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Yakovleva et al. Microbiome Res Rep 2024;3:19 https://dx.doi.org/10.20517/mrr.2023.56 Page 15 of 18

aligns with the observation that, with the increase in salt concentration, the percentage of Acetobacter
(which can be beneficial for flies) decreased, while the proportion of lactic acid bacteria (also potentially
helpful) first increased (from 0% to 4% NaCl), and then drastically decreased at 7% NaCl. It is also in line
with the presence of Starmerella yeasts in the fly microbiome at high NaCl concentrations, previously
shown to help flies survive and reproduce on the salty substrate [42,44] . These yeasts may also assist the flies in
resisting potentially pathogenic bacteria, such as Leuconostoc and Providencia, whose percentage increased
at higher salinity.

The radical rearrangement of the fly microbiome with the increase in NaCl concentration from 4% to 7%
(some correlations that are significant for 0% to 4% NaCl concentrations become insignificant when
samples 7a-7b are included in the analysis) is apparently due to the fact that extremely high salt
concentration drastically limits the growth of some groups of microorganisms, giving an advantage to other,
more salt-tolerant groups [Table 3]. Most notably, the yeast found in this study is generally more tolerant to
high salinity than most bacteria typical for the D. melanogaster gut.

The absence of S. bacillaris in lines 4a-4c and the presence of this yeast in lines 7a-7b are noteworthy. In
2017-2018, S. bacillaris dominated in lines 4a-4c [41,42] , but later, in 2019-2020 and the current study, it was no
longer found in them. This change likely represents coevolutionary dynamics or different stages of
adaptation of the holobiont (the insect host and its associated microorganisms) to the salty substrate. It can
be hypothesized that the yeast S. bacillaris tends to develop in fly lines that have recently been switched to a
saltier substrate (like lines 7a-7b) and have not yet adapted genetically. The yeasts may help the flies tolerate
[42]
high NaCl concentrations at this early adaptation stage, as shown in our previous studies . However, later,
these beneficial symbionts may be lost by flies as the insects gradually develop genetic adaptations to the
[43]
salty substrate. Further experiments are needed to test this hypothesis.

Conclusions
In summary, we observed systematic changes in the composition and structure of the bacterial and yeast
components of the D. melanogaster microbiome during the holobiont adaptation to the substrate with
increasing salinity. These results can be attributed to the higher halotolerance of yeast compared to bacteria,
leading to several changes: a gradual decrease in the proportion of beneficial bacteria and the emergence of
potentially pathogenic ones, as well as the restructuring of the yeast community. Notably, the appearance of
S. bacillaris, a yeast species, played a key role in the fruit fly’s successful adaptation to the 7% NaCl substrate.
As the salt concentration increased, the yeast seemed to replace major bacterial species, providing the flies
with beneficial metabolites and protecting them from potential pathogens.

Study limitations
The empirical results reported herein should be considered in the light of some limitations. As was
mentioned in the Materials and Methods section, the fly lines 0c, 2a-2c, and 4a-4c were reared in cylindrical
glass jars 0.25 L, and the lines 0a-0b, 7a-7b in population cages 165 mm × 165 mm × 250 mm. On the one
hand, undoubtedly, adaptation is a multi-factorial process, so a uniform setup should be kept in such
studies. On the other hand, all other experimental conditions, except the rearing in a cage or jar, were the
same, and no fluctuations were found in the data of the studied microbiota, which could be explained by
rearing flies in a jar but not in a cage, and vice versa.

DECLARATIONS
Acknowledgments
The authors would like to thank the team of the Extremophiles metabolism laboratory of Winogradsky
Institute of Microbiology, FRC Biotechnology, RAS for NGS sequencing of bacterial variable 16S rRNA gene