In this thesis I describe the identification of olfactory circuits underlying avoidance of Leptop... more In this thesis I describe the identification of olfactory circuits underlying avoidance of Leptopilina wasps in Drosophila adults and larvae. The larval olfactory sensory neuron (OSN) expresses the olfactory receptor Or49a and is tuned to the waps’ sex pheromone iridomyrmecin exclusively, while the adult OSN expressing both Or49a and Or85f detects in addition to iridomyrmecin, the wasp odors actinidine and nepetalactol. The OSN type expressing Or49a and Or85f is both necessary and sufficient to govern the parasitoid avoidance behavior, and is conserved across several Drosophila species. I also contributed to the demonstration that Drosophila adults prefer Citrus fruits as oviposition substrates. This preference is due to the high content of terpenes in the flavedo. Flies detect these terpenes via only a single class of OSNs, which in this case express the odorant receptor Or19a. This preference has likely been driven by the need to avoid parasitism from endoparasitoid wasps, since t...
Here, we show that several drosophilid flies and their larvae avoid the odors of one genus of par... more Here, we show that several drosophilid flies and their larvae avoid the odors of one genus of parasitoids. This avoidance is mediated by highly specific OSNs, which in D. melanogaster larva express only the olfactory receptor Or49a and respond to the wasps' sex pheromone iridomyrmecin, while in adult express additionally Or85f and respond to the wasps' semiochemicals iridomyrmecin, actinidine and nepetalactol. This information is transferred via projection neurons to a specific part in the lateral horn known to be involved in mediating avoidance. In both D. melanogaster adult and larva these neuron are necessary and sufficient to govern odor-driven parasitoid avoidance behavior. Built on an idea conceived by all authors. Designed experiments: behavioral experiments BSH, MK, HKMD, SAME (70%) Performed experiments: Behavioral experiments (100%) Data analyses: Behavioral experiments MK, SAME (80%).
Parasitoid wasps inflict widespread death upon the insect world. Hundreds of thousands of parasit... more Parasitoid wasps inflict widespread death upon the insect world. Hundreds of thousands of parasitoid wasp species kill a vast range of insect species. Insects have evolved defensive responses to the threat of wasps, some cellular and some behavioral. Here we find an unexpected response of adult Drosophila to the presence of certain parasitoid wasps: accelerated mating behavior. Flies exposed to certain wasp species begin mating more quickly. The effect is mediated via changes in the behavior of the female fly and depends on visual perception. The sight of wasps induces the dramatic upregulation in the fly nervous system of a gene that encodes a 41-amino acid micropeptide. Mutational analysis reveals that the gene is essential to the behavioral response of the fly. Our work provides a foundation for further exploration of how the activation of visual circuits by the sight of a wasp alters both sexual behavior and gene expression.
Tsetse flies transmit trypanosomes—parasites that cause devastating diseases in humans and livest... more Tsetse flies transmit trypanosomes—parasites that cause devastating diseases in humans and livestock—across much of sub-Saharan Africa. Chemical communication through volatile pheromones is common among insects; however, it remains unknown if and how such chemical communication occurs in tsetse flies. We identified methyl palmitoleate (MPO), methyl oleate, and methyl palmitate as compounds that are produced by the tsetse fly Glossina morsitans and elicit strong behavioral responses. MPO evoked a behavioral response in male—but not virgin female— G. morsitans . G. morsitans males mounted females of another species, Glossina fuscipes , when they were treated with MPO. We further identified a subpopulation of olfactory neurons in G. morsitans that increase their firing rate in response to MPO and showed that infecting flies with African trypanosomes alters the flies’ chemical profile and mating behavior. The identification of volatile attractants in tsetse flies may be useful for reduc...
