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Page 4 of 18 Yakovleva et al. Microbiome Res Rep 2024;3:19 https://dx.doi.org/10.20517/mrr.2023.56

METHODS
Samples selection and preparation
Adult D. melanogaster of outbred laboratory lines obtained from the wild individuals collected in southwest
Moscow (Russia) in October 2014 were used. Until January 2015, the initial fly population was cultured on
standard medium (inactivated baker’s yeast - 60 g, semolina - 35 g, sugar - 50 g, crushed raisins - 45 g, agar
CAS 9002-18-0 - 8 g, propionic acid CAS 79-09-4 - 2 g per 1 liter of medium) . Three control (0a, 0b, and
[46]
0c) and 8 “salt diet” lines were isolated then from this initial fly population to model the evolutionary
process.

Control lines were reared on a standard medium without salt. Three lines labeled 2a, 2b, and 2c were reared
on a standard medium supplemented with 2% NaCl from 2014 until the start of the study. Three lines
labeled 4a, 4b, and 4c were reared on a standard medium with 4% NaCl from 2014 until the start of the
study. Two lines labeled 7a and 7b were reared on a 4% NaCl medium until 2016, and then the salt
concentration was increased by 0.5% every 2-3 months until it reached 7%. After that, 7a and 7b lines reared
on medium with 7% NaCl medium as described previously [41,43,47] . The fly lines were reared in cylindrical
glass jars 0.25 L (0c, 2a-2c, and 4a-4c) or population cages 165 mm × 165 mm × 250 mm (0a-0b, 7a-7b) with
medium and water.

In this way, the following lines of flies were tested: 3 control lines reared on a medium without salt (0a, 0b,
and 0c), 3 lines reared on a medium with 2% NaCl (2a, 2b, 2c), 3 lines - with 4% NaCl (4a, 4b, 4c) and 2 lines
- with 7% NaCl (7a and 7b). Two and 4% NaCl concentrations - were moderate, and 7% - was high.
Additionally, five samples of substrates on which flies lived for 2 weeks were tested: s0b (substrate was
processed by flies from 0b line), s0c, s2a, s4a, and s7a. Studied substrates consisted of flies’ medium and
metabolites, on which their specific microbial community developed.

Thirty adult flies at the age of 7 days after the eclosion were taken from each fly line. This age of flies was
chosen not by chance: previously, it was shown that flies emerged from the pupa being almost sterile and
[2]
[43]
formed a microbiome typical for their line by the age of 7 days by eating previously processed substrate .
To remove the substrate particles and the microbial cells from the surface of the body, the flies were washed
in 10 mL of sterile water on a MultiReax vortex (Heidolph, Germany) at 1,700 rpm for 15 min. After that,
the flies were homogenized in 3 mL of sterile water and treated by the vortex. To study the microbial
communities associated with the substrate, 30 mg of the substrate from the surface was resuspended in 3 mL
of sterile water and homogenized in the same way as the fly samples. The taken mass of the substrate was
approximately equivalent to the mass of 30 flies taken to prepare the homogenate.

Isolation and identification of the dominant representatives of microbial groups
The total number of cultivated aerobic bacteria was determined by plating the fly homogenates using the
dilution-to-extension method on Petri dishes with MRS agar medium of the following composition, g/L:
peptone - 12.5; yeast extract - 7.5; glucose - 20.0; KH PO - 2.0; dibasic ammonium citrate - 2.0; sodium
2
4
acetate - 5.0; MgSO - 0.2; MnSO - 0.05; agar - 20.0; tap water, pH 7.0. Amphotericin B (1 mg/L) was added
4
4
to the medium to inhibit yeast growth. The inoculated Petri dishes were incubated for 5-10 days at room
temperature (20-25 °C), and after that the colonies were counted. Colony growth experiments were
performed in 3 dilutions (3 replicates each) for each sample of flies or substrate.

Colonies obtained from the highest dilutions and containing cells of different morphotypes were used for
the inoculation of a fresh MRS agar medium. DNA was isolated from the obtained pure cultures using the
Fast DNA Spin Kit for Soil (MP Biomedicals, California, USA).