Signaling Mode of the Broad-Spectrum Conserved CO2 Receptor Is One of the Important Determinants of Odor Valence in Drosophila

Abstract

Odor detection involves hundreds of olfactory receptors from diverse families, making modeling of hedonic valence of an odorant difficult, even in Drosophila melanogaster where most receptors have been deorphanised. We demonstrate that a broadly tuned heteromeric receptor that detects CO₂ (Gr21a, Gr63a) and other odorants is a key determinant of valence along with a few members of the Odorant receptor family in a T-maze, but not in a trap assay. Gr21a and Gr63a have atypically high amino acid conservation in Dipteran insects, and they use both inhibition and activation to convey positive or negative valence for numerous odorants. Inhibitors elicit a robust Gr63a-dependent attraction, while activators, strong aversion. The attractiveness of inhibitory odorants increases with increasing background CO₂ levels, providing a mechanism for behavior modulation in odor blends. In mosquitoes, valence is switched and activation of the orthologous receptor conveys attraction. Reverse chemical ecology enables the identification of inhibitory odorants to reduce attraction of mosquitoes to skin.

Keywords: Drosophila; Gr21a; Gr63a; Orco; gustatory receptor; olfaction; olfactory receptor; valence.

Figure 1. Polyamines cause attraction by inhibition of the CO₂-avoidance neuron in Drosophila

(A) Schematic of T-maze behavior assay and mean preference index for flies of indicated genotypes given a choice between solvent and 1% spermidine. (n = 8 trails per genotype, 40 flies/trial). One-way ANOVA, p <0.001. (B) In a trap assay, mean preference index for indicated genotypes for 1% spermidine. (n = 10 trials per genotype, 20 flies/trial). (C) Representative action potential trace and mean percent inhibition of ab1C activity in orco⁻ flies during 0.5-s exposures to spermidine at indicated concentrations (n = 14). (D) Mean preference of flies of indicated genotypes in a T-maze for ~0.35% CO₂ and 1% spermidine (n = 8–10 per genotype). One-way ANOVA, p <0.001. (E,F) Representative trace and mean percent inhibition of ab1C activity in orco⁻ flies during 0.5-s exposures to odorants at indicated concentrations (n = 14). (G) Model of attraction towards the source of an odorant that inhibits background firing of the aversive ab1C neuron in a dose-dependent manner. In A and D, Different letters are significantly different by Tukey’s post hoc analysis. Error bars are s.e.m.

Figure 2. Amines inhibit ab1C, and Gr63a is necessary for attraction to amines in Drosophila

(A) Chemical structures, representative traces, and mean percent inhibition of ab1C activity in orco⁻ flies during 0.5-s exposures to the monoamines at indicated concentrations. Stimulus bar is 0.5 s (n = 14 per concentration, except for trimethyamine and butylamine, n=4). (B) T-maze behavior assay: mean preference index of flies of indicated genotypes to a choice between solvent and indicated odorants at 1% concentration. N = 6–16 trails per genotype (~40 flies/trial), One-way ANOVA, p <0.001 for all, genotypes marked with different letters are significantly different by Tukey’s post hoc analysis. Error bars are s.e.m.

Figure 3. Amines inhibit the CO₂-receptor neuron in Aedes aegypti

(A, B) Representative traces and mean percent inhibition of cpA activity for A, spermidine (n = 6 per concentration), and B, spermine (n = 6 per concentration). The stimulus is room air with ambient CO₂ concentration which stimulates the ab1C neuron. (C) Mean percent inhibition of cpA activity by a panel of amines diluted to 1%. (n = 4 sensilla). (D) Representative traces and mean percent inhibition of cpA by a panel of amines (1%) when overlaid on 0.15% CO₂. (n = 5–6 sensilla). (E) Representative traces of a 10% spermidine pulse (3-s). (F) Representative traces and mean percent inhibition of cpA when a 10% spermidine pulse (1-s) is overlaid on pulse a 0.15% CO₂ (3-s), or when 0.15% CO₂ (1-s) is overlaid on 10% spermidine (3-s). (n = 4–6 sensilla). (Representative traces are shown only for trials testing CO₂ overlays onto 3-s spermidine). (G) Representative traces and mean percent inhibition of cpA by indicated concentrations of spermidine when overlaid on 1% cyclopentanone (3-s). Average response to cyclopentanone alone = 79 +/− 7.6 spikes s⁻¹. (n = 4–6 sensilla) (H) Schematic of mosquito two-choice assay. (I) Mean number of mosquitoes landing on spermidine-treated or solvent-treated netting and preference index for a 5-min two-choice assay. (n = 5 trials, 40 mosquitoes/trial). t-test, ** p < 0.01, and (J) mean preference index in the same experimental paradigm with A. aegypti wild type and orco⁵ mutant females. (n = 6 trials, 40 mosquitoes/trial). Error bars are s.e.m.

