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Yakovleva et al. Microbiome Res Rep 2024;3:19 https://dx.doi.org/10.20517/mrr.2023.56 Page 7 of 18

The diversity of bacteria in the fly lines adapted to the substrate with different salinity and in the
corresponding substrates
Bacterial communities associated with D. melanogaster differed significantly, in agreement with NaCl
concentration in the substrate. Figure 1 represents the average relative abundance of bacteria of different
taxa in the fly line denoted at the x-axis. Raw data are presented in Supplementary Table 1. In the absence of
NaCl (lines 0a-0c), as well as at 2% and 4% NaCl concentrations (lines 2a-2c and 4a-4c), bacteria of
Acetobacter and Lactiplantibacillus genera dominated in flies, and their relative abundance ranged from 69%
to 99%. However, with the increase in NaCl concentration, the relative abundance of Acetobacter decreased,
while that of Lactiplantibacillus grew, and at 4% NaCl, it became dominant. The composition of the fly
microbiome changed entirely at 7% NaCl, being dominated by bacteria of Leuconostoc and Providencia
genera.

The microbiomes of the lines reared under the same conditions were generally similar but differed in the
relative abundance of Lactiplantibacillus and Acetobacter. The Lactiplantibacillus content varied from 26%
to 82%, and Acetobacter - from 12% to 38% in control lines (0a-0c). Bacteria of Gilliamella, Leuconostoc, and
Levilactobacillus genera (16%, 12%, and 6%, respectively) were detected in line 0a, and the representatives of
the genus Fructilactobacillus (10%) were present in line 0c. Lines reared on the substrate with 2% NaCl
contained 69% to 89% lactic acid bacteria, while Acetobacter was present only in line 2a (19%). Acetobacter
was replaced by Levilactobacillus (4% and 14%) and Commensalibacter (3% and 7%) in lines 2c and 2b. In
lines reared on the substrate with 4% NaCl, Lactiplantibacillus absolutely dominated, and Leuconostoc was
found in significant amounts only in the microbiome of line 4b (18%). Thus, the main microorganisms
associated with D. melanogaster were Lactiplantibacillus and Acetobacter, and the proportion of Acetobacter
decreased to a few percent (in the case of 4% salt) with the increase in substrate salinity. Completely
different results were observed in two lines reared on the substrate with 7% NaCl. Acetobacter was absent;
Lactiplantibacillus accounted for only 1% to 12% of the total prokaryotic community. Instead, bacteria of the
genera Leuconostoc (52% and 11%), Providencia (36% and 42%), Enterococcus (4% and 19%), and in one
case, Disgonomonas (8%) dominated [Figure 1].

The bacterial composition of the substrate with different salinity partially corresponded to the composition
of microbiomes of D. melanogaster reared on this substrate, but there were some differences [Figure 1]. In
one of the samples from the control line (s0a), 21% of bacteria of the genus Leuconostoc were present and
were absent in the microbiome of the corresponding fly line (0a). The s0b substrate was dominated by
Acetobacter, while the microbiome associated with the flies (0b) contained much less Acetobacter and
included mainly lactobacilli (Lactiplantibacillus and Fructilactobacillus). Substrate and fly microbiota
composition were similar at 2 and 4% NaCl - Lactiplantibacillus dominated. The substrate with 7% NaCl
contained the genera Leuconostoc and Providencia, which were also found in flies, but representatives of the
Orbaceae family absent in fly microbial communities were detected in the substrate in significant amounts
(almost 30%).

Salt tolerance of bacterial strains associated with D. melanogaster
Three bacterial strains were isolated from the highest positive dilutions of the fly homogenate. Based on the
analysis of full-length 16S rRNA gene sequence, the strains were identified as the representatives of
Lactiplantibacillus plantarum, Leuconostoc pseudomesenteroides, and Acetobacter pasterianus.

The salinity tolerance of these isolates was tested in four replicates for each isolated strain on each salinity
level [Figure 2, Supplementary Table 2]. ANOVA showed that the salinity tolerance strongly depended on
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the isolate, salt concentration, and interaction of these factors (P < 2 × 10 in all three cases). It was found
that A. pasteurianus was the most sensitive to salt (90% growth decrease at 2% NaCl). L. plantarum was