Drosophila-associated yeast species in vineyard ecosystems

FEMS Microbiology Letters, 362, 2015, fnv170 doi: 10.1093/femsle/fnv170 Advance Access Publication Date: 21 September 2015 Research Letter

R E S E A R C H L E T T E R – Food Microbiology

Drosophila-associated yeast species in vineyard ecosystems Samuel S. T. H. Lam and Kate S. Howell∗ Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, VIC 3010, Australia ∗ Corresponding author: Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, VIC 3010, Australia. Tel: +61 (3) 90353119;

E-mail: [email protected] One sentence summary: Yeasts may be transmitted by Drosophila in the vineyard and winery. Editor: Derek Jamieson

ABSTRACT Yeast activity during wine fermentation directly contributes to wine quality, but the source and movement of yeasts in vineyards and winery environments have not been resolved. Here, we investigate the yeast species associated with the Drosophila insect vector to help understand yeast dispersal and persistence. Drosophila are commonly found in vineyards and are known to have a mutualistic relationship with yeasts in other ecosystems. Drosophilids were collected from vineyards, grape waste (marc) piles and wineries during grape harvest. Captured flies were identified morphologically, and their associated yeasts were identified. Drosophila melanogaster/D. simulans, D. hydei and Scaptodrosophila lativittata were identified in 296 captured Drosophila flies. These flies were associated with Metschnikowia pulcherrima, Hanseniaspora uvarum, Torulaspora delbrueckii and H. valbyensis yeasts. Yeast and Drosophila species diversity differed between collection locations (vineyard and marc: R = 0.588 for Drosophila and R = 0.644 for yeasts). Surprisingly, the primary wine fermentation yeast, Saccharomyces cerevisiae, was not isolated. Drosophila flies are preferentially associated with different yeast species in the vineyard and winery environments, and this association may help the movement and dispersal of yeast species in the vineyard and winery ecosystem. Keywords: Drosophila; vineyard; yeast diversity; biogeography; wine

INTRODUCTION Yeasts, a key part of the winemaking process, convert sugar into ethanol and contribute over half of the volatile compounds to the aroma and flavour of wine during fermentation (Swiegers et al. 2005). Saccharomyces cerevisiae is a predictable and controllable yeast that produces desirable flavours; it is, therefore, intentionally used in wine making. A diverse group of yeasts, collectively known as the non-Saccharomyces yeasts, also con´ tribute to wine flavour (Fleet, Lafon-Lafourcade and RibereauGayon 1984; Heard and Fleet 1985). Non-Saccharomyces yeasts are endemic in vineyard environments. Grape surfaces harbour a number of non-Saccharomyces yeasts including Candida, Metschnikowia, Hanseniaspora, Crypto-

coccus and Rhodotorula (Barata, Malfeito-Ferreira and Loureiro 2012). The yeast community can change over time depending on the stage of ripening and winemaking. Unripe grapes harbour Cryptococcus, Candida and Rhodotorula populations (Prakitchaiwattana, Fleet and Heard 2004), and yeast numbers and diversity then increase as grapes ripen to greater than 106 colony-forming units (cfu)/g of berries (Fleet et al. 2002; Barata, Malfeito-Ferreira and Loureiro 2012). Of note, S. cerevisiae is not commonly isolated from vineyard environments (Fleet 2003), and recent studies using culture-independent fungal analyses have shown that S. cerevisiae is only a minor vineyard species (Setati et al. 2012; Bokulich et al. 2014; Taylor et al. 2014), although it can remain in the vineyard for up to three years after intentional introduction (Cordero-Bueso et al. 2011). Saccharomyces cerevisiae is added into

Received: 15 June 2015; Accepted: 18 September 2015 C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]

