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

It was previously believed that the intestinal microbiota of D. melanogaster is mainly transient, entering the
[16]
gut with the substrate, and its development in the gut itself is minimal . In contrast, recent research
suggests that certain bacteria establish more complex and specific interactions with the fly gut, contributing
to a stable gut bacterial microbiota and providing benefits to the host [17-20] . Notably, L. plantarum and some
other bacterial symbionts stimulate larval growth [13,21] , increase adult longevity , and improve food resource
[22]
[23]
[1]
utilization efficiency . A. pasteurianus produces thiamine required by flies . L. brevis can influence the
motor activity of flies by altering the sugar levels and indirectly regulating neuronal activity through the
[24]
synthesis of the neurotransmitter octopamine . Given these findings, it is logical to assume that bacteria
may also influence the adaptation of flies to new conditions, including salty substrate, though the question
has not yet been thoroughly studied.

Yeasts are another indispensable component of the microbiome of D. melanogaster, playing a crucial role in
their life cycle. Yeast serves as an essential source of protein, along with other nutrients and micronutrients
required for larval development [25-32] . Larvae reared on yeast-poor substrates exhibit reduced immunity, have
a diminished ability to adapt to unfavorable conditions, and often fail to complete development [27,29-31] . On
the other hand, yeast-poor diets can enhance cold tolerance and increase the longevity of
[33]
D. melanogaster [34,35] . The yeast content in the substrate can have opposing effects on longevity, fecundity,
[36]
and mating frequency of fly females and males . In turn, flies influence the species composition of fungal
communities inhabiting forage substrates, favoring the development of some yeast species and suppressing
[37]
the growth of other yeasts and micromycetes . Larvae tend to favor yeast species that promote faster
development and increased adult body mass [4,27] . Volatile aromatic compounds released by yeasts attract
fruit flies to fermented plant substrates, which, without yeast assistance, are not as appealing to flies.
Combined with the ability of some yeasts to remain viable as they pass through the digestive tract of
D. melanogaster, this enables yeasts to utilize fruit flies for their dispersal [38-40] . Previous studies have
demonstrated that the abundance and species composition of yeasts associated with D. melanogaster may
depend on the salt concentration in the substrate. Certain yeast species, such as S. bacillaris, may contribute
to the adaptation of laboratory fly lines to salty substrates [41-44] . Evidently, the yeast community of flies adapts
to the salty substrate alongside the host organism, reflecting the adaptability of the entire symbiotic
[45]
complex. This study, along with our earlier works , highlights the role of the microbiome in general and
yeasts in particular in the adaptation of flies to salty substrate.

We did not find any studies investigating the transformation of the D. melanogaster microbiome during
adaptation to changing environmental conditions. Furthermore, the yeast and bacterial components of the
microbiome are usually studied separately, potentially leading to the oversight of significant interactions
between them. In this study, we compared the bacterial and yeast components of the microbiome of
D. melanogaster reared on substrates with different NaCl concentrations for seven years to identify trends in
microbiome changes as flies adapt to increasing salinity. Parallel studies of yeast and bacterial communities
of D. melanogaster aim to clarify the role of the microbiome in the host’s adaptation to changing
environmental conditions and shed light on the relationships within a dynamically changing microbial
community.

The primary objective of this study was to investigate the changes in the bacterial component of the fly
microbiota during host adaptation to increasing salt concentration in the substrate. The secondary, yet
crucial aim, was to elucidate the interaction between bacterial and yeast components of the fly microbiota.