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The Fungal Aroma Gene ATF1 Promotes Dispersal of Yeast Cells through Insect Vectors
Report
Graphical Abstract
Highlights
The S. cerevisiae ATF1 gene controls the production of volatile acetate esters
Aroma of ATF1 mutants elicits different neuronal activity in the fly antennal lobe
Flies are significantly more attracted to wild-type yeast than to atfl-null mutants
Authors
Joaquin F. Christiaens, Luis M. Franco.....
Emre Yaksi, Kevin J. Verstrepen
Correspondence
emre.yaksi@nerf.be (E.Y.),
kevin.verstrepen@biw.vib-kuleuven.be
(K.J.V.)
In Brief
Yeast cells produce several different volatile acetate esters. Whereas these fruity aroma compounds are key contributors to the pleasing aroma of fermented beverages like beer and wine, their physiological role for the yeast cells that produce them remains unknown. Christiaens et al. show that two acetate esters, ethyl acetate and isoamyl acetate, help to attract fruit flies that serve as vectors that promote dispersal of the yeast cells. Deletion of the yeast ATF1 gene, encoding a key acetate ester synthase, drastically reduces Drosophila attraction and therefore limits yeast dispersal.
Addition of isoamyl acetate and ethyl acetate restores attraction of Drosophila
Christiaens et al., 2014, Cell Reports 9,1-8 October 23, 2014 ©2014 The Authors http://dx.doi.org/10.1016/j.celrep.2014.09.009
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Cell Reports
Report
The Fungal Aroma Gene ATF1 Promotes Dispersal of Yeast Cells through Insect Vectors
Joaquin F. Christiaens,1'2'3 9 Luis M. Franco,3'4 5 9 Tanne L. Cools,12 Luc De Meester,6 Jan Michiels,1 Tom Wenseleers,6 Bassem A. Hassan,3 4 Emre Yaksi,5'7'8'10'* and Kevin J. Verstrepen1'2'10'*
laboratory for Genetics and Genomics, Centre of Microbial and Plant Genetics (CMPG), Department of Microbial and Molecular Systems, KU Leuven, Gaston Geenslaan 1, 3001 Leuven (Heverlee), Belgium
2VIB Laboratory of Systems Biology, Gaston Geenslaan 1, 3001 Leuven (Heverlee), Belgium 3Centre for Human Genetics, KU Leuven School of Medicine, Herestraat 49, 3000 Leuven, Belgium 4VIB Centre for the Biology of Disease, Herestraat 49, 3000 Leuven, Belgium 5Neuroelectronics Research Flanders (NERF), Kapeldreef 75, 3001 Leuven, Belgium
6Animal Ecology and Systematics Section, Department of Biology, KU Leuven, Charles Deberiotstraat 32, 3000 Leuven, Belgium
7VIB, Kapeldreef 75, 3001 Leuven, Belgium
8KU Leuven, Kapeldreef 75, 3001 Leuven, Belgium
9Co-first author
10Co-senior author
'Correspondence: emre.yaksi@nerf.be (E.Y.), kevin.verstrepen@biw.vib-kuleuven.be (K.J.V.) http://dx.doi.org/10.10167j.celrep.2014.09.009
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
SUMMARY
Yeast cells produce various volatile metabolites that are key contributors to the pleasing fruity and flowery aroma of fermented beverages. Several of these fruity metabolites, including isoamyl acetate and ethyl acetate, are produced by a dedicated enzyme, the alcohol acetyl transferase Atf1. However, despite much research, the physiological role of acetate ester formation in yeast remains unknown. Using a combination of molecular biology, neurobiology, and behavioral tests, we demonstrate that deletion of ATF1 alters the olfactory response in the antennal lobe of fruit flies that feed on yeast cells. The flies are much less attracted to the mutant yeast cells, and this in turn results in reduced dispersal of the mutant yeast cells by the flies. Together, our results uncover the molecular details of an intriguing aroma-based communication and mutualism between microbes and their insect vectors. Similar mechanisms may exist in other microbes, including microbes on flowering plants and pathogens.
INTRODUCTION
Microbes produce many secondary metabolites, several of which have strong aromas and are central contributors to thefla-vor of fermented foods and beverages such as cheese, wine, chocolate, and beer (Styger et al., 2011; Swiegers et al., 2005). Flavor formation has been extensively studied in the common brewer's yeast S. cerevisiae, which produces several volatile acetate esters such as ethyl acetate (pear aroma), isoamyl acetate (banana aroma), and phenylethyl acetate (flowery aroma; Lambrechts and Pretorius, 2000; Nordstrom, 1966; Saerens
et al., 2008; Verstrepen et al., 2003a). These acetate esters are formed in a condensation reaction, catalyzed by an alcohol acetyl transferase (AAT), between acetyl-coenzyme A and various higher alcohols derived from the central carbon and amino acid metabolism. S. cerevisiae has two different genes coding for AATases, ATF1 and ATF2, of which ATF1 controls the bulk of the acetate ester formation (Lilly et al., 2000; Ver-strepen et al., 2003b). Although many studies have focused on the optimization of aroma formation, the physiological role of these compounds remains elusive and highly debated (Mason and Dufour, 2000; Saerens et al., 2010). Some reports argue that some esters might help maintain plasma membrane fluidity in low-oxygen conditions where the synthesis of unsaturated fatty acids is impaired (Mason and Dufour, 2000). Other hypotheses suggest that ester synthesis may help tune the redox balance (Malcorps and Dufour, 1992) or that esterification of small organic acids may facilitate their removal from cells through diffusion through the plasma membrane (Nordstrom, 1964). However, no evidence has been found for any of these hypotheses, and deletion of ATF1 does not reduce fitness under laboratory conditions (Saerens et al., 2010). Moreover, the most commonly used S. cerevisiae laboratory strains show significantly lower production of aroma compounds compared to their wild and industrial relatives (Verstrepen et al., 2003b), suggesting that the synthesis of these compounds has not been selected for under laboratory culture conditions and might rather be related to survival in complex natural environments.