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grapes during the winemaking process and then transferred to the wider vineyard environment via the grape skins and waste, known as marc (Fleet 2003). Nevertheless, natural S. cerevisiae strains do reside in the vineyard environment, and S. cerevisiae has been isolated in a winery without intentional S. cerevisiae inoculation and traced to a vineyard six kilometres away (Goddard et al. 2010). Insects may disperse the yeasts, with honey bees (Goddard et al. 2010) and social wasps implicated as vectors (Stefanini et al. 2012). Drosophila are also strong candidate S. cerevisiae vectors, and S. paradoxusand S. cerevisiae-related sequences have been found in the gut and on the surface of wild Drosophila (Chandler, Eisen and Kopp 2012). Furthermore, female Drosophila prefer to oviposit on substrates colonized by fermenting yeasts (Ganter 2006; Chandler, Eisen and Kopp 2012; Palanca et al. 2013). Drosophila (Diptera: Drosophilidae; also known as the vinegar (fruit) fly) are small, 2–4 mm flies that are usually considered undesirable to winemaking as they carry acetic acidproducing bacteria that can produce undesirable aromas in wine (Amerine and Kunkee 1968; Crotti et al. 2010; Markow 2015). Drosophila are common in winery environments since they are attracted to the carbon dioxide emitted by ripe fruits and fermenting yeast (Turner and Ray 2009). It has been suggested that the yeast communities on the fruit surface ferment the flesh and produce Drosophila-attracting volatiles (Rohlfs and Kurschner ¨ 2010), with yeasts isolated from fruit and vineyard environments more attractive to Drosophila than yeasts isolated from non-fruit, non-vineyard sources (Palanca et al. 2013). Drosophila act as yeast vectors because they land on fruit and inoculate it with yeast from their body or gut to establish a new environment for future larval development (Becher et al. 2012). Once the yeast is established, further Drosophila are attracted to lay their larvae, which can spread yeast cells in the decaying tissue during burrowing and foraging (Starmer and Lachance 2011). Even at their early stages, Drosophila larvae are selective of the yeasts they feed on and, therefore, further disperse (Ganter 2006). Different Drosophila species have demonstrated specific relationships to particular yeast species depending on their biogeographic distribution and the yeast species that best supports their own development (Anagnostou, Dorsch and Rohlfs 2010). This close relationship with yeasts makes Drosophila an ideal candidate for selecting and vectoring yeasts in vineyard and winery ecosystems, which may result in yeast species selection for preferential distribution. The origin of wine-active yeasts is of renewed interest due to the promise of using nonSaccharomyces yeasts during fermentation to modify winemaking outcomes, produce novel aromas and better describe the ecosystem (Fleet 2003; Jolly, Varela and Pretorius 2014). This study describes the yeast species associated with Drosophila vectors in vineyard ecosystems. Species interactions between yeast and Drosophila are described based on their vineyard and winery location. This study of the distribution, abundance and diversity of Drosophila in vineyard ecosystems with an emphasis on their associated yeast populations can be used to better inform the production of quality wine.

MATERIALS AND METHODS Field collections of Drosophila and yeasts Drosophila flies were captured during the March and April 2013 harvest period at Mt. Langi Ghiran (Bayindeen VIC 3375 Australia) and Yering Station (Yarra Glen VIC 3775 Australia).

These sites are approximately 230 km apart; flies are, therefore, highly unlikely to move between field sites. Samples were collected at the winery, vineyard and marc pile (where the grape skins are discarded after winemaking) at each site with nets and traps (Markow and O’Grady 2008). A stocking was used to cover fermenting banana or grape bait to avoid contamination of the captured flies. Flies with Drosophila-like morphology were collected using an aspirator and placed in sterile vials, fixed with a sponge closure and stored in a portable cool box containing ice for transfer to the laboratory in under six hours. Fly activity was reduced at low temperatures, and they were revived when received in the laboratory.

