Behavioral choices expressed by flies between a control agar disk and disk treated with butyl acetate.

<p>Ordinates: ratio of the density of presence of flies on odorant-treated or non-treated food (mean±S.E.M.; number of trails for each is noted in the figure). Abscissa: Wild-type (<i>w</i>¹¹¹⁸) and flies expressing <i>Or22a</i>, <i>Or83b</i> or <i>Or22a</i>+<i>Or83b</i> under the control of <i>Gr5a</i>- or <i>Gr66a</i>-Gal4. Flies expressing both <i>Or22a</i> and <i>Or83b</i> showed an altered preference to butyl acetate as compared to the wild-type (<i>w</i>¹¹¹⁸). Depending on the GRNs that express the <i>Ors</i>, flies exhibited the opposite preferences to the same odorant. BA = Butyl acetate. * P<0.05, ** P<0.01 with the Tukey-Kramer multiple comparisons test.</p

Electrophysiological responses of <i>Drosophila</i> i-type taste sensilla to sugar and bitter substances.

<p>a. Schematic diagram of the recording setup. Electrical signals were recorded from an electrolytically sharpened tungsten electrode inserted through the cuticle at the base of a taste sensillum. To stimulate the GRNs, the tip of the sensillum was capped during 2–5 s with a glass capillary filled with a stimulating solution. The i-type sensilla house only two GRNs, which elicit spikes of different amplitudes that were separated using custom software routines (23) as shown in 1c and 1e. The sugar-sensing GRN expresses <i>Gr5a</i> and the bitter-sensitive GRN expresses <i>Gr66a </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002610#pone.0002610-Hiroi1" target="_blank">[40]</a>. b. Sample recording with 100 mM sucrose (stimulus = 2 s horizontal grey bar). c. Sucrose elicits a response only in the cell that fires spikes of smaller amplitude (lower trace); superimposed spikes (left column) and time-series extracted (central trace and bars) after software spike separation. d. Sample recording with 1 mM caffeine. e. Caffeine elicits a response only in the cell that produces spikes of larger amplitude. Vertical bars = 0.3 mV.</p

Responses to odors in taste neurons expressing <i>Ors</i> under different Gal4 drivers.

<p>a. Taste neurons expressing <i>Or22a</i>+<i>Or83b</i> in sugar-sensing neurons (driven by <i>Gr5a</i>-Gal4) respond to sugar and to butyl acetate (first two traces: smaller amplitude spikes); taste neurons expressing <i>Or22a</i>+<i>Or83b</i> in bitter-sensing neurons (driven by <i>Gr66a</i>-Gal4) respond to caffeine and to butyl acetate (lower two traces: larger amplitude spikes). Gray bar = 2 s stimulus. Horizontal black bar: 1 s; vertical bar: 0.2 mV. b. Comparison of the responses of altered GRNs to butyl acetate depending on the driver, <i>Gr5a</i>-Gal4 (“sugar cells”) or <i>Gr66a</i>-Gal4 (“bitter cells”) (mean±S.E.M.; n = 12, 17 trials). There is a small difference in response intensity to butyl acetate in <i>Gr5a</i>- and <i>Gr66a</i>-GRNs (*: P = 0.039, Student's t-test).</p

Ectopic expression of <i>Or22a</i> and <i>Or83b</i> in GRNs does not alter the projection pattern of the GRNs.

<p>a. Schematic diagram of the frontal view of a fly brain. Dorsal is to the top. Arrow heads indicate incoming fibers of GRNs in the proboscis. AL = antennal lobe, SOG = suboesophageal ganglion. b. Whole-mount of a fly brain, showing projection of <i>Gr66a</i>-positive GRNs that co-express <i>Or22a</i> and <i>Or83b</i> in the SOG. <i>Gr66a</i>-GRNs were labeled by mCD8::GFP. Areas surrounded by dotted line are ALs and SOG. This figure and the followings are single pictures from the microscope. The red signal is used here to obtain the outlines of the brain. c. Close-up view of the SOG and ALs in b. d. Projection of <i>Gr5a</i>-GRNs expressing <i>Or22a</i> and <i>Or83b</i> in the SOG. Both type of GRNs (<i>Gr66a</i> and <i>Gr5a</i>) were not affected in their projection patterns by co-expressing the Ors in their projection patterns. Scale bars in b, c and d = 50 µm.</p

The gram-negative sensing receptor PGRP-LC contributes to grooming induction in <i>Drosophila</i> - Fig 3