<p>(A) Scanning electron micrograph of a <i>G</i>. <i>morsitans</i>... more <p>(A) Scanning electron micrograph of a <i>G</i>. <i>morsitans</i> head with arrows indicating the openings of the sensory pit and sacculus. (B) Transmitted light image of an antennal cross section (coronal plane) showing the sensory pit and sacculus that open to the medial and lateral sides of the antenna, respectively. (C) Micrograph of a sectioned sensory pit with its opening to the antennal surface at the upper left. (D) Micrograph of the sensory pit, showing a top-down view of the olfactory sensilla that line the pit. (E, E’) The sensory pit is lined with basiconic Type II sensilla. The arrow in E indicates the sensillum shown in E’. (F) Dorsal chamber of the sacculus (medial is left, lateral is right) that is lined with basiconic Type III sensilla (G, G’). The arrow in G indicates the sensillum shown in G’. (H) Ventral chamber of the sacculus (medial is at left, lateral is at right) that is lined with coeloconic (I, J) and no-groove coeloconic sensilla (K). The asterisk in panel J marks an axial view of a coeloconic sensillum that had its tip cut off during cryosectioning, fortuitously revealing its inner structure. Scale bars = 200 μm for A; 5 μm for C, D, F and H; 1 μm for E, G; and 0.25 μm for E’, G’, I, J, and K.</p
<p>(A) Odorant response profile of GmmOr35 expressed in homozygous <i>Or22ab</i>... more <p>(A) Odorant response profile of GmmOr35 expressed in homozygous <i>Or22ab</i><sup><i>GAL4</i></sup> ab3A ORNs. Neuronal responses are reported in spikes/second +/- S.E.M., to individual odorants diluted 10<sup>−2</sup>. (B,C) Sample traces from ab3A empty neurons expressing the <i>GmmOr35</i> transgene in response to the paraffin oil diluent (B) and 1-hexen-3-ol (C), each presented for 0.5 seconds (black bars). (D) Dose response curve for GmmOr35 and 1-hexen-3-ol. (E) Response profile of GmmOr19. In panels A, D, E, spontaneous firing frequencies have been subtracted from all responses; responses to the paraffin oil diluent have been subtracted from the responses to all odorants. n≥5 for all stimuli tested.</p
<p>(<b>A</b>) Reconstruction of two DA2 PNs. (<b>B</b>) Reconstruct... more <p>(<b>A</b>) Reconstruction of two DA2 PNs. (<b>B</b>) Reconstruction of two DL4 PNs. (<b>C</b>) comparison of DA2 and DL4 domains after registration of datasets into a common reference space. DA2 and DL4 PNs overlap in the base of the MB and ventroposterior LH. a: anterior, d: dorsal, l: lateral, p: posterior v: ventral.</p
<p>For all images dorsal is up and medial is to left. (A) <i>GmmOrco</i>. (B) &... more <p>For all images dorsal is up and medial is to left. (A) <i>GmmOrco</i>. (B) <i>GmmOr9</i>. (C) <i>GmmOr6</i>. (D) <i>GmmOr40</i>. (E) <i>GmmOr35</i>. Some background fluorescence is visible that does not emanate from cell bodies; see sense strand control in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1008005#pgen.1008005.s001" target="_blank">S1H Fig</a>. (F) <i>GmmOr38</i>. (G) <i>GmmOr19</i>. (H) <i>GmmOr44</i>. (I) <i>GmmOr29</i>. We note that in several of these images there is background fluorescence that emanates from the cuticle, which is thicker in the antenna of <i>G</i>. <i>morsitans</i> than in <i>D</i>. <i>melanogaster</i>. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1008005#pgen.1008005.s001" target="_blank">S1 Fig</a> for sense strand controls.</p
<p>(A, B) <i>In situ</i> hybridization to <i>GmmObp76a</i> in (A) 4... more <p>(A, B) <i>In situ</i> hybridization to <i>GmmObp76a</i> in (A) 40 μm and (B) 10 μm antennal cross sections. Arrow in panel B marks a trichoid sensillum in the same plane as a cell body marked by <i>GmmObp76a</i>. (C, D) <i>GmmObp6</i> in (C) 40 μm and (D) 10 μm antennal cross sections. Arrow in D marks a basiconic sensillum in the same plane as a cell body marked by <i>GmmObp6</i>. (E, F) <i>GmmObp84a</i> in (E) 40 μm and (F) 10 μm antennal cross sections. Arrow in F marks a coeloconic sensillum in the ventral chamber of the sacculus in the same plane as a cell body marked by <i>GmmObp84a</i>. (G, H) <i>GmmObp59a</i> (red) in (G) 40 μm and (H) 10 μm antennal cross sections. Arrow in H marks a coeloconic sensillum in the same plane as a cell body marked by <i>GmmObp84a</i>.</p
<p>(<b>A</b>) Example spike traces of GC-coupled SSR with all <i>D</i&... more <p>(<b>A</b>) Example spike traces of GC-coupled SSR with all <i>D</i>. <i>melanogaster</i> OSN types and the headspace of <i>L</i>. <i>boulardi</i> (note that the amount of odors within headspace is too low to be detected and analyzed by GC, but is still detected by ab10B). FID, flame ionization detector. (<b>B</b>) GC-coupled SSR with the ab10B neuron and the wash of <i>L</i>. <i>boulardi</i> (1st panel), as well as the identified active compounds (2nd–4th panel). (<b>C</b>) SSR dose-response curves of the ab10B neuron tested with active compounds. (<b>D</b>) GC-coupled SSR with mutant ab3A neuron ectopically expressing either Or49a or Or85f. Blue, green, and red lines indicate active compounds. (<b>E</b>) Tuning breadths of Or49a and Or85f. 232 odorants are displayed along the <i>x</i>-axis according to strengths of responses they elicit from each receptor. Odorants eliciting strongest responses are placed near the center of distribution. Negative values indicate inhibitory responses. For a list of compounds, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002318#pbio.1002318.s005" target="_blank">S4 Fig</a>; for raw data see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002318#pbio.1002318.s001" target="_blank">S1 Data</a>. (<b>F</b>) Identification of glomeruli activated by parasitoid odors (-)-iridomyrmecin, (<i>R</i>)-actinidine, and nepetalactol (a mixture of 1S4aR7R7aS, 1R4aS7S7aS-nepetalactol and their enantiomers). 