Isolation and identification of yeasts and Drosophila Individual flies were transferred by aspiration onto Wallerstein Laboratory Nutrient (WLN) agar plates (Oxoid, Australia) for 24 h at 25◦ C. WLN is differential medium used for culturing beverage-associated yeasts (Pallmann et al. 2001). In this system, Drosophila walk across the solid agar medium and sample the media by regurgitation and defecation, thus depositing yeasts (Coluccio et al. 2008). Fly surface-associated yeasts are also likely to be deposited. This method of yeast isolation was chosen to mimic the fly behaviour when visiting grape berries. Drosophila were identified under a dissecting microscope using a taxonomical key from (Markow and O’Grady 2008). Characteristic yeast colonies were selected for further definitive identification. Individual yeast colonies were isolated by subculturing on fresh WLN plates. DNA was extracted from fresh colonies by heating a small amount of yeast biomass in distilled water at 94◦ C. Samples were then placed on ice for two minutes before being centrifuged for 20 seconds at a minimum of 10 000 × g. The supernatant was removed and stored in a sterile microcentrifuge tube. Yeast identification was carried out using a protocol modified from White et al. (1990). The internal transcribed spacer (ITS) region was amplified by PCR and then cleaved using specific restriction enzymes to produce species-specific patterns (Esteve-Zarzoso et al. 1999). PCR conditions, restriction digest and agarose gel resolution were as previously described (EsteveZarzoso et al. 1999). Since the restriction enzyme fragmentation pattern can be ambiguous for yeast species identification, PCR fragments from the ITS region were amplified using highfidelity Taq polymerase (iProof High-Fidelity PCR Kit; Bio-Rad; USA), cleaned using the Ultra Clean PCR Clean-Up DNA Purification Kit (Mo Bio Laboratories, USA), and sequenced using ITS1 and ITS4 primers (Australian Genome Research Facility). The aligned consensus sequences (ClustalW) were compared with known sequences using the BLAST search engine, hosted by the NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and strains with an identity score greater than 98% were collected (Clavijo, ´ and Paneque 2010). Strains identified by DNA sequencCalderon ing were then used to identify other strains with similar colony morphology and verified by comparing with the restriction enzyme profiles. Sequence data was uploaded to the Pubmed public database with Genbank accession numbers KT758304758337. Data were analysed after log (x + 1) transformation using non-metric multidimensional scaling (NMDS) based on the Bray–Curtis resemblance matrix. The aim was to determine the similarity between vineyards and locations within the vineyard to establish if yeast and Drosophila diversities were similar. An analysis of similarity (ANOSIM) was then performed to find between-group and within-group dissimilarities and

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significant differences between locations. R statistics were used instead of P values to assess the magnitude of difference, because P is sensitive to sample size and R is not (Clarke and Warwick 2001). Finally, a similarity percentage (SIMPER) analysis was performed to quantify the percentage contribution of each fly or yeast species to the differences observed between vineyard, marc or winery sites. ANOSIM and SIMPER analyses were conducted in Primer 6 (Clarke and Warwick 2001).

RESULTS AND DISCUSSION Drosophila abundance and diversity Flies were collected during the March and April 2013 harvest period at Mt. Langi Ghiran and Yering Station. In total, 82 flies were collected at Yering Station, and 214 flies were collected at Mt. Langi Ghiran (Table S1, Supporting Information). Four Drosophilid fly species were identified in this study: Scaptodrosophila lativittata, Drosophila melanogaster, D. simulans and D. hydei. Since D. simulans is morphologically similar to D. melanogaster, both species were placed into a joint category so that they could be included in the analysis. Indeed, Sc. lativittata is not true Drosophila, but, for simplicity, Drosophila and Scaptodrosophila were collectively named Drosophila in this study. Drosophila species diversity at Mt. Langi Ghiran and Yering Station were compared (Fig. 1). Here, D. melanogaster/D. simulans were the dominant species at both sites at 92.7% and 97.2%, respectively. Scaptodrosophila lativittata was more abundant at Mt. Langi Ghiran than Yering Station (6.1% versus 0.47%). In contrast, the distribution of D. hydei was similar at Mt. Langi Ghiran and Yering Station at 1.2% and 2.3% of the total population, respectively. Overall, the species distribution was similar at both sites. The distribution of fly species was grouped by vineyard, marc and winery to avoid the small sample sizes from the vineyard collection point skewing the results. The results were visualized using NDMS (Fig. 2). The close proximity of vineyard and marc data points indicates that the distributions of species were similar between these locations. However, the Drosophilids found at the two winery collection sites were not similar (Fig. 2). A differential distribution of Drosophila at a single site has previously been reported, with D. simulans the most common species in compost piles containing rotten fruit and vegetables (Oakeshott, Vacek and Anderson 1989). Here, D. simulans may have been the dominant species, but we did not differentiate between D. melanogaster and D. simulans due to the method used for identification. However, Sc. lativittata has not previously been described in vineyards, but Scaptodrosophila species have been found to survive in many different types of vegetation (van Klinken and Walter 2001). Drosophila melanogaster is closely associated with human activity, and it is often abundant in farmed areas where there is decaying fruit and vegetable matter (Umina et al. 2005). Other Drosophila species reported alongside D. melanogaster at composts containing rotting fruit or vegetables include D. simulans, D. immigrans, D. hydei and D. busckii (Oakeshott, Vacek and Anderson 1989). Fly collections from Australian vineyards have found D. hydei, D. immigrans, D. repleta and D. simulans (Rako L and Shirriffs J, pers. comm.). Drosophila species significantly differed between vineyard and marc sites (Table S2, Supporting Information). Drosophila melanogaster/D. simulans most contributed to these observed differences and explained 75% of the species diversity between

