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Increased sugar valuation contributes to the evolutionary shift in egg-laying behavior of the fruit pest Drosophila suzukii [1]

['Matthieu Cavey', 'Aix-Marseille Université', 'Cnrs', 'Ibdm', 'Institut De Biologie Du Développement De Marseille', 'Campus De Luminy Case', 'Marseille', 'Bernard Charroux', 'Solène Travaillard', 'Gérard Manière']

Date: 2023-12

Behavior evolution can promote the emergence of agricultural pests by changing their ecological niche. For example, the insect pest Drosophila suzukii has shifted its oviposition (egg-laying) niche from fermented fruits to ripe, nonfermented fruits, causing significant damage to a wide range of fruit crops worldwide. We investigate the chemosensory changes underlying this evolutionary shift and ask whether fruit sugars, which are depleted during fermentation, are important gustatory cues that direct D. suzukii oviposition to sweet, ripe fruits. We show that D. suzukii has expanded its range of oviposition responses to lower sugar concentrations than the model D. melanogaster, which prefers to lay eggs on fermented fruit. The increased response of D. suzukii to sugar correlates with an increase in the value of sugar relative to a fermented strawberry substrate in oviposition decisions. In addition, we show by genetic manipulation of sugar-gustatory receptor neurons (GRNs) that sugar perception is required for D. suzukii to prefer a ripe substrate over a fermented substrate, but not for D. melanogaster to prefer the fermented substrate. Thus, sugar is a major determinant of D. suzukii’s choice of complex substrates. Calcium imaging experiments in the brain’s primary gustatory center (suboesophageal zone) show that D. suzukii GRNs are not more sensitive to sugar than their D. melanogaster counterparts, suggesting that increased sugar valuation is encoded in downstream circuits of the central nervous system (CNS). Taken together, our data suggest that evolutionary changes in central brain sugar valuation computations are involved in driving D. suzukii’s oviposition preference for sweet, ripe fruit.

Funding: This work was supported by funds from: -French National Research Agency (EvoSugar ANR-19-CE16-0007, MC, https://anr.fr/ ) -European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013 / ERC Grant Agreement n° 615789, BP) -A*MIDEX project (n° ANR-11-IDEX-0001-02, BP) funded by the « France 2030» French Government program, managed by the French National Research Agency (ANR, https://anr.fr/ ) -France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INSB-04-01, call "France 2030", BP, https://anr.fr/ ) -Centre National de la Recherche Scientifique (CNRS, BP, YG, https://www.cnrs.fr/en ) -Université de Bourgogne (YG, https://www.u-bourgogne.fr/ ) -Conseil Régional Bourgogne Franche-Comte (PARI grant, YG, https://www.bourgognefranchecomte.fr/ ) -FEDER (European Funding for Regional Economical Development, YG, https://ec.europa.eu/regional_policy/funding/erdf_en ) -European Council (ERC starting grant, GliSFCo-311403, YG) -ANR (PEPNEURON, YG, https://anr.fr/ ) -SATT-Grand Est/Sayens (DrosoMous, YG, MBG, https://www.sayens.fr/ ) -Burgundy council (ALIMENN, GM, https://www.bourgognefranchecomte.fr/ ) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we evaluate and compare in detail the role of sugar perception in oviposition decisions in D. suzukii and D. melanogaster. Using behavioral assays and genetic manipulation of sugar gustatory perception, we show that sugar has, in fact, a higher value as an oviposition cue for D. suzukii than for D. melanogaster and that sugar perception is required to drive D. suzukii’s preference for ripe fruit substrates over fermented fruit substrates. Thus, an increased valuation of sugar has contributed to the evolutionary shift in D. suzukii. Calcium imaging experiments suggest that increased sugar valuation is not immediately encoded at the level of the sensory neurons. Our data support the idea that changes in the processing of sugar sensory information have played a central role in the behavioral divergence of D. suzukii.

Fruit sugars—mainly glucose and fructose—are common to all ripe fruits and are gradually degraded during fruit decay, making them potential indicators of fruit maturity for oviposition decisions. Interestingly, D. suzukii shows a weaker preference than D. melanogaster for sugars (glucose, fructose, and sucrose) over plain agarose in oviposition choice assays. This correlates with a reduced expression of some of the sugar-gustatory receptor genes in taste organs and reduced sensitivity of its gustatory receptor neurons (GRNs) to sucrose and fructose compared to D. melanogaster. However, GRN sensitivity to glucose is not reduced in D. suzukii compared to D. melanogaster, despite the weaker behavioral response to this sugar [ 22 ]. Thus, it remains unclear whether physiological changes in sugar-GRNs underlie the divergent behavioral responses to sugar in D. suzukii. Furthermore, whether and how these physiological differences in sugar-sensing neurons might contribute to the enhanced preference of D. suzukii for ripe fruit substrates remains to be addressed.

