Saccharomyces cerevisiae found in the crop of a Neotropical 1 Drosophila species fly collected in a natural forest remnant – 2 comments on Hoang , Kopp & Chandler ( 2015 )

3 Depto. Ciência de Alimentos, Fac. Engenharia de Alimentos, Universidade Estadual de 10 Campinas – UNICAMP, SP, Brasil. 11 12 *Corresponding author: 13 Louis Bernard Klaczko, Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, 14 Universidade Estadual de Campinas – UNICAMP, Cx. Postal 6109, Campinas, 13083-970 SP, 15 Brasil. 16 Telephone: 55+19-3521-1150; FAX: 55+19-3521-6235. 17 E-mail: lbk@unicamp.br 18


Introduction
The symbiotic association between yeast and Drosophila in natural environments has long been assessed with experiments investigating Drosophila species attraction to baits inoculated with different yeast species as well as isolating yeasts from Drosophila crops (Dobzhansky & Da Cunha, 1955;Powell, 1997;Buser et al., 2014).A number of differential attractivity experiments have used baits inoculated with various yeast species isolated from Drosophila crops and also commercial Saccharomyces cerevisiae, as a control treatment (e.g.: Da Cunha, Dobzhansky & Sokoloff, 1951;Klaczko, Powell & Taylor, 1983;Becher et al., 2012).Hoang, Kopp & Chandler (2015) criticized this approach, first, claiming that: "S.cerevisiae is rarely found with natural populations of D. melanogaster or other Drosophila species".To explain the finding of D. simulans associated with S. cerevisiae in a single study from New Zealand, they argued that it could be due to the unnatural environment (vineyard) where the flies were collected.Furthermore, they carried out a feeding preference experiment in the laboratory with D. melanogaster, when they allowed flies to choose between S. cerevisiae and another species taken from five natural yeast species.In no case, did the flies prefer S. cerevisiae over the other species.Finally, they questioned the overuse of S. cerevisiae as a model for studying the fly-yeast relationship, since it "may not be fully representative of host-microbe interactions as they operate in nature." We collected specimens of Drosophila tripunctata species group within an Atlantic Rainforest fragment.This group encompasses 80 species (Bächli, 2016) and is widely distributed over the Neotropical region (Val, Vilela & Marques, 1981;Hatadani et al., 2009).Several species that belong to D. tripunctata group are forest interior dwelling species (FIDS) of flies and use PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2090v1| CC-BY 4.0 Open Access | rec: 1 Jun 2016, publ: 1 Jun 2016 naturally-occurring fruits for feeding and breeding (Mata, Valadão & Tidon, 2015;Machado, Gottschalk & Robe, 2016).
Our objective was to carry out a pilot experiment to examine yeast species associated with Drosophila species in a natural Atlantic Rainforest fragment, especially examining, the yeast found with FIDS of the D. tripunctata group.

Materials & Methods
We sampled yeast of Drosophila crops from an Atlantic Rainforest fragment located at Itatiba, SP, Brazil (23º 00.073' S, 46º 52.917' W; altitude = 740 m) on June 29, 2015.We collected drosophilids by sweeping entomological nets over baits of mashed banana inoculated with commercial S. cerevisiae and covered with sterile tulle cloth.Flies were brought to the laboratory and dissected within one hour as suggested by Phaff et al. (1956).Wild males were identified by their external morphology and genitalia (Breuer & Rocha, 1971;Vilela & Bächli, 1990).
Before dissected in a drop of Drosophila Ringer's solution, flies were immersed in distilled water and in alcohol 70%, following the procedures described by Hamby et al. (2012).
Next, crops were streaked in formulated YM medium (1.0% glucose, 0.5% peptone A, 0.3% yeast extract, 0.3% malt extract, 2.0% agar with Chloramphenicol 1.0%) and incubated at 30°C for 48 hours.Then, genomic DNA of the colonies was extracted as described by Rosa et al. (2009).Regions ITS-D1/D2 of the 26S rRNA gene sequences were amplified according to PCR conditions and protocol described in Rosa et al. (2009).Yeast species were identified submitting the sequences to GenBank database and comparing them to entries for yeast.

Results
Twenty males of different Drosophila species had their crop dissected, but only five yeast strains were isolated from five fly specimens sampled of the Itatiba population (Table 1).
From two different D. mediopunctata males two Candida sp.strains were isolated (top BLAST identity was 97% to Candida sake strain K2.6.1 and 96% to Candida sake strain NRRL Y-1622).
A not yet identified yeast species was isolated from D. frotapessoai; from D. unipunctata a Starmerella bacillaris strain was identified with 100% identity to reference strain CBS 13663.
Finally, from D. paraguayensis crop, Saccharomyces cerevisiae was isolated and identified with 100% identity to reference strain NRRL Y-12632.

Discussion & Conclusion
Several reports show the diversity of substrates where Saccharomyces cerevisiae, Starmerella bacillaris and Candida sake have already been found.Particularly, they were found in fruits, grains and in the soil of natural environments (ARS, 2016).Barbosa et al. (2016) reported the occurrence of natural populations of S. cerevisiae associated with bark trees in several Brazilian forest ecosystems, including Atlantic Rainforest.The results of this work show that yeast populations of this species are available to Drosophila in these ecosystems.Moreover, Drosophila paraguayensis, D. mediopunctata and its cryptic sibling species D. unipunctata have been collected repeatedly in the interior of forests, and adults have emerged from naturally collected fruits (Mata, Valadão & Tidon, 2015;Machado, Gottschalk & Robe, 2016).These are good evidences that they occur naturally within the forest environment.
Experiments of differential attractiveness in the field are important for characterizing the feeding habit differentiation of Drosophila species.For example, Klaczko, Powell & Taylor (1983) collected Drosophila over baits inoculated with S. cerevisiae, Kloeckera apiculata (=Hanseniaspora uvarum) and other yeasts in James Reserve, San Jacinto Mountains, USA.
They collected fewer specimens of D. obscura group and D. melanogaster group over baits inoculated with S. cerevisiae than K. apiculata over baits (796 to 1243 respectively).Yet, flies from subgenus Drosophila, such as D. occidentalis, were more collected over S. cerevisiae baits (295 over 194).
We found a similar pattern in the Itatiba population (Batista et al., 2015).

Table 1 -
Yeast strains isolated from crops of Drosophila species belonging to the tripunctata group, yeast species with top identity compared to sequences submitted in BLAST, with identity and percentage identity to reference accession number.