1st to 3rd columns, false color-coded images showing odorant-induced calcium-dependent fluorescence changes in OSNs expressing Or49a or PNs labeled by GH-146-Gal4 at the antennal lobe (AL) level. Flies express UAS-GCaMP3.0 under control of either Or49a-Gal4, or the GH146-Gal4 driver line. (<b>G</b>) GC-coupled extracellular recordings from larval dorsal organ and wash of <i>L</i>. <i>boulardi</i>. (for more GC-SSR traces of wildtype ab10B neurons and mutant ab3A neurons expressing Or49a or Or85f see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002318#pbio.1002318.s004" target="_blank">S3 Fig</a>)</p
<p>(A) Phylogenetic tree showing the evolutionary relationship among four families within t... more <p>(A) Phylogenetic tree showing the evolutionary relationship among four families within the order Diptera: Culicidae, Drosophilidae, Glossinidae, and Muscidae. Estimated divergence times are from Wiegmann et al., 2011 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1008005#pgen.1008005.ref004" target="_blank">4</a>]. (B) Photograph of <i>D</i>. <i>melanogaster</i> and <i>G</i>. <i>m</i>. <i>morsitans</i> courtesy of Dr. Geoffrey Attardo (adapted from Sun et al. 2018 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1008005#pgen.1008005.ref027" target="_blank">27</a>]). (C) Scanning electron micrograph of the antenna of <i>G</i>. <i>morsitans</i>. Arrows indicate the third antennal segment (Ant.), arista, and sensory pit (SP). Micrographs of trichoid (D, D’), basiconic (E, E’), coeloconic (F), and intermediate (G, G’) olfactory sensilla from <i>G</i>. <i>morsitans</i>. Arrows in D, E, and G indicate the sensilla that are shown at higher magnifications in the images to the right. Scale bars = 100 μm for C; 1 μm for D, E, G; 0.5 μm for D’, E’, G’; and 0.25 μm for F.</p
<p>(A) <i>In situ</i> hybridization to <i>GmmOrco</i> (red). (B) Im... more <p>(A) <i>In situ</i> hybridization to <i>GmmOrco</i> (red). (B) Immunostaining of pan-neuronal marker Elav (green). (C) Merged image of <i>GmmOrco</i> and anti-E lav. (D) Magnification of (C).</p
<p>(A-D) <i>In situ</i> hybridization to <i>GmmOr6</i> (red) combin... more <p>(A-D) <i>In situ</i> hybridization to <i>GmmOr6</i> (red) combined with anti-Elav immunostaining (green). (A) <i>GmmOr6</i>, (B) anti-Elav, and (C) merged image of the whole antenna. (D) Magnified merged image of the sensory pit without DIC. The circular structure in the center of the image that is uniformly green is autofluorescent cuticle of the pit. Likewise, the green fluorescence in the upper right corner is cuticular autofluorescence from the sacculus. (E-H) <i>In situ</i> hybridization to <i>GmmOr9</i> (red) combined with anti-Elav immunostaining (green). (E) <i>GmmOr9</i>, (F) anti-Elav, and (G) merged image of the whole antenna. (H) Magnified merged image of the sensory pit without DIC. (I-L) Double <i>in situ</i> hybridization to <i>GmmOr6</i> and <i>GmmOr9</i> (both red) combined with anti-Elav immunostaining (green). (I) <i>GmmOr6</i> and <i>GmmOr9</i>, (J) anti-Elav, and (K) merged image of the whole antenna. (L) Magnified merged image of the sensory pit without DIC.</p
<p>(<b>A</b>) Larval choice assay and preference indices when larvae were expos... more <p>(<b>A</b>) Larval choice assay and preference indices when larvae were exposed to the wash of <i>L</i>. <i>boulardi</i>. (<b>B</b>) Different choice assays (T-maze, Trap assay, Oviposition assay) for adult flies and resulting preference indices when exposed to the wash of <i>L</i>. <i>boulardi</i>. PI = (number of larvae, flies, or eggs in odor side − number in control side) / total number. Bar plots indicate minimum and maximum values (whiskers), the upper and lower quartiles (boxes) and the median values (bold black line). Deviation of the indices against zero was tested with Wilcoxon rank sum test.</p
<p>(<b>A</b>) Preference indices of ovipositing wildtype flies, flies expressin... more <p>(<b>A</b>) Preference indices of ovipositing wildtype flies, flies expressing <i>Shibire</i><sup><i>ts</i></sup> in ab10B neuron, and corresponding parental lines at restrictive (30°C) and permissive (23°C) temperature when tested with wash of <i>L</i>. <i>boulardi</i>. (<b>B</b>) Preference indices of the same fly lines when tested in the larval assay. Attraction to ethyl butyrate (grey bars) depict that loss of odor-guided behavior in larvae expressing <i>Shibire</i><sup><i>ts</i></sup> in ab10B neuron is odorant specific. (<b>C</b>) Light preference of ovipositing wildtype flies, flies expressing channelrhodopsin in ab10B neuron, and corresponding parental lines. (<b>D</b>) Light preferences of the same fly lines when tested in the larval assay. (<b>A–D</b>) Bar plots indicate minimum and maximum values (whiskers), the upper and lower quartiles (boxes), and the median values (bold black line). Groups were compared by the Kruskal Wallis test with a Dunn’s multiple comparison for selected pairs. For calculation of preference indices, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002318#pbio.1002318.g001" target="_blank">Fig 1</a>.</p