Evolution of host preferences, for feeding or oviposition, often involves changes in sensory responses that promote behavioral tuning to host-specific chemical cues [ 1 – 11 ]. Changes in the central nervous system (CNS) affecting specific genes or wiring patterns have also been identified, but unraveling how they contribute to behavioral shifts is more challenging [ 1 , 12 – 14 ]. Natural breeding sites for fruit flies are not very well documented, but they are known to lay their eggs on decaying organic material, presumably among other substrates. Drosophila suzukii has evolved a novel preference for laying its eggs in a wide variety of ripe fruits (for instance, strawberries, raspberries, cherries…), in contrast to most other Drosophila species, such as D. melanogaster, which prefer to lay eggs on fermented and rotten fruits. This novel behavior is causing significant damage to the fruit industry as D. suzukii spreads around the world [ 15 , 16 ]. D. suzukii’s adaptation has involved both morphological evolution of its ovipositor, which allows it to pierce hard fruit skins [ 17 , 18 ], and changes in behavioral responses to mechanosensory and chemosensory cues [ 19 – 22 ]. Chemosensory cues are likely to involve multiple molecules. For instance, D. suzukii shows increased oviposition responses to olfactory cues from ripe fruit [ 19 ]. Conversely, reduced gustatory responses to bitter compounds have been proposed to alleviate a hypothetical oviposition inhibition by ripe substrates [ 20 ]. In addition, an increased behavioral response to fermentation by-products may contribute to the repulsion of D. suzukii by fermented substrates [ 21 ].

Results

Sugar has a higher value for D. suzukii’s oviposition preference on fruit substrates The fruit maturity stages preferred by D. suzukii and D. melanogaster used in previous studies are loosely defined as a fruit matures progressively from unripe, to ripe, to overripe, to fermented and rotten. This limits our ability to identify the relevant cues for interspecies differences in substrate preference. We therefore developed a controlled-fermentation protocol using industrial strawberry purée as a starting ripe substrate to which we added yeast and bacteria to deplete fruit sugars and produce fermentation products. We measured the concentration of fermentation markers (sugar, acetic acid, and ethanol) to assess the outcome of our fermentation reaction and found that sugars were effectively depleted (S1A and S1B Fig). When offered the choice of laying eggs on the ripe or fermented substrates, D. suzukii and D. melanogaster recapitulated the opposite preferences reported for whole fruit [19] (Fig 1A). Both ripe and fermented substrates were acceptable to both species when presented individually (Fig 1B), so the difference in behavior clearly reflects a divergence in preference rather than, for instance, repulsion by a substrate in one species. Strikingly, D. suzukii was one of the very few species to prefer the ripe substrate among a range of closely and more distantly related species (Fig 1C). We also verified that the divergent preferences were not due to different adaptations to the sugar-rich diet on which the flies were housed, as this could have biased chemosensory responses [23] (S1C Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Sugar is valued more highly by D. suzukii 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. https://doi.org/10.1371/journal.pbio.3002432.g001 We then investigated the contribution of specific chemical cues to this behavioral divergence. We focused on fruit sugars, glucose and fructose, which are abundant in ripe fruit, and we chose acetic acid as a fermentation marker because it is produced from sugar degradation and has previously been shown to act as an oviposition cue for D. melanogaster [24–26]. We first opposed sugar-alone (a mixture of glucose + fructose at a 1.6% (w/v) concentration, similar to our ripe strawberry substrate) to acetic acid-alone (at a 1% concentration, similar to our fermented substrate). Interestingly, there was a clear behavioral divergence between species: D. melanogaster preferred acetic acid to sugar, whereas D. suzukii showed the opposite preference (Fig 1D). Thus, sugar and acetic acid alone, when presented at concentrations corresponding to those of the ripe and fermented substrates, respectively, recapitulate quite well the species divergence observed on complex fruit substrates at different stages of maturity. This suggests that these cues may play a determinant role in species preferences on natural substrates. To test this further, we assessed the relative importance of acetic acid and sugar in oviposition decisions in the ripe versus fermented substrate choice assay. The addition of acetic acid to the ripe substrate induced a preference shift toward the ripe substrate of similar magnitude in both species (Fig 1E). Thus, under these conditions, acetic acid appears to exert a similar weight on oviposition decisions in the 2 species. In contrast, adding sugar to the fermented substrate was sufficient to abolish D. suzukii’s preference for the ripe substrate, whereas it did not increase D. melanogaster’s preference for the fermented substrate (Fig 1F). Thus, despite presumably important differences in their chemical composition, the ripe and fermented oviposition substrates are of equal value to D. suzukii as long as their sugar concentrations are the same, whereas sugar content appears to be unimportant to D. melanogaster. These results suggest that sugar plays a particularly important role in the decision of D. suzukii and has a higher value relative to the fermented substrate for this species than for D. melanogaster. This led us to focus on sugar and investigate its potential role in evolutionary changes in D. suzukii’s oviposition behavior.