In this study, we set out to investigate acetate ester production in the context of the well-established relationship between yeasts and their insect vectors (Fogleman et al., 1981; Giglioli, 1897; Phaff et al., 1956; Stefanini et al., 2012; Suh et al., 2005). Numerous studies have shown that, unlike fungi that disperse through air (Roper et al., 2010) or motile bacteria, sessile microbes like yeasts depend on animal and especially insect vectors for their dispersal (Francesca et al., 2012; Ganter et al., 1986; Goddard et al., 2010). In turn, microbes serve as a
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Figure 1. Drosophila Transport Yeast Cells in a Laboratory Setup
Yeast cells present on the front leg of a Drosophila fly. Sterile Individual flies (n = 11) were allowed to roam a plate containing a single fluorescent yeast colony (Movie S1) for 1 hr. Afterward, flies were dissected and yeast cells on the various appendages were visualized using fluorescence microscopy. To quantify the number of cells that were transported, each part of the body was washed in a peptone buffer and the buffer was subsequently plated for cell counting (Table S1).
protein-rich food source for these insects and may even boost their immunocompetence (Anagnostou et al., 2010; Gibson and Hunter, 2010; Sang, 1956; Stamps et al., 2012). Previous studies have demonstrated that fermentation can increase the attractiveness of natural substrates toward Drosophila (Becher et al., 2012), even though there appears to be a variability in different yeast strains' ability to do so (Palanca et al., 2013). Additionally, multiple insect species are attracted to fruit and plant aroma mixtures that contain acetate esters (Boch et al., 1962; Galizia et al., 1999; Grosjean et al., 2011; Nojima et al., 2003). Finally, a few seminal studies have demonstrated that Drosophila antennae possess specific receptors for certain acetate esters (Elmore et al., 2003; Hallem and Carlson, 2006; Vosshall et al., 2000). Given this complex mutualism between microbes and insects, it seems likely that mechanisms evolved to facilitate these interactions. In this study, we show that a specific yeast gene, ATF1, produces acetate esters that change the neural representation of yeast odor in the Drosophila antennal lobe and help to attract insect vectors.
RESULTS
Yeast Strain Characterization
Experiments to investigate whether acetate esters help to attract insect vectors were carried out using two biological model systems: the fruit fly Drosophila melanogaster and the brewer's yeast Saccharomyces cerevisiae. To test the suitability of these
organisms for our research, we demonstrated that Drosophila do transport yeast cells in a laboratory setup and can indeed act as a vector (Figure 1; Movie S1; Table S1). However, the most commonly used S. cerevisiae lab strain, S288c, produces much less aroma compared to wild and industrial S. cerevisiae strains (Verstrepen et al., 2003b), making it a poor reference strain for this study. Hence, in order to select a suitable strain, we performed fermentations with 285 genetically diverse Saccharomyces sensu lato strains and analyzed their aroma profile (Figure S1). From this collection, we selected a representative diploid strain, coded Y182, for our experimental setup. This S. cerevisiae strain was isolated from a vineyard and has an average production of both isoamyl as well as ethyl acetate. Moreover, the strain also shows a relatively high efficiency for genetic transformation, which enabled us to use genetic engineering to create ATF1 deletion mutants. We deleted both alleles of ATF1 in this diploid strain and subsequently analyzed the concentration of aroma compounds in the fermentation products of the wild-type and null mutant strains, focusing on higher alcohols, acetate esters, and medium-chain fatty acid esters, as well as ethanol and acetic acid levels, compounds that have been proposed to attract Drosophila (Hallem and Carlson, 2006; Hallem et al., 2004). These analyses confirmed that the levels of various acetate esters were severely reduced or even completely abolished in the atf1~ mutant strain, whereas no significant differences in the concentration of other volatile compounds such as acetaldehyde or higher alcohols was observed (Table 1). The effect of the presence of ATF1 on the yeast's fitness was investigated using an automated multimode plate reader as well as automated time-lapse microscopy. In keeping with previous studies (Saerens et al., 2010), no difference in the growth rate between the wild-type (WT) and atf1~ mutant was observed in liquid or on solid media (see Supplemental Information for more information).