<p>A) Scanning electron micrograph of the <i>G</i>. <i>morsitans</i>... more <p>A) Scanning electron micrograph of the <i>G</i>. <i>morsitans</i> antenna. The white square is centered on the opening of the sensory pit. B) Magnified image of the opening to the sensory pit. The arrow indicates the approximate position of a recording electrode, which is inserted through the opening of the sensory pit in order to pierce a basiconic Type II sensillum for electrophysiological recording. Scale bar is 5 μm. (C) Odorant response profile of sensory pit B neurons. Odorants were diluted 10<sup>−2</sup> in paraffin oil. n≥5 for all stimuli tested. (D,E) Sample traces from sensory pit sensilla in response to paraffin oil diluent (D) and 2-propanol (E) presented for 0.5 seconds (black bars). The B neuron, represented by the smaller spikes, shows a weak response to paraffin oil, a response that is also observed in some <i>Drosophila</i> neurons [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1008005#pgen.1008005.ref046" target="_blank">46</a>], and a strong response to 2-propanol. The A neuron, represented by large spikes, may be inhibited by 2-propanol. (F) The T-maze paradigm. (G) Behavioral responses of <i>G</i>. <i>morsitans</i>. Means were compared using one-way ANOVA, followed by Tukey’s test for pairwise comparison against paraffin oil for all odorants. **, p<0.01. n = 10 replicates for all odorants; n = 5 for the paraffin oil diluent. (H) Responses of <i>G</i>. <i>fuscipes</i>. t-test; p<0.05. n = 14 replicates.</p
<p>(A) Genomic region of <i>Or22a</i> and <i>Or22b</i> before and a... more <p>(A) Genomic region of <i>Or22a</i> and <i>Or22b</i> before and after it is targeted for CRISPR/Cas9-mediated homology-directed repair to knock-in the <i>Gal4</i> transcription factor and DsRed eye marker genes. The formal designation of this stock is <i>Df(2L)Or22ab</i>, <i>TI{GAL4}Or22ab</i> but for convenience it is indicated as <i>Or22ab</i><sup><i>GAL4</i></sup>. (B) GFP signal from antennal cross sections from <i>D</i>. <i>melanogaster</i> carrying the <i>Gal4</i> knock-in <i>(Or22ab</i><sup><i>GAL4</i></sup><i>)</i>, the <i>UAS-mcd8</i>:<i>GFP</i> transgene on the second chromosome, or both. (C, D) Odorant response profiles from (C) wild type control ab3A and (D) homozygous <i>Or22ab</i><sup><i>GAL4</i></sup> ab3A ORNs. (E, F) Odorant response profiles from <i>D</i>. <i>melanogaster UAS-Or</i> transgenes previously shown [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1008005#pgen.1008005.ref015" target="_blank">15</a>] to confer strong responses to E2-hexenal (Or7a) (E) or methyl salicylate (Or10a) (F), expressed in homozygous <i>Or22ab</i><sup><i>GAL4</i></sup> ab3A olfactory receptor neurons. In panels C-F neuronal responses are reported in spikes/second +/- S.E.M., using individual odorants diluted at 10<sup>−2</sup> and presented for 0.5 seconds. n≥5 for all odorants tested. Spontaneous firing frequencies have been subtracted from all responses; responses to the paraffin oil diluent have been subtracted from the responses to all odorants.</p
<p>(A) Odorant response profile of GmmOr9 expressed in homozygous <i>Or22ab</i>... more <p>(A) Odorant response profile of GmmOr9 expressed in homozygous <i>Or22ab</i><sup><i>GAL4</i></sup> ab3A. (B-E) Sample traces. Black bars represent 0.5 second stimuli. (F) Dose response curves for GmmOr9. Error bars indicate +/- S.E.M. n≥5 for all stimuli tested.</p
Salt taste is one of the most ancient of all sensory modalities. However, the molecular basis of ... more Salt taste is one of the most ancient of all sensory modalities. However, the molecular basis of salt taste remains unclear in invertebrates. Here, we show that the response to low, appetitive salt concentrations in Drosophila depends on Ir56b, an atypical member of the ionotropic receptor (Ir) family. Ir56b acts in concert with two coreceptors, Ir25a and Ir76b. Mutation of Ir56b virtually eliminates an appetitive behavioral response to salt. Ir56b is expressed in neurons that also sense sugars via members of the Gr (gustatory receptor) family. Misexpression of Ir56b in bitter-sensing neurons confers physiological responses to appetitive doses of salt. Ir56b is unique among tuning Irs in containing virtually no N-terminal region, a feature that is evolutionarily conserved. Moreover, Ir56b is a "pseudo-pseudogene": its coding sequence contains a premature stop codon that can be replaced with a sense codon without loss of function. This stop codon is conserved among many Drosophila species but is absent in a number of species associated with cactus in arid regions. Thus, Ir56b serves the evolutionarily ancient function of salt detection in neurons that underlie both salt and sweet taste modalities.
