Raw data for oviposition experiments displayed in Fig 2.

Egg-laying rate of each replicate, on each egg-laying substrate for two-choice assays (Fig 2A, 2B, 2E and 2F) or on the single substrate for oviposition stimulation experiments (Fig 2C and 2D). (XLSX)</p

Control experiments with the ripe and fermented substrates.

Controlled fermentation of a ripe strawberry purée with Saccharomyces cerevisiae and Acetobacter pomorum provides a model substrate for fermented fruit. (A) Glucose, fructose, acetic acid, and ethanol levels measured in g/L (mean + standard deviation) in the ripe and fermented purées before dilution in the oviposition substrates. Fermentation effectively depletes sugars and produces acetic acid and ethanol. (B) Mean final concentrations after dilution in the oviposition substrates. (C) Diet does not significantly alter the species’ oviposition preferences for ripe vs. fermented substrates. The 2 species were allowed to develop on Nutrifly medium during the larval stages and either maintained on this medium during adulthood (black) or switched to standard cornmeal medium at eclosion (gray). D. melanogaster was also grown directly on standard medium during the larval stages (light blue), but we were unable to grow D. suzukii under these conditions. (Left) The oviposition preferences are independent of diet; (right) the total oviposition rate is significantly reduced for D. suzukii when aged on standard medium (n = 50, 30, 30, 50, 48). We therefore performed all experiments with flies reared on Nutrifly medium, which, unlike the standard medium, elicits an equivalent oviposition rate in both species. The data underlying this figure can be found in S1 Data. (EPS)</p

Raw data from Fig 2A and 2E: Egg-laying rates on individual substrates in two-choice assays opposing varying sugar concentrations to plain agar.

(A) Raw data from Fig 2A: egg-laying rate on individual substrates from two-choice oviposition assays opposing 1.6% glucose + fructose to plain agar in 3 experimental setups of different sizes. For each replicate, the egg-laying rate is shown for the agar side (indicated by empty boxes at the bottom) and the sugar side (pink boxes). While D. melanogaster lays almost no eggs on the plain agar side, D. suzukii lays a significant number of eggs, resulting in a reduced strength of preference for sugar compared to D. melanogaster. (B) Oviposition preference in two-choice assays under conditions previously shown to induce sugar rejection in D. melanogaster. D. melanogaster avoids laying on sucrose and chooses plain agarose when acetic acid and ethanol are present on both sides in experimental setups corresponding to our small chambers [29,30]. We repeated these experiments with D. melanogaster and D. suzukii, opposing 200 mM sucrose to plain agarose in the presence of 0.8% acetic acid and 1.6% ethanol (orange boxes) on both sides, both substrates with a hardness of 1%, in small chambers. Consistent with published results, D. melanogaster rejects sucrose in the presence of acetic acid and ethanol, as does D. suzukii. However, both species oviposit on the sucrose side in the absence of acetic acid and ethanol, showing that sugar rejection under these conditions is due to an interaction with fermentation products. (C) Raw data from Fig 2E: egg-laying rate on individual substrates from two-choice assays opposing increasing concentrations of glucose (0.01% to 1%) to plain agar. The data underlying this figure can be found in S2 Data. (EPS)</p

Raw data for S1 Fig.

S1A: glucose, fructose, acetic acid, and ethanol levels (g/L) measured in ripe (after ½ dilution in water) and fermented purées. S1B: final glucose, fructose, acetic acid, and ethanol levels (expressed as % w/v) in the oviposition substrates used in behavioral assays. S1C: Egg-laying rate of each replicate, on each egg-laying substrate of two-choice assays. (XLSX)</p

S4B: Raw data for GCaMP experiments displayed in S4B Fig: normalized peak ΔF/F₀ data for each brain for each condition and genotype.

S4C: Egg-laying rate of each replicate for oviposition stimulation assay displayed in S4C Fig. S4D: GCaMP peak ΔF/F0 data for each brain of each genotype. (XLSX)</p

<i>D</i>. <i>suzukii</i> responds to lower concentrations of sugar than <i>D</i>. <i>melanogaster</i> in oviposition assays.

(A) Two-choice oviposition assays with sugar at the concentration of the ripe strawberry substrate (1.6% glucose + fructose) versus plain agar (empty box at the bottom) in 3 different experimental setups (see Methods for details). Both wild-type D. melanogaster and D. suzukii prefer sugar in each experimental setup, but the preference is more pronounced in D. melanogaster (n = 12, 20, 16, 30, 31, 48; see raw data in S2A Fig). (B) Two-choice assays opposing different concentrations of glucose. D. suzukii does not discriminate between concentrations when both are higher than approximately 1% glucose, whereas D. melanogaster always shows preference for the higher concentration (n = 35, 45, 23, 30, 43, 44, 29, 30). (C, D) Oviposition stimulation assays (no-choice) on increasing concentrations of (C) glucose, fructose, and sucrose and (D) glucose with 2 other wild-type strains of D. melanogaster (iso1) and D. suzukii (AM). Data are shown as the mean (dots + lines) +/− standard deviation (shaded areas). Sugar concentration (x-axis) is on a logarithmic scale. The estimated EC50s are shown at the bottom and with the dashed lines. The EC50 is consistently lower for D. suzukii compared to D. melanogaster (n = 30 for each condition). (E) Two-choice assays with low concentrations of glucose versus plain agar. The proportion of replicates choosing glucose (defined as preference index >0.2) is shown by blue/red bars, the proportion of replicates choosing agar (preference index D. suzukii replicates choose sugar at low concentrations (0.05% to 0.5%) compared to D. melanogaster (n = 45 for each condition; see raw data in S2C Fig). (F) Two-choice assay opposing sugar concentrations corresponding to those of the ripe and fermented substrates (1.6% vs 0.2% glucose + fructose, respectively). D. suzukii shows a significant preference for the higher concentration substrate (n = 33, 45). The data underlying this figure can be found in S2 Dataset.</p

