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Page 10 of 18 Yakovleva et al. Microbiome Res Rep 2024;3:19 https://dx.doi.org/10.20517/mrr.2023.56

For comparison, the number of yeasts, both in D. melanogaster homogenates and on the substrate,
increased with the rise in salinity to 2% and 4% but decreased at a salt concentration of 7% [Table 1,
[44]
Supplementary Table 4] .

In flies from lines 0a-0c, 4a-4c, and 7a-7b, the number of bacteria was 1-3 orders of magnitude higher than
that of yeast. Conversely, in three lines 2a-2c, there were significantly more yeasts than bacteria (2-19 times).

Quantification of similarities between flies and substrates microbial communities
We quantified the similarity of the microbial communities of flies and their substrates by the coefficient of
biocenotic similarity [Table 2].

The bacterial communities of the fly line and corresponding substrate were most similar at a salinity of 4%
(for s4a and 4a, the similarity score was 0.98), mainly due to the predominance of species of the genus
Lactiplantibacillus in both samples. The lowest similarity (0.25) was observed at 7% salinity (s7a and 7a),
probably reflecting the difference between the conditions on a high salty substrate and the fly body as
habitats. An intermediate level of similarity was observed at salt concentrations of 0% (for 0b and s0b, 0c
and s0, the similarity score was 0.48 and 0.26, respectively) and 2% (for s2a and 2a, the similarity score was
0.34). The similarity scores were different in the yeast part of the microbial community. The coefficients of
similarity of the yeast community in control flies and their substrates were equal to zero. The similar index
at 7% NaCl was 0.09 (very low). The similarity of the yeast community of the 4% salinity substrate and flies
was low (0.33), while for the 2% salinity substrate and flies, it was the highest (0.74).

The diversity of yeast and bacterial microbiota in the fly gut
The diversity of bacterial and yeast microbiota was assessed by the Shannon index. The diversity of the
bacterial community decreased on average with the increase of salinity from 0% to 4% and rose significantly
with an increase in salt concentration to 7% [Figure 3A, diamonds]. The bacterial microbiota of flies was
more diverse than the substrate microbiota for lines 0b and 4a, and the opposite was the case for the other
fly lines studied [Figure 3B, diamonds].

The yeast communities of the flies, as well as the corresponding substrates, were less diverse than the
bacterial ones [Figure 3, triangles], except for the s0c sample. The diversity of yeasts in the substrate with 2%
and 4% NaCl was lower than in the corresponding flies. However, with an increase in salt concentration to
7%, the yeast community of the substrate became much more diverse than that of the flies.

Relative microbial abundance in one fly to 1 mg of substrate
The relative bacterial and yeast growth efficiencies on the substrate with different salinity and in the
corresponding flies were estimated [Figure 4]. The similar ratio for yeast did not increase with an increasing
salt concentration in the substrate - at salt concentrations from 2% to 7%, one fly contained about the same
amount of yeast as 1 mg of the substrate.

Pairwise correlations between different microbial parameters
To assess the possible interrelation between the bacterial and yeast components of the fly microbiome for
the corresponding 11 samples, we tested all possible pairwise correlations between the following five
variables: salt concentration, bacterial abundance, yeast abundance, bacterial diversity, and yeast diversity
(10 pairwise comparisons). At the 5% significance level, only the following two correlation coefficients were
nonzero: salt concentration - yeast diversity (positive correlation) and yeast abundance - bacteria diversity
(negative correlation). All other correlation coefficients were insignificant [Table 3].