In this thesis I describe the identification of olfactory circuits underlying avoidance of Leptop... more In this thesis I describe the identification of olfactory circuits underlying avoidance of Leptopilina wasps in Drosophila adults and larvae. The larval olfactory sensory neuron (OSN) expresses the olfactory receptor Or49a and is tuned to the waps’ sex pheromone iridomyrmecin exclusively, while the adult OSN expressing both Or49a and Or85f detects in addition to iridomyrmecin, the wasp odors actinidine and nepetalactol. The OSN type expressing Or49a and Or85f is both necessary and sufficient to govern the parasitoid avoidance behavior, and is conserved across several Drosophila species. I also contributed to the demonstration that Drosophila adults prefer Citrus fruits as oviposition substrates. This preference is due to the high content of terpenes in the flavedo. Flies detect these terpenes via only a single class of OSNs, which in this case express the odorant receptor Or19a. This preference has likely been driven by the need to avoid parasitism from endoparasitoid wasps, since t...
Here, we show that several drosophilid flies and their larvae avoid the odors of one genus of par... more Here, we show that several drosophilid flies and their larvae avoid the odors of one genus of parasitoids. This avoidance is mediated by highly specific OSNs, which in D. melanogaster larva express only the olfactory receptor Or49a and respond to the wasps' sex pheromone iridomyrmecin, while in adult express additionally Or85f and respond to the wasps' semiochemicals iridomyrmecin, actinidine and nepetalactol. This information is transferred via projection neurons to a specific part in the lateral horn known to be involved in mediating avoidance. In both D. melanogaster adult and larva these neuron are necessary and sufficient to govern odor-driven parasitoid avoidance behavior. Built on an idea conceived by all authors. Designed experiments: behavioral experiments BSH, MK, HKMD, SAME (70%) Performed experiments: Behavioral experiments (100%) Data analyses: Behavioral experiments MK, SAME (80%).
Parasitoid wasps inflict widespread death upon the insect world. Hundreds of thousands of parasit... more Parasitoid wasps inflict widespread death upon the insect world. Hundreds of thousands of parasitoid wasp species kill a vast range of insect species. Insects have evolved defensive responses to the threat of wasps, some cellular and some behavioral. Here we find an unexpected response of adult Drosophila to the presence of certain parasitoid wasps: accelerated mating behavior. Flies exposed to certain wasp species begin mating more quickly. The effect is mediated via changes in the behavior of the female fly and depends on visual perception. The sight of wasps induces the dramatic upregulation in the fly nervous system of a gene that encodes a 41-amino acid micropeptide. Mutational analysis reveals that the gene is essential to the behavioral response of the fly. Our work provides a foundation for further exploration of how the activation of visual circuits by the sight of a wasp alters both sexual behavior and gene expression.
Tsetse flies transmit trypanosomes—parasites that cause devastating diseases in humans and livest... more Tsetse flies transmit trypanosomes—parasites that cause devastating diseases in humans and livestock—across much of sub-Saharan Africa. Chemical communication through volatile pheromones is common among insects; however, it remains unknown if and how such chemical communication occurs in tsetse flies. We identified methyl palmitoleate (MPO), methyl oleate, and methyl palmitate as compounds that are produced by the tsetse fly Glossina morsitans and elicit strong behavioral responses. MPO evoked a behavioral response in male—but not virgin female— G. morsitans . G. morsitans males mounted females of another species, Glossina fuscipes , when they were treated with MPO. We further identified a subpopulation of olfactory neurons in G. morsitans that increase their firing rate in response to MPO and showed that infecting flies with African trypanosomes alters the flies’ chemical profile and mating behavior. The identification of volatile attractants in tsetse flies may be useful for reduc...