The Presence of ATF1 Leads to Increased D. melanogaster Attraction
To evaluate whether changes in the yeast's aroma profile caused by the deletion of the ATF1 gene lead D. melanogaster to prefer one strain over the other, we used an olfactory behavioral assay in a specially constructed arena (Figure 2). In this computer-controlled system, different airstreams can be released independently from each other out of the four corners of the isolated arena and cleared through a vacuum, applied in the center of the arena (Figure 2A). In this setup, the aromas from two different fermentations are delivered from opposing corners, whereas odorless air is streamed from the two remaining corners. For each olfactory behavioral assay, 50 flies were released in the arena and their positions were recorded with a camera placed above the arena. Subsequently, automated image analysis was used to quantify the number of flies in each of the four quadrants, before, during, and after odor delivery. Based on these numbers, a preference index was calculated for each input (see Figure 2 for details). During the first 2 min of each experiment, odorless air was delivered from all four corners of the arena. During this period, flies did not show preference for any specific quadrant. However, as soon as the airflow was switched so that two out of four corners contained the aroma of WT and atf1~ mutants
Table 1. Aroma Production by WT and ATF1 Strain
Compound (ppm) WT-Y182 SD atfí- SD p Value
Acetaldehyde Ethyl acetate Ethyl propionate 8.98 14.54 0.08 2.73 1.53 0.01 9.67 6.45 0.09 2.89 0.43 0.01 0.74 5.20 X 10 5a 0.19
Ethyl isobutyrate Propyl acetate Isobutyl acetate Ethyl butyrate Isobutanol 0.02 0.03 0.09 0.00 0.01 0.03 0.02 ND ND 0.00 ND ND 0.90 a a
20.24 13.38 3.26 2.57 20.40 13.31 2.23 1.60 0.94 0.96
Isoamyl acetate 0.82 0.20 0.13 0.06 5.68 X 104a
Butanol 55.45 2.21 57.67 5.22 0.53
Isoamyl alcohol 37.71 5.69 41.54 3.92 0.31
Ethyl hexanoate 0.13 0.03 0.18 0.01 0.04
Ethyl octanoate Phenyl ethyl acetate Phenyl ethanol 1.08 0.10 0.23 0.02 1.14 ND 0.13 ND 0.66 a
0.13 0.06 0.08 0.03 0.16
Ethanol (% v/v) 6.50 0.05 6.46 0.02 0.14
Acetate (g/l) 24.75 6.25 22.15 3.69 0.32
Concentrations of aroma compounds (in parts per million) produced by the diploid WTY182 strain and the isogenic ATF1 double-deletion mutant during fermentation (see Figure S1 for more information on Y182). For each strain, four fermentations were performed and each was analyzed in four technical replicates. Acetate esters are displayed in bold. These results demonstrate that the only compounds significantly affected by the ATF1~ deletion are acetate esters (ND, not detected). Production of these aroma compounds by non-Saccharomyces strains may be found in Table S2.
aSignificant difference between the WT and deletion strain (p < 0.05).
(each in one opposing corner), the flies significantly preferred the quadrant with the WT aroma over the aroma of atf1~ mutants (p value <1 x 10~4). Afterward, when the airstream at all corners was switched back to odorless control, the flies no longer preferred a specific quadrant and were once again distributed randomly (Figure 2; Movies S2 and S3).
These results indicate that Atf1 activity has a significant effect on the attraction of Drosophila by the yeast cells. To verify whether the observed behavioral differences are indeed due to the lower acetate ester levels produced by the atf1~ mutant, we supplemented the atf1~ fermentation medium with phenylethyl acetate, isoamyl acetate, and ethyl acetate (the three important acetate esters most affected by the ATF1 deletion), so that the final concentration of each ester was equal to that in the WT fermentation medium. We then repeated the olfactory preference tests with this supplemented medium pitted against the fruity WT medium, and no significant difference in preference for either medium was observed, clearly illustrating the central role of phenylethyl, isoamyl, and ethyl acetate in the altered attraction phenotype (Figures 2C and 2D). To further tease apart the specific contribution of each ester, we supplemented the atf1~ medium with each ester separately and tested these against the WT medium. These experiments revealed that ethyl acetate is the most important compound, because addition of this ester is sufficient to
make the atf1~ medium as attractive as the WT medium in our olfactory behavioral assay. Addition of isoamyl acetate resulted in increased attraction compared to the unsupple-mented atf1~ medium, but the WT attraction was not fully restored. Finally, phenylethyl acetate did not significantly affect Drosophila behavior.
To further confirm the role of the yeast ATF1 gene in Drosophila attraction, we set up a second series of preference experiments, this time in larger cages (30 x 30 x 20 cm), allowing flies more freedom of movement in three dimensions, which has been implicated to alter their behavior compared to setups where their movement is more restricted (Becher et al., 2010; Lebreton et al., 2012). Analogous with the olfactory assay in the smaller arena, we placed traps containing either WT or atf1~ mutant fermentation medium in opposing corners of the larger cages and two traps containing only water in the two remaining corners. To account for position effects, the traps were switched across different repeats. Our results further confirmed a clear preference of D. melanogaster for the WT strain, attracting 2-fold more flies than the atf1~ strain (see Figure S2 for details).
