Page 27 - Read Online

Yakovleva et al. Microbiome Res Rep 2024;3:19 https://dx.doi.org/10.20517/mrr.2023.56 Page 13 of 18

sensitivity of A. pasteurianus to salinity, as confirmed experimentally [Figure 2]. The results obtained on a
substrate with extremely high salinity (7%) show significant changes in the fly-associated bacterial
microbiome. The potentially beneficial symbionts, Lactoplantibacillus and Acetobacter, were replaced by
[59]
other bacteria, including Providencia, which can be pathogenic for D. melanogaster , and moderately salt-
tolerant Leuconostoc and Enterococcus strains. Representatives of those two genera are typical symbionts of
[18]
the human intestine and are also sometimes found in the intestines of wild fruit flies .
The detailed description of the yeast component of the microbiome of the same D. melanogaster lines was
[44]
published earlier , allowing for a comparison of the two components [Figure 5]. Yeast populations of flies
reared on low salinity substrates (0%-4%) consist almost entirely of one species, Pichia occidentalis, except
for one fly line in which no yeast was detected (0c). However, at 2% and 4% salt concentrations, three other
species (Candida californica, Zygosaccharomyces bailii, and P. membranifaciens) appeared as minor
components. At the extremely high salt concentration (7%), Starmerella bacillaris became the dominant
yeast species in flies, while the percentage of P. occidentalis drastically decreased. The minor components
also changed - C. californica and Z. bailii were not detected, while S. etchellsii and Geotrichum candidum
were observed.

The yeast populations of the substrate and flies differed significantly at the minimal (0%) and maximal (7%)
salt concentrations but were similar at intermediate levels (2% and 4%) [Figure 5]. The substrate s0b was
inhabited only by the yeasts Z. bailii, while only P. occidentalis was found in the flies that lived on this
substrate (0b). No yeasts were found in the 0c fly line, but their substrate (s0c) was inhabited by
P. occidentalis, Z. bailii, and C. californica (55%, 43%, and 2%, respectively). P. occidentalis (67%) dominated
the substrate (s7a) at 7% NaCl, but this species was scarce in flies. S. bacillaris was predominant in fly line 7a
but comprised only 23% of the yeast community in the substrate (s7a) [Supplementary Table 4].

Overall, the composition of the fly-associated yeast microbiome undergoes significant changes as the salt
concentration in the substrate increases. The diversity and abundance of yeasts in flies tend to increase with
the rising salt concentration [Figure 3A, triangles; Table 1]. This is in accordance with previous findings that
certain yeast species may help D. melanogaster adapt to elevated salt concentrations .
[41]
All identified yeast species are osmotolerant. The yeasts C. californica, P. membranifaciens, P. occidentalis,
S. bacillaris, S. etchellsii, and Z. bailii can grow in a medium with a salt concentration of up to 10% . The
[60]
yeast-like fungus Geotrichum candidum is known to be sensitive to salinity. It can grow at a salt
concentration of 7%, but the growth rate drops 14-fold compared to the salt-devoid medium [61,62] .

The substrate processed by flies for two weeks serves as a reservoir of microorganisms that enter the fly
intestines and are subsequently excreted. Our comparison of bacterial diversity in substrate and flies did not
reveal a general regularity [Figure 3B, diamonds]. In line 0b, the diversity of bacteria in the substrate was
slightly higher than in the flies (due to the presence of Leuconostoc). In the other control line, 0c, the
bacterial substrate community was almost a monoculture (99% Acetobacter), possibly because the yeast
community was one of the most diverse [Figure 3]. At the same time, the bacterial community of the
corresponding flies was quite diverse (perhaps due to the absence of yeast). The diversity of the bacterial
communities in the substrates with 2% and 7% NaCl was lower than that of the corresponding flies,
whereas, at a salt concentration of 4%, the situation was the opposite.

In all samples except for s0c, the diversity of the yeast microbiota was lower than that of the bacteria. Just as
for bacteria, no clear relationship between the yeast diversity of flies and substrates was found. The