<p>(A) Scanning electron micrograph of a <i>G</i>. <i>morsitans</i>... more <p>(A) Scanning electron micrograph of a <i>G</i>. <i>morsitans</i> head with arrows indicating the openings of the sensory pit and sacculus. (B) Transmitted light image of an antennal cross section (coronal plane) showing the sensory pit and sacculus that open to the medial and lateral sides of the antenna, respectively. (C) Micrograph of a sectioned sensory pit with its opening to the antennal surface at the upper left. (D) Micrograph of the sensory pit, showing a top-down view of the olfactory sensilla that line the pit. (E, E’) The sensory pit is lined with basiconic Type II sensilla. The arrow in E indicates the sensillum shown in E’. (F) Dorsal chamber of the sacculus (medial is left, lateral is right) that is lined with basiconic Type III sensilla (G, G’). The arrow in G indicates the sensillum shown in G’. (H) Ventral chamber of the sacculus (medial is at left, lateral is at right) that is lined with coeloconic (I, J) and no-groove coeloconic sensilla (K). The asterisk in panel J marks an axial view of a coeloconic sensillum that had its tip cut off during cryosectioning, fortuitously revealing its inner structure. Scale bars = 200 μm for A; 5 μm for C, D, F and H; 1 μm for E, G; and 0.25 μm for E’, G’, I, J, and K.</p
<p>(A) Odorant response profile of GmmOr35 expressed in homozygous <i>Or22ab</i>... more <p>(A) Odorant response profile of GmmOr35 expressed in homozygous <i>Or22ab</i><sup><i>GAL4</i></sup> ab3A ORNs. Neuronal responses are reported in spikes/second +/- S.E.M., to individual odorants diluted 10<sup>−2</sup>. (B,C) Sample traces from ab3A empty neurons expressing the <i>GmmOr35</i> transgene in response to the paraffin oil diluent (B) and 1-hexen-3-ol (C), each presented for 0.5 seconds (black bars). (D) Dose response curve for GmmOr35 and 1-hexen-3-ol. (E) Response profile of GmmOr19. In panels A, D, E, spontaneous firing frequencies have been subtracted from all responses; responses to the paraffin oil diluent have been subtracted from the responses to all odorants. n≥5 for all stimuli tested.</p
<p>(<b>A</b>) Reconstruction of two DA2 PNs. (<b>B</b>) Reconstruct... more <p>(<b>A</b>) Reconstruction of two DA2 PNs. (<b>B</b>) Reconstruction of two DL4 PNs. (<b>C</b>) comparison of DA2 and DL4 domains after registration of datasets into a common reference space. DA2 and DL4 PNs overlap in the base of the MB and ventroposterior LH. a: anterior, d: dorsal, l: lateral, p: posterior v: ventral.</p
<p>For all images dorsal is up and medial is to left. (A) <i>GmmOrco</i>. (B) &... more <p>For all images dorsal is up and medial is to left. (A) <i>GmmOrco</i>. (B) <i>GmmOr9</i>. (C) <i>GmmOr6</i>. (D) <i>GmmOr40</i>. (E) <i>GmmOr35</i>. Some background fluorescence is visible that does not emanate from cell bodies; see sense strand control in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1008005#pgen.1008005.s001" target="_blank">S1H Fig</a>. (F) <i>GmmOr38</i>. (G) <i>GmmOr19</i>. (H) <i>GmmOr44</i>. (I) <i>GmmOr29</i>. We note that in several of these images there is background fluorescence that emanates from the cuticle, which is thicker in the antenna of <i>G</i>. <i>morsitans</i> than in <i>D</i>. <i>melanogaster</i>. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1008005#pgen.1008005.s001" target="_blank">S1 Fig</a> for sense strand controls.</p
<p>(A, B) <i>In situ</i> hybridization to <i>GmmObp76a</i> in (A) 4... more <p>(A, B) <i>In situ</i> hybridization to <i>GmmObp76a</i> in (A) 40 μm and (B) 10 μm antennal cross sections. Arrow in panel B marks a trichoid sensillum in the same plane as a cell body marked by <i>GmmObp76a</i>. (C, D) <i>GmmObp6</i> in (C) 40 μm and (D) 10 μm antennal cross sections. Arrow in D marks a basiconic sensillum in the same plane as a cell body marked by <i>GmmObp6</i>. (E, F) <i>GmmObp84a</i> in (E) 40 μm and (F) 10 μm antennal cross sections. Arrow in F marks a coeloconic sensillum in the ventral chamber of the sacculus in the same plane as a cell body marked by <i>GmmObp84a</i>. (G, H) <i>GmmObp59a</i> (red) in (G) 40 μm and (H) 10 μm antennal cross sections. Arrow in H marks a coeloconic sensillum in the same plane as a cell body marked by <i>GmmObp84a</i>.</p
<p>(<b>A</b>) Example spike traces of GC-coupled SSR with all <i>D</i&... more <p>(<b>A</b>) Example spike traces of GC-coupled SSR with all <i>D</i>. <i>melanogaster</i> OSN types and the headspace of <i>L</i>. <i>boulardi</i> (note that the amount of odors within headspace is too low to be detected and analyzed by GC, but is still detected by ab10B). FID, flame ionization detector. (<b>B</b>) GC-coupled SSR with the ab10B neuron and the wash of <i>L</i>. <i>boulardi</i> (1st panel), as well as the identified active compounds (2nd–4th panel). (<b>C</b>) SSR dose-response curves of the ab10B neuron tested with active compounds. (<b>D</b>) GC-coupled SSR with mutant ab3A neuron ectopically expressing either Or49a or Or85f. Blue, green, and red lines indicate active compounds. (<b>E</b>) Tuning breadths of Or49a and Or85f. 232 odorants are displayed along the <i>x</i>-axis according to strengths of responses they elicit from each receptor. Odorants eliciting strongest responses are placed near the center of distribution. Negative values indicate inhibitory responses. For a list of compounds, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002318#pbio.1002318.s005" target="_blank">S4 Fig</a>; for raw data see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002318#pbio.1002318.s001" target="_blank">S1 Data</a>. (<b>F</b>) Identification of glomeruli activated by parasitoid odors (-)-iridomyrmecin, (<i>R</i>)-actinidine, and nepetalactol (a mixture of 1S4aR7R7aS, 1R4aS7S7aS-nepetalactol and their enantiomers). 