Higher oviposition responses to sugar in D. suzukii Our results contrast with a previous report suggesting that sugar perception may not be critical for D. suzukii’s oviposition decisions, based on the observation that D. suzukii’s preference for sugar was weaker than that of D. melanogaster in two-choice assays against plain agarose [22]. We therefore decided to reexamine thoroughly D. suzukii’s oviposition responses to sugar in different types of assays. Since the oviposition response of D. melanogaster to sugar has been shown to be context dependent and can range from sugar preference to sugar rejection [27,28], we tested different experimental conditions (chamber size and substrate composition). When sugar was opposed to plain agar, both species preferred to lay on the sugar side, and this was true in all 3 different experimental contexts (Fig 2A, raw egg-laying rate data in S2A Fig). Sugar preference was reversed for both species in the presence of fermentation cues (S2B Fig) as previously reported for D. melanogaster [29,30]. Consistent with published data [22], we observed weaker preferences for sugar in D. suzukii compared to D. melanogaster (Fig 2A), and D. suzukii showed poor discrimination ability when faced with 2 different sugar concentrations compared to D. melanogaster (Fig 2B). However, we found that D. suzukii is naturally stimulated to lay eggs by plain agar, whereas D. melanogaster is not (S2A Fig and see below). This effectively reduces D. suzukii’s apparent preference for sugar in these assays, complicating the interpretation of the two-choice experiments. PPT PowerPoint slide

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TIFF original image Download: Fig 2. D. suzukii responds to lower concentrations of sugar than D. melanogaster 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 <−0.2) in dark gray, and the proportion with no oviposition response (egg-laying rate <5 eggs) or no choice in light gray. A greater proportion of 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. https://doi.org/10.1371/journal.pbio.3002432.g002 To address this issue, we directly measured the potency of individual sugars in stimulating oviposition for both species using no-choice assays over a wide range of concentrations for the 3 sugars, glucose, fructose, and sucrose. Both species responded positively to sugar in a dose-dependent manner, with D. suzukii exhibiting a higher basal egg-laying rate on plain agar, as we have previously noted (Fig 2C and 2D). Strikingly, however, D. suzukii began to respond to sugar (i.e., inflection of the curve from basal levels) and reached its maximum oviposition rate at lower sugar concentrations than D. melanogaster. Potency can be quantified by the effective concentration 50 (EC50; sugar concentration that induces half-maximal oviposition rate), which corrects for differences in basal oviposition rates. The EC50s were consistently lower for D. suzukii compared to D. melanogaster for all 3 sugars tested (4- to 10-fold lower). Similar differences were observed with other wild-type strains of these species (Fig 2D). D. suzukii is thus more responsive to sugar stimulation than D. melanogaster, and these results suggest that its weaker preference for sugar observed in two-choice assays (Fig 2A) may not simply reflect a lower valuation of the stimulus of interest. Furthermore, these results suggest that the lower ability of D. suzukii to discriminate between sugar concentrations (Fig 2B) may, in fact, be due to a higher behavioral responsiveness to sugar rather than a lower detection sensitivity. To further confirm these results, we decided to perform additional two-choice assays using very low concentrations of sugar against plain agar to determine at what concentrations oviposition preference on sugar was first detectable in the 2 species. As there were too few replicates with enough eggs on the lowest sugar concentration substrates, we could not rely on the oviposition preference index. Instead, for each condition, we counted the number of replicates showing a clear positive response to sugar compared to replicates showing either sugar rejection (agar choice) or no choice or no oviposition response (no eggs). Strikingly, D. suzukii showed a marked increase in the proportion of positive responses to glucose at lower concentrations than D. melanogaster (from 0.05% glucose for D. suzukii compared to 0.5% for D. melanogaster; Fig 2E, raw data in S2C Fig). These results are consistent with the dose–response experiments and suggest that sugar detection at low concentrations is more likely to trigger oviposition in D. suzukii than in D. melanogaster. Taken together, our results suggest that the weaker preference of D. suzukii for sugars observed in specific experimental contexts must be interpreted with caution and does not rule out a role for this cue in biasing D. suzukii’s preference for sugar-rich substrates in natural contexts. In line with this idea, we asked whether D. suzukii was able to discriminate between the sugar concentrations present in our ripe (1.6% glucose + fructose) and fermented (approximately 0.2%) strawberry substrates. Indeed, D. suzukii chose the higher concentration substrate (Fig 2F) and could therefore, in principle, use this information when choosing between ripe and fermented substrates.