Figure 1. Distribution of Drosophilids captured at (A) Mt. Langi Ghiran and (B) Yering Station sites.

vineyard and marc sites (Table S3, Supporting Information). This difference can be explained by collection site differences: vineyards and marc piles are very different in form and function, with the marc pile a large source of food that would be expected to be more attractive to Drosophila species than the vineyard. Species that cannot compete with D. melanogaster and D. simulans would be more likely to be found outside the marc pit, as observed here at both sites. Drosophila melanogaster and D. simulans dominated in the marc piles at both vineyards and all locations. At Mt. Langi Ghiran, Sc. lativittata and D. hydei were found outside the marc area in the vineyard and winery. While this suggested that D. melanogaster/D. simulans were well established in the vineyard, this dominance may have only been limited to the marc pile. Some laboratory studies have shown that interspecies competition exists that can alter the composition of Drosophila populations (Barker, Krebs and Davies 2005). Alternatively, different Drosophila species might prefer different environments. For instance, Oakeshott, Vacek and Anderson (1989) proposed that fruit resources may be partitioned by Drosophila, since each species has a different microbiota composition. Therefore, we next quantified fly-associated yeasts to look for significant associations.

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Figure 2. 2D NMDS ordination figure for Drosophila species at Yering Station and Mt. Langi Ghiran compressed into vineyard, winery and marc collection points. Different numbers in the figure refer to different collections within the vineyard, winery and marc groupings. Circles denote the separation between groups, which is quantified by the R value in Table S5 (Supporting Information).

Yeast abundance and diversity The ‘fly walk’ plates proved to be a good method for isolating fly body and digestive tract-associated yeasts, since both surfaceassociated and regurgitated/defecated yeasts were transferred to the media. Individual colonies were observed on the agar plates, and contaminating mould growth was minimal. The number of viable yeast colonies per captured fly was calculated and was quite variable, ranging from 2 cfu to over 600 cfu per fly (Fig. S1a and b, Supporting Information). Average counts approached 300 colonies per fly. There were no significant differences in cfu between collection site or fly species or sex (D. melanogaster/simulans). It is likely that these numbers are an overestimation of the microbial populations present, since this technique collected body surface and regurgitated and defecated yeasts and, since the flies were on the plates for 24 h, it is possible that yeast cells were physically redistributed by fly movement over this time. Yeasts were collected from WLN agar plates and subcultured on fresh plates to obtain pure cultures for colony morphology and identification analysis. The culture-based methodology was chosen because the yeasts were needed in pure culture form for further experiments: in total, 86 yeast colonies were subcultured and grouped by similarity for further identification. Since ITS-PCR with restriction enzyme digestion did not give reliable gel resolution and band sizes were difficult to compare (EsteveZarzoso et al. 1999), yeast colonies were identified using a combination of ITS-PCR and sequencing. Six different yeast species, Torulaspora delbrueckii, Pichia membranifaciens, Metschnikowia pulcherrima, Hanseniaspora uvarum, H. valbyensis and Lachancea thermotolerans were identified using a combination of PCR, sequencing and colony morphology. Sequencing data were correlated with the ITS-PCR/digest results to confirm that all yeast species with the same colony morphology were the same (data not shown). Colonies morphotypes were then counted from all plates. Many flies carried more than one yeast species. Hanseniaspora spp. yeasts were most common, with M. pulcherrima also frequently isolated (Table S4, Supporting Information). Interest-