WT and atfl Aromas Are Perceived Differently by Drosophila
To further evaluate the effect of isoamyl and ethyl acetate on the Saccharomyces-Drosophila interaction, we used in vivo calcium imaging to measure the neuronal responses to atf1~ and WT yeast aroma in the antennal lobe of a ;Gal4-GH146/UAS-GCaMP6m; Drosophila strain expressing the transgenic calcium indicator GCaMP6m in its projection neurons (Figures 3A and S3A; Chen et al., 2013). These measurements show a clear alteration in the response of projection neurons, which receive direct olfactory input from olfactory sensory neurons, to the mutant yeast strain compared to the WT strain (Figure 3B). To quantify the differences in the olfactory representation of the different yeast strains, we compared the odor-induced neuronal responses using pairwise Pearson's correlations (Figure 3C). Moreover, we further analyzed these activity maps by comparing the autocorrelation of the repetition of the WT yeast odor across different trials to the cross correlation of the odor representations of WT yeast against atf1~ yeast and atf1~ yeast supplemented with the missing esters (Figure S3E). Both analyses revealed that the WT and atf1~ yeast odor are represented by significantly different activity patterns in the fly antennal lobe. Moreover, we observed that the addition of each ester in physiological concentrations to the atf1~ mutant yeast medium shifts the evoked antennal lobe activity more toward the WT activity pattern, as evident by the significant increase in correlations between WT and atf1~ yeast odors when esters are supplemented. Furthermore, the principal component analysis of the antennal lobe odor responses shows that supplementing atf1~ with esters shifts the representation of atf1~ yeast odor toward WT yeast odor (Figure 3F). In summary, all these analyses confirms that atf1~ yeast odor and the WT yeast odor are represented differently in the D. melanogaster antennal lobe and the odor representations of atf1~ yeast become more similar to WT yeast when each ester is supplemented to the same levels as found in the WT medium.
Expression of ATF1 Results in Increased Yeast Dispersal
The previous experiments revealed that the ATF1 -mediated production of acetate esters has a significant impact on the attractiveness of yeast cells toward D. melanogaster. However, this does not necessarily imply a benefit for the yeast, because increased attraction of flies might merely result in more yeast cells being consumed by the flies. However, we hypothesized that increased fly attraction might also result in increased dispersal of the yeast cells through their insect vectors. To study whether expression of ATF1 affects yeast dispersal by fruit flies, both the WT and atf1~ mutant strain were fluorescently labeled
Figure 2. Volatile Acetate Ester Production Affects the Attractiveness of Yeast Cells for Drosophila melanogaster
(A) Experimental arena populated with fifty 20 hr-starved D. melanogaster CS10 flies. Air is flown from the four corners of the arena, with a common outlet in the center, generating four quadrants that each have their own air profile. Fly movements were recorded during 10 min experiments (odorless air for 2 min from each of the four corners, odors for 3 min from two opposing corners, and odorless air from the other two corners, and finally 5 min of odorless air from all four corners; see also main text for details and Movies S2 and S3).
(B) Preference index defined by the fraction of flies being present in the corresponding odorized quadrant during the last minute of odor exposure. The odors of WT yeast and atf1~ yeast supplemented with ethyl acetate (EA) and isoamyl acetate (IA) (at concentrations that match the levels present in the WT yeast) exhibit a higher preference index than atf1~ yeast odor whereas atf1~ supplemented with phenylethyl acetate (PA) does not (paired t test; *p < 0.02; **p < 0.001). No significant difference was found when WT yeast odor was competed against atf1~ yeast odor supplemented with all three esters or with ethyl acetate and iso-amyl acetate or with ethyl acetate. The error bars represent SEM. n.s., not significant.
(C) Temporal course of number of flies per quadrant for the different experimental conditions. Colors correspond to the respective media (air shown in black/gray). Shadowed traces represent SEM for the number of flies (n = 200 flies; four experimental sessions; three trials/odor/session). The gray rectangle indicates the time of odor exposure. Inset figures correspond to snapshots taken at the end of the odor delivery period (5th min). For the large cage setup, see Figure S2.
by overexpressing either yECitrine or mCherry. These labeled strains were inoculated on one of two designated spots on a specially constructed YPD plate, equidistant from the center of the plate (Figure S4). For each strain, the inoculum contained 1 x 106 cells, corresponding to the amount of Saccharomyces cells that may be found on infected grapes (Mortimer and Polsinelli, 1999). Subsequently, a fly was allowed to roam around the test environment overnight in complete darkness, after which it was removed. Then, the spots containing the initial inocula were removed as well so that only dispersed yeast cells remained and the plates were incubated to allow growth of these cells. After 48 hr, the plates were washed and the relative presence of each strain was quantified using a flow cytometer. Because there is no growth difference between the WT and mutant strains (Supplemental Information), the relative presence of each strain indicates the ratio in which the two strains were transported by the fly. Analysis of 100 independent experiments revealed that the fruity WT
Figure 3. WT and atf1~ Yeast Odors Are Represented Differently in the Antennal Lobe of Drosophila melanogaster
(A) Experimental setup depicting a fly expressing the transgenic calcium indicator GCaMP6 in projection neurons. Briefly, the fly is fixed to an aluminum chamber with wax and the cuticle above the antennal lobes is removed, which allows imaging of the antennal lobes from the dorsal side. A tube is placed in front of its antennae for odors to be delivered (carrier stream flow rate = 2,000 ml/min).
