Odors alone initiate PER

(A) Schematic for recording odor-evoked PER. (B) Six points on the head tracked by DeepLabCut. The movement of the proboscis was characterized by the positions of three segments of the proboscis and the angles between the segments, namely the rostrum angle (R), haustellum angle (H), and labellum angle (L). (C) Example odor-evoked PER. Top, images of a proboscis in a retracted (0.2 s), fully extended (3.1 s) and partially extended (5.5 s) state. Bottom, the three angles over time. Green bar indicates an odor application period. Odor is ethyl butyrate. (D) PER to nine odors in an example fly. Each row corresponds to a trial and each tick mark indicates the timing of PER. See Materials and Methods for the abbreviations of odors. (E) PER probability in wild type flies in response to different concentrations of odors. The same stimulus was used for air, mineral oil, and water controls. n = 21, 26, 27, and 28 flies for 10−4, 10−2, 10−1, and 0.5 concentration groups, respectively. Error bar, standard error of the mean. (F) PER duration in wild type flies. The black lines indicate the mean and the shaded areas indicate the 95% confidence interval. n = 28 flies. (G) Integrated PER duration in wild type flies in response to different concentrations of odors. The data is from the same flies as in E. Box plots indicate the median (gray line), mean (black dot), quartiles (box), and 5-95% range (bar).

Odors evoke PER through GRNs

(A) Integrated PER duration in intact (n = 28) and olfactory organs removed (n = 32) wild type flies. Flies with olfactory organs removed show less PER compared to control flies. p = 0.005. (B-G) Integrated PER duration in control (UAS-Kir2.1/+, n = 20, B), Orco ORN silenced (Orco-Gal4>UAS-Kir2.1, n = 24, C), and GRN silenced (Gr5a-Gal4>UAS-Kir2.1, n = 25, D; Gr66a-Gal4>UAS-Kir2.1, n = 24, E; Ppk28-Gal4>UAS-Kir2.1, n = 28, F; Ir94e-Gal4>UAS-Kir2.1, n = 23, G) flies. Each silenced line was compared to the control in B. p = 0.06, 1e-6, 0.39, 0.10 and 0.12 for C-G. Silencing of Orco ORNs and Gr5a GRNs reduce odor-evoked PER. (H) Integrated PER duration in control (UAS-Kir2.1/+, n = 21) and Gr66a GRN silenced (Gr66a-Gal4>UAS-Kir2.1, n = 21) flies in a fed state. Silencing of Gr66a GRNs enhances odor-evoked PER in a fed state. Odor concentration was 10−1. p = 0.002. (I) Integrated PER duration in control (n = 29) and Gr5a mutant (n = 29) flies. PER is severely reduced in mutant flies. p = 1.0e-5. (J-L) Integrated PER duration in control and Obp RNAi flies. Obp57d/e RNAi flies show reduced (n = 33 and 25 for control and RNAi), Obp49a RNAi flies show enhanced (n = 24 and 20 for control and RNAi), and Obp19b RNAi flies show similar level of PER (n = 24 and 21 for control and RNAi) as compared to their respective controls. p = 0.006, 0.005, and 0.24 for J-L. Scheirer–Ray–Hare test. Box plots indicate the median (gray line), mean (black dot), quartiles (box), and 5-95% range (bar).

Sweet- and bitter-sensing GRNs directly respond to odors

(A) Schematic for two photon calcium imaging of GRN axon termini in the subesophageal zone. The top image shows an anterior view of a brain of Gr5a-Gal4>UAS-GCaMP6s fly. White rectangle indicates the target region of calcium imaging. Scale bar: 50 μm. (B) Example response (ΔF/F) to ethyl butyrate. Green bar indicates an odor application period. (C) Example responses of Gr5a GRNs to nine odors. Green bar indicates an odor application period. ΔF/F of GCaMP6s fluorescence is color coded according to the scale bar. (D) Trial averaged peak responses of Gr5a GRNs to individual odors. Covering the labella (n = 4, p = 2e-13) but not maxillary palps (n = 4, p = 0.15) with glue reduces odor responses as compared to control (n = 5). Two-way ANOVA. (E, F) Same as in C, D, but for Gr66a GRNs. As in Gr5a GRNs, covering the labella (n = 5, p = 9e-08) but not maxillary palps (n = 5, p = 0.56) with glue reduces odor responses as compared to control (n = 6). Two-way ANOVA. (G) A linear combination of Gr5a and Gr66a GRN responses well predicted the magnitude of odor-evoked PER. Each dot represents an odor. Coefficient of determination = 0.81. (H) Odor responses are severely reduced in Gr5a GRNs in a Gr5a receptor mutant background (n = 6 and 8 for control and mutant, p = 5e-06, two-way ANOVA). Box plots indicate the median (gray line), mean (black dot), quartiles (box), and 5-95% range (bar).