1st to 3rd columns, false color-coded images showing odorant-induced calcium-dependent fluorescence changes in OSNs expressing Or49a or PNs labeled by GH-146-Gal4 at the antennal lobe (AL) level. Flies express UAS-GCaMP3.0 under control of either Or49a-Gal4, or the GH146-Gal4 driver line. (<b>G</b>) GC-coupled extracellular recordings from larval dorsal organ and wash of <i>L</i>. <i>boulardi</i>. (for more GC-SSR traces of wildtype ab10B neurons and mutant ab3A neurons expressing Or49a or Or85f see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002318#pbio.1002318.s004" target="_blank">S3 Fig</a>)</p
<p>(A) Phylogenetic tree showing the evolutionary relationship among four families within t... more <p>(A) Phylogenetic tree showing the evolutionary relationship among four families within the order Diptera: Culicidae, Drosophilidae, Glossinidae, and Muscidae. Estimated divergence times are from Wiegmann et al., 2011 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1008005#pgen.1008005.ref004" target="_blank">4</a>]. (B) Photograph of <i>D</i>. <i>melanogaster</i> and <i>G</i>. <i>m</i>. <i>morsitans</i> courtesy of Dr. Geoffrey Attardo (adapted from Sun et al. 2018 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1008005#pgen.1008005.ref027" target="_blank">27</a>]). (C) Scanning electron micrograph of the antenna of <i>G</i>. <i>morsitans</i>. Arrows indicate the third antennal segment (Ant.), arista, and sensory pit (SP). Micrographs of trichoid (D, D’), basiconic (E, E’), coeloconic (F), and intermediate (G, G’) olfactory sensilla from <i>G</i>. <i>morsitans</i>. Arrows in D, E, and G indicate the sensilla that are shown at higher magnifications in the images to the right. Scale bars = 100 μm for C; 1 μm for D, E, G; 0.5 μm for D’, E’, G’; and 0.25 μm for F.</p
<p>(A) <i>In situ</i> hybridization to <i>GmmOrco</i> (red). (B) Im... more <p>(A) <i>In situ</i> hybridization to <i>GmmOrco</i> (red). (B) Immunostaining of pan-neuronal marker Elav (green). (C) Merged image of <i>GmmOrco</i> and anti-E lav. (D) Magnification of (C).</p
<p>(A-D) <i>In situ</i> hybridization to <i>GmmOr6</i> (red) combin... more <p>(A-D) <i>In situ</i> hybridization to <i>GmmOr6</i> (red) combined with anti-Elav immunostaining (green). (A) <i>GmmOr6</i>, (B) anti-Elav, and (C) merged image of the whole antenna. (D) Magnified merged image of the sensory pit without DIC. The circular structure in the center of the image that is uniformly green is autofluorescent cuticle of the pit. Likewise, the green fluorescence in the upper right corner is cuticular autofluorescence from the sacculus. (E-H) <i>In situ</i> hybridization to <i>GmmOr9</i> (red) combined with anti-Elav immunostaining (green). (E) <i>GmmOr9</i>, (F) anti-Elav, and (G) merged image of the whole antenna. (H) Magnified merged image of the sensory pit without DIC. (I-L) Double <i>in situ</i> hybridization to <i>GmmOr6</i> and <i>GmmOr9</i> (both red) combined with anti-Elav immunostaining (green). (I) <i>GmmOr6</i> and <i>GmmOr9</i>, (J) anti-Elav, and (K) merged image of the whole antenna. (L) Magnified merged image of the sensory pit without DIC.</p
<p>(<b>A</b>) Larval choice assay and preference indices when larvae were expos... more <p>(<b>A</b>) Larval choice assay and preference indices when larvae were exposed to the wash of <i>L</i>. <i>boulardi</i>. (<b>B</b>) Different choice assays (T-maze, Trap assay, Oviposition assay) for adult flies and resulting preference indices when exposed to the wash of <i>L</i>. <i>boulardi</i>. PI = (number of larvae, flies, or eggs in odor side − number in control side) / total number. Bar plots indicate minimum and maximum values (whiskers), the upper and lower quartiles (boxes) and the median values (bold black line). Deviation of the indices against zero was tested with Wilcoxon rank sum test.</p
<p>(<b>A</b>) Preference indices of ovipositing wildtype flies, flies expressin... more <p>(<b>A</b>) Preference indices of ovipositing wildtype flies, flies expressing <i>Shibire</i><sup><i>ts</i></sup> in ab10B neuron, and corresponding parental lines at restrictive (30°C) and permissive (23°C) temperature when tested with wash of <i>L</i>. <i>boulardi</i>. (<b>B</b>) Preference indices of the same fly lines when tested in the larval assay. Attraction to ethyl butyrate (grey bars) depict that loss of odor-guided behavior in larvae expressing <i>Shibire</i><sup><i>ts</i></sup> in ab10B neuron is odorant specific. (<b>C</b>) Light preference of ovipositing wildtype flies, flies expressing channelrhodopsin in ab10B neuron, and corresponding parental lines. (<b>D</b>) Light preferences of the same fly lines when tested in the larval assay. (<b>A–D</b>) Bar plots indicate minimum and maximum values (whiskers), the upper and lower quartiles (boxes), and the median values (bold black line). Groups were compared by the Kruskal Wallis test with a Dunn’s multiple comparison for selected pairs. For calculation of preference indices, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002318#pbio.1002318.g001" target="_blank">Fig 1</a>.</p
