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Page 2 of 18 Yakovleva et al. Microbiome Res Rep 2024;3:19 https://dx.doi.org/10.20517/mrr.2023.56

abundance of Acetobacter, potentially beneficial to D. melanogaster, decreased as NaCl concentration increased,
whereas the relative abundance of the more halotolerant lactobacilli first increased, peaking at 4% NaCl, and then
declined dramatically at 7%. At this salinity level, potentially pathogenic bacteria of the genera Leuconostoc and
Providencia were dominant. The yeast microbiome of D. melanogaster also undergoes significant changes with an
increase in salt concentration in the substrate. The total yeast abundance undergoes nonlinear changes: it is lowest
at 0% salt concentration and highest at 2%-4%. At a 7% concentration, the yeast abundance in flies and their
substrate is lower than at 2%-4% but significantly higher than at 0%.

Conclusions: The abundance and diversity of bacteria that are potentially beneficial to the flies decreased, while
the proportion of potential pathogens, Leuconostoc and Providencia, increased with an increase in salt concentration
in the substrate. In samples with a relatively high abundance and/or diversity of yeasts, the corresponding
indicators for bacteria were often lowered, and vice versa. This may be due to the greater halotolerance of yeasts
compared to bacteria and may also indicate antagonism between these groups of microorganisms.

Keywords: Fruit fly, holobiont, adaptation, high salinity substrate, Acetobacter, lactobacteria, bacterial and yeast
microbiota

INTRODUCTION
Drosophila melanogaster (D. melanogaster) is a classical object used to study the microbiome and its role in
the host’s life. The fly microbiome consists of two main components: yeasts and bacteria. Its composition is
less diverse compared to mammals, which makes it easier to trace the influence of various external factors,
making D. melanogaster a convenient model object for studying the relationship between the host and its
microbiome .
[1-7]
In addition, D. melanogaster is a convenient object for experimental studies of the organism’s adaptation to
new conditions, including its response to a substrate with high NaCl content. High salinity serves as a
convenient and frequently used factor for studying the adaptation process. The adaptation of
D. melanogaster to a substrate with high salinity aligns with the objectives of evolutionary studies, as this
factor is atypical for wild Drosophila. It not only represents a novel environment for them but also an
unfavorable one. It was found that a substrate with a salt content exceeding 2% increased the mortality of
D. melanogaster larvae or significantly delayed their development. At a concentration above 4%, the flies
[8,9]
usually died within 4-5 days, accompanied by a sharp decrease in the number of eggs laid . However, after
several decades of generations living on a diet with a progressively increasing salinity, laboratory
populations became adapted to the substrate with 6%-7% NaCl [8,10] and even 7%-8% [11,12] .

According to previous studies, the bacterial microbiota of the fly gut is primarily dominated by
representatives of one, two, or three taxa: the order Lactobacillales (phylum Bacillota) and the families
Enterobacteriaceae and Acetobacteraceae (phylum Pseudomonadota). In laboratory-reared D. melanogaster,
the bacterial component of the microbiome is generally less diverse than in wild populations and, in most
[13]
cases, is represented by two physiologically distinct groups . The first group includes obligately aerobic
members of the Acetobacter genus, mainly A. pomorum and A. pasteurianus. The second group is
comprised of aerotolerant bacteria of the family Lactobacillaceae, previously classified in the genus
[14]
Lactobacillus but now reclassified into representatives of the genera Lactiplantibacillus (L. plantarum) and
Levilactobacillus (L. brevis). The specific composition of the fly microbiota varies and is highly dependent
on the environmental conditions, including their diet .
[15]