<p>(A) Schematic indicating how the Imd pathway is activated by Gram-negative bacteria. Grooming behavior induced by LPS contact on wings in (B) black square: w;;<i>PGRP-LC^E12</i> with Cantonized 2nd chromosome, green square: y,w,<i>PGRP-LE¹¹²</i> and grey circle: w;;PGRP-LBΔ (C) black square: w;;PGRP-LC^E12 and orange square: w;P[acman]-<i>PGRP-LC;PGRPLC^E12</i> rescue flies. Standard LPS was used as a stimulus. A significant increase in response from that of the control (water) is indicated by asterisks: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 (Dunnett’s test); n = 40 (n = 20 for each sex). Data represents mean +/- SE, analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185370#pone.0185370.g001" target="_blank">Fig 1</a>. (D) shows results of Chi-square test on grooming induction. Mutants, which showed significant concentration-dependent behavioral increase were in white zone, and no-significance in gray zone. White zone in a table shows significant difference (p < 0.05), and grey zone indicates no-significance (p > 0.05).</p

L-canavanine aversion is reduced when bitter-sensitive taste neurons express a RNAi construct against Gαo47A or a dominant negative form of Gαo47A.

<p>Two-choice feeding test experiments showing preference index (PI) for the blue solution of different genotypes. Control (white bars) and 30 mM L-canavanine (black bars) indicate that no drug or 30 mM L-canavanine was added to the blue solution, respectively. A complete preference or aversion is indicated by a PI value of 1 or 0, respectively. The down regulation of Gαo47A by RNA interference (Gr66a-Gal4/+;UAS-RNAiGo/+) and the inhibition of Gαo47A by using a dominant negative construct (Gr66a-Gal4/UAS-Go^GDP) reduced the aversion to L-canavanine compared to controls (wild-type, Gr66a-Gal4/+, UAS-RNAiGo/+ and UAS-Go^GDP/+). Note that all genotypes did not show any defect for sugar detection. Error bars indicate SEM. Asterisks indicate significant differences by Unpaired Student's <i>t</i> test (** p<0.01, *** p<0.001).</p

Sequence alignment of the intracellular loops of mGluRs and DmXR.

<p><i>i1</i>, <i>i2</i>, and <i>i3</i> correspond to the first, second, and third intracellular loops of mGluRs and DmXR, respectively. Residues conserved in mGluRs coupled to phospholipase C (mGluR1 and 5) are boxed in grey, and the corresponding residues in most adenylyl cyclase coupled mGluRs (mGluR2, 3, 4, 6, 7 and 8) and DmXR are boxed in black, respectively.</p

The GPCR DmX has the best coupling with Gαo protein subtype in HEK transfected cells.

<p>L-canavanine induced-inositol phosphate (IP) production was measured from HEK cells co-expressing the DmX receptor and the indicated Gα protein. As a control, we used HEK cells transfected with DmXR expression vector alone (called ‘No G’). Basal and 10 mM L-canavanine were used for all stimulations, indicated by white and black bars, respectively. IP stimulation was calculated relatively to IP production in basal conditions. HEK cells co-expressing DmXR and Gα₁₅, Gα_qi9 or Gα_qo5 produced IP after L-canavanine stimulation, indicating that these Gα proteins can efficiently couple to DmXR, the best coupling being observed with Gα_qo5. No such effect was observed with HEK cells co-expressing DmXR and Gα₁₆, Gα_q or Gα_qz5. Experiments done with Gα₁₅ could be considered as a positive control because Gα₁₅ protein is known to couple with most GPCRs. Data are means +/− SEM from triplicate experiments. IP production was compared with basal activity using Unpaired Student's <i>t</i> test (* p<0.05, ** p<0.01, *** p<0.001).</p

Proboscis extension reflex (PER) responses to sucrose (SUC) of males pre-exposed to gustatory and olfactory stimuli.

<p><b>A</b>) pre-exposure to SUC and QUI, test with 0.03 M SUC. <b>B</b>) pre-exposure to SUC ipsi- or contralateral antenna, test with 0.03 M SUC. <b>C</b>) pre-exposure to PHE, test with 0.03 M SUC. <b>D</b>) responses of males to SUC after a non-specific mechanical stimulus, test with 0.1 M SUC. Columns show the percentage of males extending the proboscis when one of their antennae was contacted with a toothpick soaked with a SUC solution. Within each frame (A, B, C, D), the percentage of PER responses were significantly different between columns with different letters (Chi-Square, p<0.05). Numbers at the bottom of bars indicate numbers of tested males. Sensitivity to SUC was intra-modally increased by pre-exposure to SUC and QUI, and cross-modally increased by pre-exposure to PHE.</p

Proboscis extension reflex (PER) responses to different test-concentrations of sucrose (SUC) of males pre-exposed to sucrose (1 M SUC) and quinine (0.1 M QUI).

<p>Each point represents the percentage of males extending their proboscis when their antennae were contacted with different concentrations of SUC. The number of tested males is n = 40 for each data point. Dashed boxes enclose values that do not differ significantly (Chi-Square tests, p<0.05). Sensitivity to SUC was higher in males pre-exposed to SUC and QUI, showing a stronger difference with naïve males at low doses.</p