<p>A) Scanning electron micrograph of the <i>G</i>. <i>morsitans</i>... more <p>A) Scanning electron micrograph of the <i>G</i>. <i>morsitans</i> antenna. The white square is centered on the opening of the sensory pit. B) Magnified image of the opening to the sensory pit. The arrow indicates the approximate position of a recording electrode, which is inserted through the opening of the sensory pit in order to pierce a basiconic Type II sensillum for electrophysiological recording. Scale bar is 5 μm. (C) Odorant response profile of sensory pit B neurons. Odorants were diluted 10<sup>−2</sup> in paraffin oil. n≥5 for all stimuli tested. (D,E) Sample traces from sensory pit sensilla in response to paraffin oil diluent (D) and 2-propanol (E) presented for 0.5 seconds (black bars). The B neuron, represented by the smaller spikes, shows a weak response to paraffin oil, a response that is also observed in some <i>Drosophila</i> neurons [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1008005#pgen.1008005.ref046" target="_blank">46</a>], and a strong response to 2-propanol. The A neuron, represented by large spikes, may be inhibited by 2-propanol. (F) The T-maze paradigm. (G) Behavioral responses of <i>G</i>. <i>morsitans</i>. Means were compared using one-way ANOVA, followed by Tukey’s test for pairwise comparison against paraffin oil for all odorants. **, p<0.01. n = 10 replicates for all odorants; n = 5 for the paraffin oil diluent. (H) Responses of <i>G</i>. <i>fuscipes</i>. t-test; p<0.05. n = 14 replicates.</p
<p>(A) Genomic region of <i>Or22a</i> and <i>Or22b</i> before and a... more <p>(A) Genomic region of <i>Or22a</i> and <i>Or22b</i> before and after it is targeted for CRISPR/Cas9-mediated homology-directed repair to knock-in the <i>Gal4</i> transcription factor and DsRed eye marker genes. The formal designation of this stock is <i>Df(2L)Or22ab</i>, <i>TI{GAL4}Or22ab</i> but for convenience it is indicated as <i>Or22ab</i><sup><i>GAL4</i></sup>. (B) GFP signal from antennal cross sections from <i>D</i>. <i>melanogaster</i> carrying the <i>Gal4</i> knock-in <i>(Or22ab</i><sup><i>GAL4</i></sup><i>)</i>, the <i>UAS-mcd8</i>:<i>GFP</i> transgene on the second chromosome, or both. (C, D) Odorant response profiles from (C) wild type control ab3A and (D) homozygous <i>Or22ab</i><sup><i>GAL4</i></sup> ab3A ORNs. (E, F) Odorant response profiles from <i>D</i>. <i>melanogaster UAS-Or</i> transgenes previously shown [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1008005#pgen.1008005.ref015" target="_blank">15</a>] to confer strong responses to E2-hexenal (Or7a) (E) or methyl salicylate (Or10a) (F), expressed in homozygous <i>Or22ab</i><sup><i>GAL4</i></sup> ab3A olfactory receptor neurons. In panels C-F neuronal responses are reported in spikes/second +/- S.E.M., using individual odorants diluted at 10<sup>−2</sup> and presented for 0.5 seconds. n≥5 for all odorants tested. Spontaneous firing frequencies have been subtracted from all responses; responses to the paraffin oil diluent have been subtracted from the responses to all odorants.</p
<p>(A) Odorant response profile of GmmOr9 expressed in homozygous <i>Or22ab</i>... more <p>(A) Odorant response profile of GmmOr9 expressed in homozygous <i>Or22ab</i><sup><i>GAL4</i></sup> ab3A. (B-E) Sample traces. Black bars represent 0.5 second stimuli. (F) Dose response curves for GmmOr9. Error bars indicate +/- S.E.M. n≥5 for all stimuli tested.</p
Salt taste is one of the most ancient of all sensory modalities. However, the molecular basis of ... more Salt taste is one of the most ancient of all sensory modalities. However, the molecular basis of salt taste remains unclear in invertebrates. Here, we show that the response to low, appetitive salt concentrations in Drosophila depends on Ir56b, an atypical member of the ionotropic receptor (Ir) family. Ir56b acts in concert with two coreceptors, Ir25a and Ir76b. Mutation of Ir56b virtually eliminates an appetitive behavioral response to salt. Ir56b is expressed in neurons that also sense sugars via members of the Gr (gustatory receptor) family. Misexpression of Ir56b in bitter-sensing neurons confers physiological responses to appetitive doses of salt. Ir56b is unique among tuning Irs in containing virtually no N-terminal region, a feature that is evolutionarily conserved. Moreover, Ir56b is a "pseudo-pseudogene": its coding sequence contains a premature stop codon that can be replaced with a sense codon without loss of function. This stop codon is conserved among many Drosophila species but is absent in a number of species associated with cactus in arid regions. Thus, Ir56b serves the evolutionarily ancient function of salt detection in neurons that underlie both salt and sweet taste modalities.
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