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Yakovleva et al. Microbiome Res Rep 2024;3:19 https://dx.doi.org/10.20517/mrr.2023.56 Page 5 of 18

For amplification of the 16S rRNA gene, biomass of pure bacterial culture grown for 2-3 days was subjected
to boiling in Tris-EDTA buffer (pH 8.0) which contained 5% Triton X-100, and homogenization by
mechanical destruction with sterile glass beads (250-300 μm in diameter) using a Homogenizer Minilys
(Bertin Instruments, Montigny-le-Bretonneux, France) at 5,000 rpm for 30 s. The resulting homogenate was
centrifuged, and the resulting supernatant was used as a DNA template for PCR .
[48]
For amplification of the gene of interest, the primers 27f (5’-AGAGTTTGATCCTGGCTCAG-3’) and
Un1492r (5’-ACGGYTACCTTGTTACGACTT-3’) were used [49,50] . PCR amplification program was 94 °C
3:00, 55 °C 0:30, 72 °C 1:00, 94 °C 0:30, for 37 cycles, following 55 °C 0:30, 72 °C 5:00.

The PCR products were purified and sequenced by the Research and Production Company “Evrogen”
[51]
(Moscow, Russia) using the 1100 r . The editing of the nucleotide sequences was carried out using
Chromas Lite 2.01. The Clustal Omega and the BLAST algorithm from the GenBank database were used to
align, compare, and identify nucleotide sequences. The obtained bacterial nucleotide sequences were
deposited to NCBI GenBank under accession numbers OR272523-OR272526.

The yeast component of fly microbiota was described earlier in the paper by Dmitrieva et al. .
[44]

Bacterial diversity identification and estimation
Estimation of bacterial microbiota composition was performed by NGS sequencing of variable V4 region of
16S rRNA gene. DNA isolation from flies and substrate homogenates was performed using the DNeasy
PowerLyzer Microbial Kit (Qiagen, Hilden, Germany). Amplification of the V4 region of the 16S rRNA gene
was carried out using two primers consisting of the Illumina TruSeq sequencing primer adapters and 515F/
Pro-mod-805R primer sequences: Forward 515F (5’-GTGBCAGCMGCCGGGTAA-3’) and Reverse Pro-
[52]
mod-805R (5’-GACTACNVGGGTMTCTAATCC-3’) . PCR amplification was performed as follows: 32
[53]
cycles of denaturation at 95 °C for 25 s; primer annealing at 56 °C for 20 s; DNA synthesis at 72 °C for 30 s,
and a final elongation at 72 °C for 20 min . High-throughput sequencing of the libraries was performed
[54]
with MiSeq Reagent Micro Kit v2 (300-cycles) MS-103-1002 (Illumina, USA) on a MiSeq sequencer
(Illumina, USA) according to the manufacturer’s instructions. The raw reads were processed as described by
Gavrilov et al. and analyzed using the SILVAngs service with default parameters (SILVA138.1 SSU
[55]
database, https://ngs.arb-silva.de/silvangs/). The obtained nucleotide sequences were deposited to NCBI
BioProject under accession numbers PRJNA999597.

For each sample, profiling was made in two replicates, and the percentage of bacteria of each genus was
averaged over them. Wolbachia, an intracellular symbiont of D. melanogaster (on average 15% in each
sample), was excluded from the further analyses. Bacterial taxa whose percentage did not exceed 5% in any
of the lines were assigned to the “Other” section.

Assessment of the salt tolerance of the dominant bacterial groups
To assess salt tolerance, we grew the isolated strains in liquid MRS medium with 0%, 2%, 4%, and 7% NaCl
stirring on a shaker (200 rpm); 4 replicates for each isolated strain on each salinity level. Strains’ growth was
evaluated by measuring the optical density on a KFK-3-01 30M3 photoelectric colorimeter at a wavelength
of 600 nm. Salt tolerance was evaluated as the ratio (in percentage) of the optical density of a culture grown
on a medium with NaCl to the optical density of a culture grown on a medium without NaCl.

Data analyses
The diversity of microbiota in each sample was assessed using the Shannon species diversity index .
[56]