GRNs integrate odor-taste multisensory input to drive PER

(A) Schematic for examining PER in response to odor-taste multimodal stimuli. Stimuli are presented to the labellum with a wick (arrowhead) immersed in a sucrose solution with or without odors. A constant air stream was applied from behind the wick. (B) PER probability in response to sucrose at different concentrations with (green) or without (black) banana odor in wild type flies. Odor concentration was 10−4. Banana odor enhances sucrose-evoked PER even in the absence of olfactory organs (n = 13 and 11, p = 0.002 and 0.04 for intact and olfactory organ removed flies). Scheirer–Ray–Hare test. Error bar, standard error of the mean. (C) Summary of mechanisms underlying PER in a multisensory environment.

Genotypes used to generate data presented in each figure. Examination of odor-evoked PER

Characteristics of PER in wild type flies

(A) Latency to the first odor-evoked PER in wild type flies. Green bar indicates an odor application period. p = 1.0e-4, one-way ANOVA. Box plots indicate the median (gray line), mean (black dot), quartiles (box), and 5-95% range (bar). (B) K-medoid clustering of odor- and sucrose-evoked proboscis extensions based on the rostrum and haustellum angles over time in wild type flies. All the trajectories during PER and partial proboscis extensions were clustered into five clusters (color coded), and the representative trajectory closest to each cluster medoid was plotted for each stimulus. The green trajectories represent full extensions that qualify the definition of PER (see Methods). Note that these green trajectories are observed in response to sucrose and all the odors (except for the controls), suggesting that the movement of proboscis during odor-evoked PER is similar to that during sucrose-evoked PER.

Dependence of odor-evoked PER on feeding state

(A) Integrated PER duration in starved (n = 28) and fed (n = 19) wild type flies. PER is enhanced in the starved state. p = 0.88, Scheirer–Ray–Hare test. (B) Same as in a but for odor concentration of 10−1. p = 1e-6, Scheirer–Ray–Hare test. Box plots indicate the median (gray line), mean (black dot), quartiles (box), and 5-95% range (bar).

Wings and legs are not required for odor-evoked PER

(A) Integrated PER duration in wing-removed Orco-Gal4>UAS-Kir2.1 flies (n = 26). (B) Integrated PER duration in wing and leg-removed, Orco-Gal4>UAS-Kir2.1 flies (n = 27). These results suggest that GRNs in wings and legs are dispensable for odor-evoked PER. Box plots indicate the median (gray line), mean (black dot), quartiles (box), and 5-95% range (bar).

Contributions of OBPs to odor-evoked PER

(A, B) Integrated PER duration in control and Obp56g RNAi flies. These flies show similar level of PER (n = 24 and 23 for control and RNAi). p = 0.58, Scheirer–Ray–Hare test. (C-H) Integrated PER duration in control and OBP RNAi flies. Obp18a RNAi and Obp57e RNAi flies show reduced level of PER as compared to control. n = 33, 25, 22, 25, 27, and 28 flies for C-H. p = 0.05, 0.36, 0.08, 0.05, and 0.90 for D-H, Scheirer–Ray–Hare test. Box plots indicate the median (gray line), mean (black dot), quartiles (box), and 5-95% range (bar).

Responses of GRNs to odors and sucrose

(A) Image of a brain of Gr5a-Gal4>UAS-GCaMP6s fly, stained with anti-GFP to reveal Gr5a neurons (green) and nc82 to reveal synapses (magenta). Scale bar, 50 μm. (B) Baseline florescence intensity of GCaMP6s in Gr5a GRNs. Fluorescence (arbitrary unit) is color coded according to the scale bar. (C) Peak florescence intensity of GCaMP6s in the same fly as shown in B after stimulation with 4-methylcyclohexanol. Fluorescence is color coded according to the scale bar. The ROI used for ΔF/F calculation is indicated by a white rectangle. Scale bar, 20 μm. (D-F) Same as in A-C, but for Gr66a-Gal4>UAS-GCaMP6s fly and benzaldehyde stimulation. (G) Trial averaged peak responses of Gr5a GRNs to different concentrations of ethyl butyrate (EBR) and benzaldehyde (BNZ). n = 5 flies. (H) Same as in G, but for Gr66a GRNs. (I) Trial averaged peak responses of Ppk28 GRNs to individual odors. These neurons do not respond to odors. n = 5 flies. (J) Same as in I, but for Ir94e GRNs. n = 9 flies. Box plots indicate the median (gray line), mean (black dot), quartiles (box), and 5-95% range (bar).

Multisensory PER in flies with intact or removed olfactory organs

(A) Integrated PER duration in response to a banana odor presented at various concentrations in wild type flies. n = 21 flies. Box plots indicate the median (gray line), mean (black dot), quartiles (box), and 5-95% range (bar). (B) Trial averaged peak responses of Gr5a GRNs in response to various concentrations of banana odor in Gr5a-Gal4>UAS-GCaMP6s flies. n = 5 flies. (C) PER probability in response to sucrose with or without odors in wild type flies in which olfactory organs are intact or have been removed. Odor is EBR. Intact, n = 25, p = 0.005. Olfactory organs removed, n = 25, p = 0.007. Wilcoxon signed-rank test. (D) Same as in C, but for BNZ. Intact, n = 21, p = 0.007. Olfactory organs removed, n = 36, p = 0.0007. Wilcoxon signed-rank test. The concentration of odors and sucrose was 10−4 and 0.25%, respectively. Error bar, standard error of the mean.