Multisensory interactions regulate feeding behavior in Drosophila

Abstract

The integration of two or more distinct sensory cues can help animals make more informed decisions about potential food sources, but little is known about how feeding-related multimodal sensory integration happens at the cellular and molecular levels. Here, we show that multimodal sensory integration contributes to a stereotyped feeding behavior in the model organism Drosophila melanogaster Simultaneous olfactory and mechanosensory inputs significantly influence a taste-evoked feeding behavior called the proboscis extension reflex (PER). Olfactory and mechanical information are mediated by antennal Or35a neurons and leg hair plate mechanosensory neurons, respectively. We show that the controlled delivery of three different sensory cues can produce a supra-additive PER via the concurrent stimulation of olfactory, taste, and mechanosensory inputs. We suggest that the fruit fly is a versatile model system to study multisensory integration related to feeding, which also likely exists in vertebrates.

Keywords: Drosophila; mechanosensation; multisensory integration; olfaction; taste.

Fig. 1.

Yeast odors enhance sugar-induced PER. (A) Schematic of the PER assay. Flies were attached to a glass slide and a tastant droplet was applied to a foreleg. (B) PER evoked by sucrose or a yeast suspension. n = 8 to 17. (C) PER after ablation of the olfactory organs (third antennal segments or maxillary palps). n is indicated in the bar. (D) PER in Orco-null flies. n is indicated in the bar. (E) A modified PER assay. While a tastant droplet was applied to a foreleg, an odor source was brought near the flies without contact. n is indicated in the bar. All data are presented as means ± SEM. One-way ANOVAs with Tukey post hoc tests or Kruskal–Wallis with Mann–Whitney U tests were used for C–E. The asterisks indicate statistically significant differences from wild-type flies, unless otherwise indicated. *P < 0.01, **P < 0.001.

Fig. 2.

Or35a ORNs mediating yeast odor-enhanced PER. (A) GC-MS analysis for yeast-derived volatile compounds. Volatile compounds are indicated in the order of abundance. (B) ORN screen for yeast odor-enhanced PER. The modified PER analysis was performed in flies expressing Kir2.1 in a subset of ORNs. n = 6 to 13. (C and D) Modified PER in Or35a or Orco mutant flies. The genotypes are indicated. n = 6 to 14. (E) Modified PER using volatile compounds found in yeast culture. n = 6. (F) PER upon optogenetic activation of Or35a ORNs. Or35a>ReaChR flies were tested in the absence or presence of all transretinal. n = 7 to 11. All data are presented as means ± SEM. Unpaired Student’s t tests or Mann–Whitney U tests were used for B–E. One-way ANOVAs with Tukey post hoc tests or Kruskal–Wallis with Mann–Whitney U tests were used for F. The asterisks indicate statistically significant differences from PER induced by sucrose alone, unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

Mechanosensation is required for yeast odor-enhanced PER. (A and B) Modified PER induced by touching a foreleg (A) or the labellum (B) in conjunction with yeast odor. n = 4 to 8 and n = 8 for legs and labellum, respectively. (C) Modified PER induced by touching a foreleg with 10 mM sucrose in solutions of differing viscosity and yeast odor. Methyl cellulose was added to change viscosity. n = 6 to 9. The centipoise (cP) values for 10 mM sucrose and 30% yeast are 1.05 cP and 1.62 cP, respectively. (D) Mechanosensory screen. Modified PER was performed using 10 mM sucrose and yeast odor. n = 8 to 18. All data are presented as means ± SEM. Unpaired Student’s t tests or Mann–Whitney U tests. *P < 0.01, **P < 0.001.

Fig. 4.

Hair plates are required for yeast odor-enhanced PER. (A) Expression pattern of split^HP-GAL4 (split^HP-GAL4 line intersection strategy on the Left). Confocal images of leg, VNC, and brain expressing mCD8::GFP driven by split^HP-GAL4. VNC and brain were stained with a rabbit anti-GFP (green) and nc82 (magenta). VNC image merged with bright field. The dashed lines and arrows indicate attached coxae and cxHP8, respectively. The hair plate image (Upper Left) is a higher magnification of the marked area on the coxa image (Bottom Right). Magenta in the leg images is cuticle autofluorescence. (Scale bars for brain, VNC, and leg, 100 μm; for hair plate, 25 μm.) (B) Modified PER upon chronic silencing of split^HP neurons. n = 6 to 9. (C) Modified PER upon acute silencing of split^HP neurons. n = 6 to 12. (D) Modified PER of flies with knockdown of mechanosensory genes in split^HP neurons. n = 7 to 10. (E) Modified PER with piezo^K.O flies expressing piezo cDNA in split^HP neurons. n = 7 to 8. (F) Optogenetic activation of split^HP-GAL4-labeled neurons. Modified PER was performed using 5% methylcellulose containing 10 mM sucrose and yeast odor. Fly collars were used to prevent light leakage. A red light was applied via a glass fiber to either the body or the head. R81. (R81E10)>ReaChR flies were used as controls to confirm optogenetic activation of neurons in the brain. n = 7 to 11. (G) PER with vibration stimuli on the coxa hair plate. The coxa was immobilized using glue. PER was induced by 10 mM sucrose with isoamyl alcohol in either 0.2% or 5% methyl cellulose. Vibration was applied to activate coxa hair plate MNs. n = 8 to 13. All data are presented as means ± SEM. Unpaired Student’s t tests or Mann–Whitney U tests were used for B–E. One-way ANOVAs with Tukey post hoc tests or Kruskal–Wallis tests with Mann–Whitney U tests were used for F–G. The asterisks indicate statistically significant differences from PER evoked by sucrose alone, unless otherwise indicated. *P < 0.01, **P < 0.001.

Fig. 5.

Concurrent input of three different sensory cues enhances PER. (A) PER enhancement by multisensory integration. split^HP>ReaChR;piezo^K.O flies were tested. 10 mM sucrose was used as a taste stimulus, yeast odor as an olfactory stimulus, and red light as a mechanical stimulus (optogenetic). n = 8 to 10. (B) Modified PER upon inhibition of hair plate neurons. split^HP neurons expressing GtACR1 were inhibited optogenetically. n = 8 to 10. All data are presented as means ± SEM. Kruskal–Wallis tests with Mann–Whitney U tests were used. The asterisks indicate statistically significant differences from PER evoked by sucrose alone, unless otherwise indicated. *P < 0.001.