ingly, colonies with Saccharomyces-like morphology were not observed on any of the plates, and ITS region sequencing of candidate yeasts did not identify Saccharomyces spp. As discussed by Barata, Malfeito-Ferreira and Loureiro (2012), S. cerevisiae is infrequently isolated from vineyards, with only 0.05–0.1% of sound berries carrying this important genus. Recent pyrosequencing studies have isolated S. cerevisiae in only 0.0005% of the total fungal population in vineyard samples in New Zealand (Taylor et al. 2014) and 4% of the fungal population in California (Bokulich et al. 2014). A recent study of D. simulans captured in New Zealand vineyards found that 1% of the flies carried S. cerevisiae (Buser et al. 2014). It might be expected that S. cerevisiae would be more frequent at winery and marc locations, since it is primarily responsible for the alcohol fermentations that occur at these sites. The yeast species distribution was compared between the two vineyard sites. There was a higher number of H. valbyensis and M. pulcherrima at Mt. Langi Ghiran, with 62.9% of flies carrying H. valbyensis and 22.9% of flies carrying M. pulcherrima (Fig. 3). Hanseniaspora uvarum was carried by 8.6% of flies, and P. membranifaciens was carried by 5.7% of flies. Flies collected at Yering Station were associated with two species not found at Mt. Langi Ghiran: T. delbrueckii, and L. thermotolerans. Torulaspora delbrueckii and L. thermotolerans formed 10.2% of the total yeast species at Yering Station and were predominantly found at the marc piles. Hanseniaspora species yeasts were also isolated and were most common, with H. uvarum present in 35.5% of the total number of flies. Similar to Mt. Langi Ghiran, P. membranifaciens was infrequent at Yering Station (2.9%). Yeast distribution data were examined according to winery, vineyard and marc pile collection points. The yeast species isolated from marc pile Drosophila were more diverse than those isolated from flies captured in the vineyard (Fig. 4). The yeast species isolated from flies at the vineyard and marc collection points were significantly different (Table S5, Supporting Information), with H. valbyensis (34%) contributing most to the difference and P. membranifaciens and M. pulcherrima responsible for about 20% each (Table S6, Supporting Information).

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Table 1. The frequency (number of isolates/number of flies sampled) of the most common yeasts associated with Drosophilids captured in vineyard and winery sites. Yeast/fly H. uvarum H. valbenysis M. pulcherrima P. membranefaciens T. delbrueckii

D. mel/sim

D. hydei

S. lattivitata

0.48 0.50 0.36 0.05 0.12

0.67 0.00 0.17 0.00 0.17

0.17 0.67 0.50 0.00 0.00

winemaking, S. cerevisiae would be expected to be isolated in all of these situations. Two Hanseniaspora species were more common in flies from the marc pile at Yering Station, which may suggest that this species was outcompeting other species or was more attractive to Drosophila. The species diversity of yeasts was higher at Yering Station, which might be explained by the vineyard’s layout. Yering Station has two marc piles, one red and one white. The yeast species are different in each marc pile. Red and white grape varieties are handled differently during winemaking: red skins in the marc pile have all the yeasts from fermentation because the grape juice is fermented with the skins (Rankine 2004), while in white winemaking the skins are pressed for juice and immediately discarded in the marc pit; therefore, it only contains yeasts originally present on the grapes and those inoculated by the winemaking equipment. Therefore, large numbers of (spent) fermenting yeasts would not expected in grape skins discarded in the white marc pit. Figure 3. Distribution of yeast species on Drosophilids captured at (A) Mt. Langi Ghiran and (B) Yering Station sites.