Sugar is valued more highly by <i>D</i>. <i>suzukii</i> in two-choice oviposition assays on natural fruit substrates.

(A) Oviposition substrate preference in a two-choice assay in a large chamber (see Methods for details) opposing a ripe strawberry purée (indicated by the red box above) and the same purée fermented for 3 d under controlled conditions (brown box below; see S1 Fig for additional information and controls). Preference is quantified by a preference index (see Methods). Filled circles in this and the following graphs indicate a significant preference for one of the 2 substrates (Mann–Whitney paired test); open circles indicate no significant preference for either substrate. Shaded bars: mean, error bars: standard deviation. D. melanogaster (blue) and D. suzukii (red) show opposite preferences for these substrates. Species preferences are significantly different from each other; Mann–Whitney U test, p-values indicated on the graph (n = 35, 30 replicates). (B) Stimulation (no-choice) assays with these substrates (indicated by red and brown boxes, respectively) show that neither is repulsive to the 2 species, both stimulate oviposition to a similar extent when presented alone (n = 20, 20, 20, 20). (C) Oviposition assay with ripe vs. fermented substrates for several Drosophila species (for some species, multiple wild-type strains were used; see Methods for species names; n = 50, 20, 20, 30, 20, 20, 20, 18, 20, 20, 20, 15, 15, 20, 20, 49, 17, 20, 20, 18, 20). (D) Two-choice assays opposing sugar alone (glucose + fructose at the concentration found in the ripe substrate, i.e.: 1.6%, indicated by the pink box above the graph) versus agar or agar with acetic acid (1%, similar to fermented substrates, orange boxes below the graph). D. melanogaster and D. suzukii show opposite preferences for sugar vs. acetic acid (n = 36, 39, 40, 40). (E, F) Relative value of acetic acid and sugar in the ripe and fermented substrates. (E) Adding 1% acetic acid to the ripe substrate shifts oviposition preferences toward acetic acid to a similar extent in both species (n = 40, 40, 40, 39). (F) Adding 1.6% sugar to the fermented substrate abolishes the preference of D. suzukii for the ripe substrate but does not shift D. melanogaster’s preference for the fermented substrate (n = 30, 30, 30, 30). The data underlying this figure can be found in S1 Dataset.</p

Raw data for oviposition experiments displayed in S2 Fig.

Egg-laying rate of each replicate, on each egg-laying substrate. (XLSX)</p

Comparative calcium imaging of sugar responses in the PNS.

(A) GCaMP7s imaging in the synaptic terminals of sugar-GRNs in the SEZ upon stimulation of the proboscis with glucose. Example images are shown for the 2 species 15 s before stimulation, at the peak response, and 15 s after stimulation. Fluorescence intensity is color coded (scales on the side). ROIs used for quantification are indicated (dashed circles). (B, C) Stimulation with water, glucose, fructose, sucrose, and KCl. (B) ΔF/F0 traces of individual ROIs plotted as heat maps (color scale at bottom right) for the different conditions. The period of water or sugar stimulation is indicated by gray rectangles at the bottom. (C) Distribution of peak ΔF/F0 for each condition (shaded area: mean, error bars: standard deviation). The magnitude of the calcium response is generally higher for D. melanogaster at high sugar concentrations (Mann–Whitney U test, p-values indicated on the graph). p-Values in color below the graph indicate statistical comparisons with water controls (n = 9, 11, 10, 10, 8, 9, 8, 10, 10, 9, 8, 7, 8, 6, 7, 6, 8, 8, 22, 23 brains). See S4 Fig for additional information. (D, E) Manipulation of sugar perception in D. melanogaster shifts its oviposition preference in ripe versus fermented substrate assays. (D) Increasing sugar input to the CNS via UAS-NaChBac expression in sugar GRNs significantly increases the value of the ripe substrate. Compared to parental controls (gray and black), Gr64af>NaChBac females (green) show a shift in preference toward the ripe substrate (n = 30, 30, 30, 30). (E) Increasing the sugar concentration of the ripe substrate increases its value relative to the fermented substrate for D. melanogaster. One dose of glucose + fructose (1.6%) added to the ripe substrate (which already contains 1.6% sugar) eliminates the preference for the fermented substrate, whereas the addition of 2 doses (3.2%) reverses the preference in favor of the sweeter ripe substrate (n = 30 for each condition). The data underlying this figure can be found in S4 Dataset.</p

Raw data for oviposition experiments displayed in Fig 3D and 3E.

Egg-laying rate of each replicate, on each egg-laying substrate. (XLSX)</p