Sugar perception is required for ripe substrate preference in D. suzukii To formally test the hypothesis that sugar perception is required to guide oviposition choice in D. suzukii, we generated genetic tools to manipulate sugar perception in D. suzukii. Sugars are sensed in D. melanogaster by a family of 9 partially redundant gustatory receptors expressed in approximately 100 GRNs on different body parts and internally (reviewed in [31,32]). We generated a pan-sugar-GRN Gal4 line in D. suzukii homologous to the DmelGr64af-Gal4 line previously shown to drive Gal4 expression in almost all sugar-GRNs [33–35]. Our DsuzGr64af-Gal4 line labels neurons in the main gustatory organs, the labellum and tarsi, in a highly reproducible pattern reminiscent of D. melanogaster (Fig 3A and 3B). Axonal projection patterns in the central brain are also very similar between species (S3A Fig). Neuron counts from our Gal4 line agree very well with those obtained from electrophysiological recordings in D. suzukii [22] and collectively show an overall reduction in sugar-GRNs in D. suzukii compared to D. melanogaster on the proboscis and forelegs (Fig 3C). PPT PowerPoint slide

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TIFF original image Download: Fig 3. Sugar sensing is required for ripe substrate preference in D. suzukii. DsuzGr64af-Gal4 expression in female proboscis (A) and legs (B) observed with a UAS-GCaMP7s-T2A-Tomato reporter. The mean number of positive neurons ± standard deviation is indicated for each organ on the upper side and each tarsus (n = 14 proboscises, forelegs, midlegs, and hindlegs imaged; see also S3A Fig for additional characterization of the DsuzGr64af-Gal4 line). (C) Comparative sugar GRN counts in D. melanogaster (blue) and D. suzukii (red) females determined from Gr64af-Gal4 reporter expression and electrophysiogical recordings of sensilla showing a consistent reduction in D. suzukii proboscis and forelegs compared to D. melanogaster (athis study; b[22]; c[33]; d[34]; e[35]; nd: not determined). (D) Two-choice oviposition assay with 1.6% glucose + fructose vs. plain agar. Sugar-GRN inhibition via UAS-Kir2.1 expression reduces—but does not completely abolish—the response to sugar to a similar extent in both species (n = 25, 18, 34, 56, 35, 46, 30). (E) Oviposition choice assay on ripe vs. fermented substrate. Sugar-GRN inhibition does not alter the preference of D. melanogaster but significantly reduces the preference of D. suzukii for the ripe substrate (n = 30, 33, 33, 34, 33, 35, 26, 34). The data underlying this figure can be found in S3 Dataset. https://doi.org/10.1371/journal.pbio.3002432.g003 Next, we functionally validated our DsuzGr64af-Gal4 line with a hyperpolarizing UAS-Kir2.1 transgene [36] we generated in D. suzukii. Silencing sugar-GRNs with Kir2.1 significantly reduced sugar preference of both species to a similar extent in two-choice oviposition assays (Fig 3D). However, in both species, sugar preference was not completely abolished, suggesting an incomplete inhibition of sugar perception (which could, for example, be related to insufficient strength of transgene expression). In conclusion, our DsuzGr64af-Gal4 line appears to target the full complement of sugar-GRNs in D. suzukii and produces similar effects in behavioral assays to existing tools in D. melanogaster. We then functionally tested the contribution of sugar sensing to oviposition decisions on complex fruit substrates in D. melanogaster and D. suzukii. Remarkably, while silencing sugar-GRNs had no effect on D. melanogaster’s preference for the fermented substrate over the ripe one, it significantly reduced D. suzukii’s preference for the ripe substrate over the fermented substrate (Fig 3E). This manipulation pushed D. suzukii close to indifference between the 2 substrates and closer to the D. melanogaster state. Since a subset of sugar-GRNs in D. melanogaster have been shown to respond to the fermentation product acetic acid, which can act as an oviposition cue [24–26], we asked whether defects in acetic acid perception could contribute to this phenotype. However, silencing of sugar-GRNs did not affect the oviposition responses of D. suzukii (or D. melanogaster) to acetic acid at concentrations similar to those of our fermented substrate (0.5% to 1%; S3B Fig). In conclusion, our results show that sugar perception in D. suzukii significantly contributes to oviposition preference toward a ripe substrate, confirming that sugar is one of the major determinants of preference on natural substrates in D. suzukii.

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