(B) Temporal course of the average fluorescence signal in response to different odors in the entire antennal lobe. Shadowed traces represent SEM for the change in fluorescence. The blue rectangle depicts the 2 s time window after response onset, used for quantitative analyses. Inset bar plot shows that pure atf1~ yeast odor and atf1~ odor supplemented with PA elicit significantly weaker responses than WT yeast odor and atf1~ yeast odor supplemented with EA and IA, respectively (n = 14 flies; *p < 0.02; paired t test).
(C) Representative antennal lobe activity maps corresponding to different yeast and ester odors as well as paraffin oil as control, respectively. Calcium responses were averaged during the 2 s window shown in (B).
(D) Regions of interest manually assigned to each responsive glomerulus based on activity maps for the fly shown in (C). On the right, the corresponding glomerulus-based response profiles for the different odors and control are shown. Profiles were constructed for the 2 s window shown in (B).
(E) Correlation matrix representing the pairwise similarities for the different odor response profiles (n = 14 flies). Note that atf1~ yeast odors supplemented with esters are more correlated to WT yeast odor than atf1~ yeast odor alone.
(F) Principal component analysis of the odor response profiles. The first three components are shown (total variances explained by first three principle components [PCs] = 79.6%; PC1 = 59.4%; PC2 = 12.8%; PC3 = 7.4%). Upon addition of more esters, the atf1~ yeast odor representations are progressively pushed in the direction of the representations of the WT yeast odor, indicating a more-similar response.
Additional analyses are in Figure S3.
strain was on average transported four times more than the atf1 ~ mutant (Figure 4). A separate mirror experiment (n = 100) demonstrated that the nature of the fluorescent tag did not influence the dispersal of either strain (Supplemental Information).
DISCUSSION
Together, these results demonstrate that expression of the fungal alcohol acetyl transferase gene ATF1 promotes attraction
of D. melanogaster, which in turn promotes dispersal of the yeast cells by the insect vector. Using a combination of molecular, behavioral, and neurobiological tests, we show that acetate esters produced by yeasts change the antennal lobe odor representations of Drosophila melanogaster and greatly increase its attraction to yeast. Moreover, we identify two acetate esters, ethyl acetate and isoamyl acetate, as the main signaling molecules associated with this phenotype.
It is interesting to note that compounds like ethyl acetate and isoamyl acetate render ripening fruits their typical aroma (Ver-meiretal., 2009). It is therefore tempting to speculate that yeasts
Figure 4. Fruity Yeast Strains Get Dispersed More by D. melanogaster
Two fluorescently labeled strains (green or red) were plated equidistantly from the center of a YPD agar plate (black circles; see also Figure S4 for details). A 20 hr-starved CS10 fly was introduced to the plate, which was closed and placed in a container, shielded from external light. After 14 hr, the fly was sedated and removed from the plate, after which the plate was incubated for 48 hr at 30°C to allow growth of dispersed yeast cells. Then, the spots from the initial inoculum were removed and the remaining cells were washed off the plate and analyzed using a flow cytometer.
(A) Image of a representative plate after 48 hr of incubation, before washing. The WT strain is yECitrine-labeled (green-yellow) and the atf1~ strain mCherry-labeled (red). The image clearly shows that the atf1~ (red) strain shows reduced dispersal by the flies.
(B) Violin plot of the fraction of dispersed WT cells (n = 95, excluding five samples where no cells were dispersed); black lines indicate quartiles and median. Fruity WT cells were dispersed significantly more than atf1~ cells, shown by the fact that the estimated fraction (gray band = 95% confidence interval) is significantly higher than the 50% expected if dispersal was equal for both (binomial mixed model; p value < 1 x 10~10; details in Experimental Procedures).
have evolved the capacity to synthesize these esters to mimic this aroma in order to attract flies. However, it is difficult to show that the yeast ATF1 gene specifically evolved to stimulate the production of aroma compounds with the aim of attracting insects. Nonetheless, there are compelling arguments for this hypothesis, because several reports demonstrate the intricate mutualism between yeasts and flying insects like fruit flies (Ganter et al., 1986; Giglioli, 1897; Gilliam et al., 1974; Phaff et al., 1956; Reuter et al., 2007; Starmer et al., 1986; Stefanini et al., 2012). For the yeast, dispersal is essential to reach new niches, especially when nutrient levels are running low. Under these conditions, the center of yeast colonies is expected to be a hypoxic environment (Cap et al., 2012) and hypoxic conditions may increase ATF1 expression (Fujii et al., 1997), which in turn results in increased acetate ester formation (Lilly et al., 2000; Malcorps and Dufour, 1992; Verstrepen et al., 2003b). Hence, the production of aroma compounds like isoamyl acetate could help alert flies to the presence of yeast cells, a vital component of their diet. Whereas some of the yeast cells are consumed by the insects, a fraction of cells will stick to the fly body and get dispersed to a different environment. Furthermore, yeasts might reap more benefits from this interaction than mere dispersal. Low nutrient levels trigger the formation of spores that can survive passage through the fly gut and promote outbreeding and thus genetic variability (Freese et al., 1982; Reuter et al., 2007). Additionally, it has been demonstrated that certain insects, such as wasps, act as a reservoir for yeasts when environmental conditions are harsh, for example, during a cold winter (Stefanini etal., 2012).
