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Page 6 of 18 Yakovleva et al. Microbiome Res Rep 2024;3:19 https://dx.doi.org/10.20517/mrr.2023.56

The similarity of flies- and substrate-associated microbial communities was assessed using the coefficient of
[57]
biocenotic similarity (K) , which considers both the abundance and species composition of
microorganisms in the compared samples [Equation (1)].

Where K is the species composition similarity coefficient [Equation (2)], K is the coefficient of relative
s
u
species abundance similarity [Equation (3)].

(1)
(2)
Where S , S are the numbers of species in the first and second samples, respectively, S is the number of
u
species common for compared samples.
To calculate the coefficient of relative species abundance similarity, we calculate the percentage of each
species of microorganisms in each sample, then select a smaller value for the species common to both
samples and sum up the selected values:

Where m is the number of common species in two samples, N and N are the total numbers of
(1)
(2)
microorganisms in the first and second samples, respectively, n and n are the numbers of the i-th
(2)
(1)
i
i
species of microorganism in the first and second samples, respectively.
The difference in salt tolerance was examined with an analysis of variance (ANOVA). Multiple comparisons
were made with Tukey’s HSD test. To plot the figures, the values for the replicates of plating experiments
were averaged. The possible interrelation between the bacterial and yeast components of the fly microbiome
was checked by pairwise correlations between the following five variables: salt concentration, bacterial
abundance, yeast abundance, bacterial diversity, and yeast diversity (10 pairwise comparisons). The
Spearman and Kendall correlation coefficients were calculated because the relationships between the
variables are rather nonlinear. The results obtained based on both coefficients were very similar; the
Spearman coefficients are given below. The significance of the correlation coefficient was checked using t-
test.

RESULTS
Bacterial communities associated with D. melanogaster adapted to the substrate with different salinity were
studied through growth experiments and the analysis of the variable V4 region of the 16S rRNA gene. The
samples represented the following study variants - flies reared on the substrate without NaCl (3 lines), with
2% and 4% NaCl (3 lines each), with 7% NaCl (2 lines), and samples of the substrate processed by flies from
5 lines. The results revealed that the quantitative and qualitative composition of communities varied among
different lines, sometimes significantly, even within replicates (in fly lines reared on the substrate with the
same salinity), but several general trends were observed.