Special relationships between Drosophila and yeasts

Hanseniaspora valbyensis and M. pulcherrima were dominant at Mt. Langi Ghiran (Fig. 3). Metschnikowia pulcherrima is common on pressed grapes (Settanni et al. 2012), and M. pulcherrima and H. uvarum have been isolated without S. cerevisiae (Settanni et al. 2012). This is interesting, because, as the primary yeast added in

Drosophilids are known to carry different yeast species (Ganter 2006; Chandler, Eisen and Kopp 2012). Here, we associated the yeast species isolated on solid media with the Drosophila species to investigate potential interactions in the winery and vineyard ecosystem (Table 1). It is important to note

Figure 4. 2D NMDS ordination figure for yeast species at Yering Station and Mt. Langi Ghiran compressed into vineyard, winery and marc collection points. Different numbers in the figure refer to different collections within the vineyard, winery and marc groupings. Circles denote the separation between groups, which is quantified by the R value in Table S2 (Supporting Information).

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that, in contrast to recent studies, our method isolated both surface-associated yeasts and those regurgitated or defecated by the flies. Most flies carried Hanseniaspora spp., with a high proportion of flies carrying H. uvarum (Table 1). This is in agreement with Chandler, Eisen and Kopp (2012), who reported that the gutassociated yeasts from a wide variety of Drosophila species were often Hanseniaspora spp. This genus produces aromas that are attractive to D. melanogaster (Palanca et al. 2013). The attractiveness of this yeast to D. suzukii could be exploited in traps to monitor this horticultural pest (Hamby et al. 2012). It is noteworthy that H. valbenysis was not associated with D. hydei, which may suggest that this yeast is specifically excluded by this species. Further experiments should confirm this observation, as very few D. hydei were captured in this study. Metschnikowia pulcherrima was present in many flies in this study (Table 1) but was not a major isolate in the gut-associated culture-independent study of Chandler, Eisen and Kopp (2012). It may be that M. pulcherrima is found on the fly surface or is readily cultured on yeast isolation media and was thus captured in our study. The feeding and oviposition preferences of D. melanogaster adults and larvae have previously been tested against a panel of yeasts including M. pulcherrima (Anagnostou, Dorsch and Rohlfs 2010). Although the flies did not have a preference for this or any other yeast, M. pulcherrima was unfavourable to larval fitness. Yeasts are thought to provide vitamins and amino acids to Drosophila and perhaps detoxify their environment (Ganter 2006), but the role played by M. pulcherrima is unclear. Fly gender may affect the yeast choice made by Drosophila (Becher et al. 2012). Our data indicate that male and female D. melanogaster/D. simulans had a similar preference for yeasts at each location. This is in contrast to Becher et al. (2012), who reported that female flies and larvae determined and maintained specific yeast communities. Here, only D. simulans/melanogaster flies were sex typed, and there were no significant associations with yeast species (data not shown). The relationship between yeast type and fly gender requires further study. Consistent with our findings, flies reared or captured on similar food sources often have the same microbial partners (Chandler, Eisen and Kopp 2012; Chandler et al. 2014). Furthermore, our evidence suggests that flies living in different regions of the vineyard might inhabit different ecological niches and remain quite separate. Drosophila and yeast are associated due to their mutualistic relationship, where Drosophila rely on yeast as food and yeast may benefit from dispersion on Drosophila to different environments (Becher et al. 2012; Palanca et al. 2013; Buser et al. 2014). Supporting this, the yeast ATF1 gene promotes dispersal by insect vectors, since the volatiles emitted by yeasts are attractive to insects and may aid distribution (Christiaens et al. 2014). In conclusion, we isolated Drosophila from different locations within two geographically distinct vineyards and identified distinct yeast and fly species at different sites. Drosophila flies may be preferentially associated with different and specific yeast species in the vineyard environment. This finding is of industrial importance since yeast species and strain control in wineries impacts on the flavour, and thus the value, of wine.

SUPPLEMENTARY DATA Supplementary data are available at FEMSLE online.

ACKNOWLEDGEMENTS The authors thank Jen Shirrifs, Lea Rako, and Michele Schiffer for help with fly capture and identification. Damien Sheehan (Mt Langi Ghiran) and Andrew Clarke (Yering Station) provided invaluable assistance with the field work.

FUNDING This project was supported by Australia’s grapegrowers and winemakers through their investment body, the Grape and Wine Research and Development Corporation, with matched funds from the Federal Government. Conflict of interest. None declared.

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