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Page 12 of 18 Yakovleva et al. Microbiome Res Rep 2024;3:19 https://dx.doi.org/10.20517/mrr.2023.56

Table 4. Pairwise Spearman correlations calculated for 9 samples of flies reared on the substrate with 0%, 2%, and 4% NaCl
Variables Salt concentration Bacterial abundance Yeast abundance Bacterial diversity Yeast diversity
Salt concentration 1
Bacterial abundance +0.15 1
Yeast abundance +0.74 * -0.27 1
Bacterial diversity -0.79 * -0.10 -0.66 ** 1
Yeast diversity +0.80 * -0.29 +0.70 * -0.73 * 1
* **
5% significance level; 10% significance level.

Figure 4. The ratio of the microorganism abundance in one fly to 1 mg of substrate. Sample sizes for studying bacteria were 30 flies or 3
mL of the substrate, 3 dilutions, 3 replicates each; for yeasts - 30 flies or 3 mL of the substrate, 10 replicates.

DISCUSSION
The microbiome of D. melanogaster is a classical subject of genetic and microbiological research. However,
most studies of fly-associated microbes focus either on bacteria or on yeast, whereas attempts to study both
components simultaneously are quite rare and vulnerable to criticism. For example, using the same selective
medium for plating both groups of microorganisms has been a source of critique . Our study confirmed
[33]
the low diversity of fly-associated bacteria, predominantly represented by culturable and well-studied
species. The bacterial microbiome of the control lines is dominated by L. plantarum and A. pasteurianus,
the two most common species associated with laboratory-grown fruit flies. Other species include
L. pseudomesenteroides, a species reported earlier from D. melanogaster microbiota, and Giliamella sp.,
unusual for fruit flies but known as symbionts of honeybees and bumblebees’ intestines . D. melanogaster,
[58]
like bees, are insects closely associated with sugar-rich plant substrates; therefore, their gut microbiome
might be similar in some ways, especially on the control and low salinity substrate (2%), as observed in this
study. Moreover, our results are compatible with the idea that the fly microbiome is not entirely transient,
as its composition, in most cases, differs significantly between the flies and the substrate they inhabit.

We found that an increase in substrate salinity to 2%-4% results in a decline in diversity of the fly-associated
bacterial microbiome [Figure 3A, diamonds], as well as in a decrease of Acetobacter to lactic acid bacteria
ratio both in the flies and in the corresponding substrates. This may be partially caused by the high