Detection of sweet tastants by a conserved group of insect gustatory receptors

Abstract

Sweet taste cells play critical roles in food selection and feeding behaviors. Drosophila sweet neurons express eight gustatory receptors (Grs) belonging to a highly conserved clade in insects. Despite ongoing efforts, little is known about the fundamental principles that underlie how sweet tastants are detected by these receptors. Here, we provide a systematic functional analysis of Drosophila sweet receptors using the ab1C CO2-sensing olfactory neuron as a unique in vivo decoder. We find that each of the eight receptors of this group confers sensitivity to one or more sweet tastants, indicating direct roles in ligand recognition for all sweet receptors. Receptor response profiles are validated by analysis of taste responses in corresponding Gr mutants. The response matrix shows extensive overlap in Gr-ligand interactions and loosely separates sweet receptors into two groups matching their relationships by sequence. We then show that expression of a bitter taste receptor confers sensitivity to selected aversive tastants that match the responses of the neuron that the Gr is derived from. Finally, we characterize an internal fructose-sensing receptor, Gr43a, and its ortholog in the malaria mosquito, AgGr25, in the ab1C expression system. We find that both receptors show robust responses to fructose along with a number of other sweet tastants. Our results provide a molecular basis for tastant detection by the entire repertoire of sweet taste receptors in the fly and lay the foundation for studying Grs in mosquitoes and other insects that transmit deadly diseases.

Keywords: chemoreceptor; chemosensory; heterologous expression.

Fig. 1.

An in vivo ectopic expression system for analysis of individual Grs. (A) Schematic of ectopic expression in the ab1C neuron with trace from wild-type ab1 sensillum depicting activities of the four ab1 neurons. Glass micropipettes for tastant recordings contain sensillum lymph ringer (SLR) control (gray) or stimulus in SLR (blue). (B) Sample ab1 recordings in flies expressing Gr5a in ab1C neurons (ab1C:Gr5a-2x). Black dots indicate ab1C spikes. (C) Mean responses of ab1C:Gr5a-1x and ab1C:Gr5a-2x neurons. Baseline activity to SLR is not subtracted from stimulus-evoked activity. Sugars were tested at a concentration of 100 mM. Letters indicate statistical significance (P < 0.001; one-way ANOVA with Tukey’s post hoc test; n = 6–12). (D) Dose-dependent response to trehalose (n = 6–12). (E) Mean responses of ab1C:Gr5a neurons generated with Gr21a– or Gr63a–GAL4 as indicated to 100 mM sugars (n = 6). (F) Mean responses of ab1C:Gr5a-2x neurons in wild-type (+Gr63a) or ΔGr63a (–Gr63a) flies to 100 mM sugars (n = 10–14). All genotypes in E and F were compared with each other by using two-way ANOVA with Tukey’s post hoc test. Only genotypes with one copy of UAS–Gr5a (E) are significantly different from genotypes with two copies of UAS–Gr5a (F) (P < 0.05).

Fig. 2.

Tastant response profiles of sweet taste receptors. (A) Mean electrophysiological responses of ab1C:GrX neurons. All sugars were tested at a concentration of 100 mM, except maltotriose at 250 mM and glycerol at 10% (vol/vol). For each data point, n = 6–14. *P < 0.05; **P < 0.001 [vs. control ab1C flies (w¹¹¹⁸)]. (B) Mean electrophysiological responses of Gr5a expressed alone (–) or with the indicated receptor in ab1C neurons. For each data point, n = 6–7. *P < 0.05; **P < 0.001 [vs. Gr5a alone (–)]. (C) Phylogenetic tree of sweet Grs (Left) and heat map of mean neuronal responses of ab1C:GrX neurons to indicated sweet tastants. Data are the same as in A. Heat map was made with PAST (http://folk.uio.no/ohammer/past).

Fig. 3.

Sweet taste responses in Gr mutants. (A) Mean responses of sweet taste neurons in L-type sensilla to indicated tastants. Indicated genotypes were: w¹¹¹⁸ (wild-type), ΔEP(X)-5 (ΔGr5a), Gr64f^MB12243 (ΔGr64f), Gr64e^MB03533 (ΔGr64e), Gr61a¹ (ΔGr61a), and Gr64a¹ (ΔGr64a). All stimuli were tested at a concentration of 100 mM, except glycerol (10%). *P < 0.05; **P < 0.001 (one-way ANOVA with one-tailed Dunnett’s t test vs. wild-type; n = 6–22). (B) Heat maps of ab1C:GrX responses (Upper) and percent reduction in taste neuron responses in corresponding GrX mutants (Lower); the latter only includes data points significantly different from wild-type in C. Percent loss of response was calculated by using [(wild type – mutant)/wild type] x 100. Heat maps were made by using JMP 10 (www.jmp.com). (C) Scatter plot of percent loss of response in Gr mutant and ab1C:GrX response (gain) for each GrX–ligand combination. Filled circles indicate taste neuron responses that are significantly reduced in mutant flies (ΔGrX); open circles indicate those that are not. Shaded area indicates ab1C:GrX responses that are not statistically significant.

Fig. 4.

Functional analysis of other Drosophila and mosquito Grs. (A) Mean response of ab1C neurons in control w¹¹¹⁸ flies (ab1C) and flies expressing Gr59c (ab1C:Gr59c). *P < 0.05 (vs. control; n = 6–12). Sucrose was tested at a concentration of 100 mM and bitter compounds at 10 mM. (B) Dose-dependent response of ab1C:Gr59c (n = 6–12). (C) Sample recordings and mean responses of ab1C:Gr59c in wild-type (+Gr63a) and ΔGr63a (–Gr63a) flies to indicated stimuli (10 mM). Genotypes are not significantly different (P > 0.05; n = 6). Concentrations were as in A. (D) Dose-dependent response of ab1C:DmGr43a (n = 6–11). (E) Mean responses of ab1C neurons in control w¹¹¹⁸ flies (ab1C) and flies expressing Drosophila Gr43a (ab1C:DmGr43a) or its mosquito ortholog (ab1C:AgGr25) to indicated stimuli tested at a concentration of 100 mM, except maltotriose (250 mM) and glycerol (10%). *P < 0.05; **P < 0.001 (vs. ab1C; n = 6–12).