A last important question is whether aroma-based attraction of insects is limited to S. cerevisiae. In a first preliminary assay, we observed that many different yeasts can be isolated from the body of Drosophila isolated from natural environments and that the vast majority of these yeasts produced aroma-active esters (Table S2). Moreover, we also isolated several strong aroma-producing yeasts from flowers. These preliminary results suggest that aroma production is not restricted to S. cerevisiae and may be a much more general theme in microbe-insect inter-
actions. This is further supported by other studies that reported the isolation of many different yeast species from various insects and the discovery that receptors for acetate esters are widespread in insects (Galizia et al., 1999; Phaff et al., 1956). The formation of fungal aroma compounds may even play a role in mimicry and the relation between plants and their pollinators (Dentinger and Roy, 2010; Stokl et al., 2010).
Lastly, our study also highlights the limitations of using standard laboratory conditions with pure single-species cultures. Clearly, studying model organisms in a more complex ecological context will increase our understanding of physiology and may help to reduce the number of genes with unknown functions.
EXPERIMENTAL PROCEDURES Strains
Both ATF1 alleles In the Y182 strain were deleted using deletion cassettes based on pUG6, conferring resistance to either Hygromycin B or G-418 disulfate. Both markers were removed through the Cre/LoxP technique using pSH65. Deletions as well as marker removal were confirmed through (lack of) growth on selective media, as well as PCR (primers in Table S3). Fermentations performed with WT and mutant strains were analyzed using a head-space-gas chromatography-flame ionization detector system; ethanol and acetate levels were determined using enzymatic kits. Growth rates in liquid YPD were measured in an automated multimode plate reader, whereas growth rates on solid media were determined using automated time-lapse microscopy (New et al., 2014). Differences between strains were analyzed using Mann-Whitney U tests.
Olfactory Preference Assays
Prior to testing, 50 CS10 flies were starved for 20 hr at ~25°C in scintillation vials containing a wet filter paper and then placed in the four-input arena. A camera placed above the arena filmed each trial, each lasting 10 min (2 min, odorless air; 3 min, odor exposure; 5 min, odorless air); each set of 50 flies was tested three times. The number of flies in each quadrant was detected by an automated MATLAB algorithm, based on binarized images. Paired t tests were used for statistical comparisons. For the assays in large cages, 20 CS10 flies were starved for 20 hr at 25°C in vials containing 2% agarose. Tests lasted 5 hr, after which all flies were sedated and the number of flies in each trap was counted. Statistical analyses were performed using a generalized linearmixed model (GLMM), available in R-package lme4. For the calcium
imaging experiments (Yaksi and Wilson, 2010), principle-component analyses and Pearson's correlations were calculated on the antennal lobe images obtained during 2 s after response onset windows. For statistical analyses, paired t tests were performed on antennal lobe peak responses and MannWhitney for correlation coefficients comparisons.
Dispersal Experiments
Flies were starved for 20 hr at 25°C in vials containing 2% agarose. Dispersal experiments were performed with custom-made plates (see Figure S4) filled with YPD-2% agar. Statistical analyses of the dispersal of both strains were performed using a GLMM, available in R-package lme4. Significance of deviation from 50:50 was tested using a Wald z test, also available in lme4.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures, four figures, three tables, and three movies and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2014.09.009.
AUTHOR CONTRIBUTIONS
J.F.C. and L.M.F. designed, performed, and analyzed experiments and wrote the manuscript. T.L.C. performed experiments. L.D.M., J.M., T.W., and B.A.H. helped with design and interpretation of results and wrote the manuscript. K.J.V. conceived the study, and E.Y. and K.J.V. designed and interpreted experiments and wrote the manuscript.
ACKNOWLEDGMENTS
The authors thank Zeynep Okray; Ine Maes; and all K.J.V., E.Y., and B.A.H. lab members as well as CMPG members for their valuable help and suggestions. Research in B.A.H. lab is supported by VIB; the WiBrain Interuniversity Attraction Pole (BELSPO IUAP) network; and Fonds Wetenschappelijke Onderzoeks (FWO) grants G.0543.08, G.0680.10, G.0681.10, and G.0503.12. Research in the lab of E.Y. is supported by ERC Starting Grant 335561, FWO, VIB, and NERF funding. Research in the lab of K.J.V. is supported by ERC Starting Grant 241426, HFSP program grant RGP0050/2013, VIB, EMBO YIP program, FWO, and IWT.
Received: April 7, 2014 Revised: July 23, 2014 Accepted: September 3, 2014 Published: October 9, 2014
REFERENCES
Anagnostou, C., LeGrand, E., and Rohlfs, M. (2010). Friendly food for fitter flies? Influence of dietary microbial species on food choice and parasitoid resistance in Drosophila. Oikos 119, 533-541.
Becher, P.G., Bengtsson, M., Hansson, B.S., and Witzgall, P. (2010). Flying the fly: long-range flight behavior of Drosophila melanogaster to attractive odors. J. Chem. Ecol. 36, 599-607.
Becher, P.G., Flick, G., Rozpedowska, E., Schmidt, A., Hagman, A., Lebreton, S., Larsson, M.C., Hansson, B.S., Piskur, J., Witzgall, P., et al. (2012). Yeast, not fruit volatiles mediate Drosophila melanogaster attraction, oviposition and development. Funct. Ecol. 26, 822-828.