Figure 4. Attractiveness of an inhibitor is modulated by background level of CO₂

(A) Representative traces and mean activity of ab1C during 0.5 s exposures to ethyl pyruvate at indicated concentrations. (n=5 sensilla per concentration) (B) In a T-maze, mean preference of flies of indicated genotypes for indicated concentrations of ethyl pyruvate. (n = 8–10 trials per genotype, 40 flies/trial). (B) Schematic of T-maze assay. Prior to testing, CO₂ was injected into both arms of the T-maze to elevate background levels. (D) Preference in a T-maze of wildtype flies for 1 % ethyl pyruvate in the presence of CO₂ elevated to indicated concentrations. (n = 4–6 per concentration, 40 flies/trial).

Figure 5. T-maze behavior and receptor dependency in Drosophila

(A) Bar graph of olfactory behavior for the indicated odorants (1% concentration) and genotypes in a T-maze: n = 6–16 trials per odorant per genotype. (B) Shown are the first two principle components on a 24 dimensional Odorant receptor response space for 54 odorants from Halem and Carlson, 2006. Numbers in parenthesis indicate the fraction of variance in the data represented in each axis. (C) Scatter plot of mean preference index from T-maze assays of each of the 54 odorants (10⁻² dilution) vs. Principle component 1 values for each odorant. R = Pearson correlation coefficient. (D) Mean preference index in a T-maze behavior assay for the indicated odorants (10⁻² dilution) in wildtype and orco− D. melanogaster Error bars are s.e.m. t-test; p*<0.05, **<0.005, ***<0.001.

Figure 6. Broadly tuned CO₂ receptor neuron activity inversely correlates with behavior

(A) Mean ab1C electrophysiology activity to a panel of the indicated odorants (10⁻² dilution) normalized from +1 to −1 using 1-Butanol response, and “0” as background activity. Recordings were performed in orco− background for ease of counting ab1C action potentials (n= 5–14). Mean ab1C electrophysiology activity is overlaid with the mean preference index of flies in a T-maze of indicated genotype (from Figure 5D). (B,C) Data from (A) shown as a scatter plots of ab1C ligand activity vs. mean preference index in T-maze behavior for D. melanogaster that are wild-type (B), and orco− mutant (C). Pearson’s correlation coefficient (R) is indicated for each plot showing significant correlation. (D The evolutionary rate (top) of receptors obtained from OrthoDB from Dipteran inclusive orthologs. All receptors from the Or, Ir and Gr family that are known to play a role in olfaction or expressed in the olfactory system are included that appear in the Dipteran inclusive ortholog search. Red arrowheads indicate known co-receptors. (Bottom) Percentage amino acid identity of the top BLASTP hit for the indicated D. melanogaster receptor in the two indicated mosquito genomes. (F) Model: In Drosophila melanogaster activation in a dose-dependent manner of the CO₂ receptor neuron leads to avoidance, and inhibition in a dose-dependent manner leads to attraction.

Figure 7. CO₂ receptor neuron activity is required for prediction of odor valence

A) Sample workflow of the modeling approach. The T-maze preference index for 54 odor × 24 Or-response matrix was used to predict the PI; this Or-only model was initially fit using OLS regression and was then retested for fit after adding ab1C activity for the 54 odorants. Uninformative predictors were removed and the reduced model was validated. B) Tabulated measures of fit are shown for the labeled model on the original data. C) Predicted PI was plotted as a function of the observed PI for the 24OR+ ab1C model; the red line depicts the linear trend while the overlaying gray band is the standard error for the fit. D) Predictors that are selected most frequently and their selection rates, across 5000 iterations of stepwise regression, resampling the 54-odorant set on each run. The black vertical line is the empirically determined threshold for consistent selection out of 5000 iterations. E) Linear equation of the optimal predictors. Units for the coefficients reflect the Z transformed spikes/s. F) Average performance on 1000 cross-validation test folds is shown for two models. To ensure optimal performance and stability of the larger Or-only model, the test average is shown for ridge regression and compared to ab1C alone using OLS regression.