastants and odorants are altered in accordance with feeding state. For example, the taste of acetic acid is aversive to fed flies but attractive to starved flies, revealing a hunger-dependent switch. Similarly, hexanoic acid evokes activity in both sweet and bitter-sensing taste neurons, and the activity of bitter taste neurons is dependent on different receptors from those involved in the appetitive response. Further, hunger enhances activity in sweet taste circuits and suppresses that of bitter taste circuits, providing a mechanism for complex state-dependent modulation of taste response that increase activity of both appetitive and deterrent neurons (Brown, 2021).

    The neural circuits that are required for aversive taste memory have been well defined for sugar, yet little is known about how fatty acid taste is conditioned. The pairing of sugar with bitter quinine results in aversive memory to sugar. Optogenetic activation of bitter taste neurons that are activated by quinine, in combination with the presentation of sugar, is sufficient to induce sugar avoidance. Further studies have elucidated that aversive taste memories are dependent on mushroom body neurons that form the gamma and alpha lobes, the PPL1 cluster of dopamine neurons, and alpha lobe output neurons, revealing a circuit regulating taste memory that differs from that controlling appetitive olfactory memory. It will be interesting to determine whether shared components regulate conditioning to fatty acids or whether distinct mushroom body circuits regulate sweet and fatty acid taste conditioning. Further, examination of the central brain circuits that regulate aversive taste conditioning to different classes of fatty acids will provide insight into how taste discrimination is processed within the brain (Brown, 2021).

    Mechanisms of Carboxylic Acid Attraction in Drosophila melanogaster

    Sour is one of the fundamental taste modalities that enable taste perception in animals. Chemoreceptors embedded in taste organs are pivotal to discriminate between different chemicals to ensure survival. Animals generally prefer slightly acidic food and avoid highly acidic alternatives. 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 (Shrestha, 2021).

    Ir76b is a Co-receptor for Amine Responses in Drosophila Olfactory Neurons

    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 (Vulpe, 2021).


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    Zygotically transcribed genes

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