Boch, R., Shearer, D.A., and Stone, B.C. (1962). Identification of isoamyl acetate as an active component in the sting pheromone of the honey bee. Nature 195, 1018-1020.
Cap, M., Stepanek, L., Harant, K., Vachova, L., and Palkova, Z. (2012). Cell differentiation within a yeast colony: metabolic and regulatory parallels with a tumor-affected organism. Mol. Cell 46, 436-448.
Chen, T.W., Wardill, T.J., Sun, Y., Pulver, S.R., Renninger, S.L., Baohan, A., Schreiter, E.R., Kerr, R.A., Orger, M.B., Jayaraman, V., et al. (2013). Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295-300.
Dentinger, B.T.M., and Roy, B.A. (2010). A mushroom by any other name would smell as sweet: Dracula orchids. McIlvainea 19, 1-13. Elmore, T., Ignell, R., Carlson, J.R., and Smith, D.P. (2003). Targeted mutation of a Drosophila odor receptor defines receptor requirement in a novel class of sensillum. J. Neurosci. 23, 9906-9912.
Fogleman, J.C., Starmer, W.T., and Heed, W.B. (1981). Larval selectivity for yeast species by Drosophila mojavensis in natural substrates. Proc. Natl. Acad. Sci. USA 78, 4435-4439.
Francesca, N., Canale, D.E., Settanni, L., and Moschetti, G. (2012). Dissemination of wine-related yeasts by migratory birds. Environ. Microbiol. Rep. 4, 105-112.
Freese, E.B., Chu, M.I., and Freese, E. (1982). Initiation of yeast sporulation of partial carbon, nitrogen, or phosphate deprivation. J. Bacteriol. 149,840-851. Fujii, T., Kobayashi, O., Yoshimoto, H., Furukawa, S., and Tamai, Y. (1997). Effect of aeration and unsaturated fatty acids on expression of the Saccharo-myces cerevisiae alcohol acetyltransferase gene. Appl. Environ. Microbiol. 63, 910-915.
Galizia, C.G., Sachse, S., Rappert, A., and Menzel, R. (1999). The glomerular code for odor representation is species specific in the honeybee Apis mellifera. Nat. Neurosci. 2, 473-478.
Ganter, P.F., Starmer, W.T., Lachance, M.-A., and Phaff, H.J. (1986). Yeast communities from host plants and associated Drosophila in southern Arizona: new isolations and analysis of the relative importance of hosts and vectors on comunity composition. Oecologia 70, 386-392.
Gibson, C.M., and Hunter, M.S. (2010). Extraordinarily widespread and fantastically complex: comparative biology of endosymbiotic bacterial and fungal mutualists of insects. Ecol. Lett. 13, 223-234. Giglioli, I. (1897). Insects and yeasts. Nature 56, 575-577. Gilliam, M., Wickerham, L.J., Morton, H.L.,and Martin, R.D. (1974). Yeasts isolated from honey bees, Apis mellifera, fed 2,4-D and antibiotics. J. Invertebr. Pathol. 24, 349-356.
Goddard, M.R., Anfang, N., Tang, R., Gardner, R.C., and Jun, C. (2010). A distinct population of Saccharomyces cerevisiae in New Zealand: evidence for local dispersal by insects and human-aided global dispersal in oak barrels. Environ. Microbiol. 12, 63-73.
Grosjean, Y., Rytz, R., Farine, J.P., Abuin, L., Cortot, J., Jefferis, G.S., and Benton, R. (2011). An olfactory receptor for food-derived odours promotes male courtship in Drosophila. Nature 478, 236-240.
Hallem, E.A., and Carlson, J.R. (2006). Coding of odors by a receptor repertoire. Cell 125, 143-160.
Hallem, E.A., Ho, M.G., and Carlson, J.R. (2004). The molecular basis of odor coding in the Drosophila antenna. Cell 117, 965-979.
Lambrechts, M.G.,and Pretorius, I.S. (2000). Yeast and its importance to wine aroma: A review. S. Afr. J. Enol. Vitic. 21, 97-129.
Lebreton, S., Becher, P.G., Hansson, B.S., and Witzgall, P. (2012). Attraction of Drosophila melanogaster males to food-related and fly odours. J. Insect Physiol. 58, 125-129.
Lilly, M., Lambrechts, M.G., and Pretorius, I.S. (2000). Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates. Appl. Environ. Microbiol. 66, 744-753.
Malcorps, P., and Dufour, J.P. (1992). Short-chain and medium-chain aliphatic-ester synthesis in Saccharomyces cerevisiae. Eur. J. Biochem. 210, 1015-1022.
Mason, A.B., and Dufour, J.P. (2000). Alcohol acetyltransferases and the significance of ester synthesis in yeast. Yeast 16, 1287-1298. Mortimer, R., and Polsinelli, M. (1999). On the origins of wine yeast. Res. Microbiol. 150, 199-204.
New, A.M., Cerulus, B., Govers, S.K., Perez-Samper, G., Zhu, B., Boogmans, S., Xavier, J.B., and Verstrepen, K.J. (2014). Different levels of catabolite repression optimize growth in stable and variable environments. PLoS Biol. 12, e1001764.
Nojima, S., Linn, C., Jr., and Roelofs, W. (2003). Identification of host fruit volatiles from flowering dogwood (Cornus florida) attractive to dogwood-origin Rhagoletis pomonella flies. J. Chem. Ecol. 29, 2347-2357. Nordstrom, K. (1964). Formation of esters from acids by brewer's yeast. IV. Effect of higher fatty acids and toxicity of lower fatty acids. Jnl. Institute Brewing 68, 398-407.
Nordstrom, K. (1966). Formation of esters from acids by Brewer's yeast: formation from unsaturated acids. Nature 210, 99-100. Palanca, L., Gaskett, A.C., Gunther, C.S., Newcomb, R.D., and Goddard, M.R. (2013). Quantifying variation in the ability of yeasts to attract Drosophila mela-nogaster. PLoS ONE 8, e75332.
Phaff, H.J., Miller, M.W., and Shifrine, M. (1956). The taxonomy of yeasts isolated from Drosophila in the Yosemite region of California. Antonie van Leeuwenhoek 22, 145-161.
Reuter, M., Bell, G., and Greig, D. (2007). Increased outbreeding in yeast in response to dispersal by an insect vector. Curr. Biol. 17, R81-R83. Roper, M., Seminara, A., Bandi, M.M., Cobb, A., Dillard, H.R., and Pringle, A. (2010). Dispersal of fungal spores on a cooperatively generated wind. Proc. Natl. Acad. Sci. USA 107, 17474-17479.
Saerens, S.M., Delvaux, F., Verstrepen, K.J., Van Dijck, P., Thevelein, J.M., and Delvaux, F.R. (2008). Parameters affecting ethyl ester production by Saccharomyces cerevisiae during fermentation. Appl. Environ. Microbiol. 74, 454-461.
Saerens, S.M., Delvaux, F.R., Verstrepen, K.J., and Thevelein, J.M. (2010). Production and biological function of volatile esters in Saccharomyces cerevisiae. Microb. Biotechnol. 3, 165-177.
Sang, J.H. (1956). The quantitative nutritional requirements of Drosophila mel-anogaster. J. Exp. Biol. 33, 45-72.
Stamps, J.A., Yang, L.H., Morales, V.M., and Boundy-Mills, K.L. (2012). Drosophila regulate yeast density and increase yeast community similarity in a natural substrate. PLoS ONE 7, e42238.
Starmer, W.T., Barker, J.S., Phaff, H.J., and Fogleman, J.C. (1986). Adaptations of Drosophila and yeasts: their interactions with the volatile 2-propanol
in the cactus-microorganism-Drosophila model system. Aust. J. Biol. Sci. 39, 69-77.
Stefanini, I., Dapporto, L., Legras, J.L., Calabretta, A., Di Paola, M., De Filippo, C., Viola, R., Capretti, P., Polsinelli, M., Turillazzi, S., and Cavalieri, D. (2012). Role of social wasps in Saccharomyces cerevisiae ecology and evolution. Proc. Natl. Acad. Sci. USA 109, 13398-13403.
Stokl, J., Strutz, A., Dafni, A., Svatos, A., Doubsky, J., Knaden, M., Sachse, S., Hansson, B.S., and Stensmyr, M.C. (2010). A deceptive pollination system targeting drosophilids through olfactory mimicry of yeast. Curr. Biol. 20, 18461852.
Styger, G., Prior, B., and Bauer, F.F. (2011). Wine flavor and aroma. J. Ind. Microbiol. Biotechnol. 38, 1145-1159.
Suh, S.O., McHugh, J.V., Pollock, D.D., and Blackwell, M. (2005). The beetle gut: a hyperdiverse source of novel yeasts. Mycol. Res. 109, 261-265.
Swiegers, J.H., Bartowsky, E.J., Henschke, P.A., and Pretorius, I.S. (2005). Yeast and bacterial modulation of wine aroma and flavour. Aust. J. Grape Wine Res. 11, 139-173.
Vermeir, S., Hertog, M.L.A.T.M., Vankerschaver, K., Swennen, R., Nicolai, B.M., and Lammertyn, J. (2009). Instrumental based flavour characterisation of banana fruit. LWT - Food Sci. Technol. 42, 1647-1653.
Verstrepen, K.J., Derdelinckx, G., Dufour, J.P., Winderickx, J., Thevelein, J.M., Pretorius, I.S., and Delvaux, F.R. (2003a). Flavor-active esters: adding fruiti-ness to beer. J. Biosci. Bioeng. 96, 110-118.
Verstrepen, K.J., Van Laere, S.D., Vanderhaegen, B.M., Derdelinckx, G., Dufour, J.P., Pretorius, I.S., Winderickx, J., Thevelein, J.M., and Delvaux, F.R. (2003b). Expression levels of the yeast alcohol acetyltransferase genes ATF1, Lg-ATF1, and ATF2 control the formation of a broad range of volatile esters. Appl. Environ. Microbiol. 69, 5228-5237.
Vosshall, L.B., Wong, A.M., and Axel, R. (2000). An olfactory sensory map in the fly brain. Cell 102, 147-159.
Yaksi, E., and Wilson, R.I. (2010). Electrical coupling between olfactory glomeruli. Neuron 67, 1034-1047.
