# Distinct communities and differing dispersal routes in bacteria and fungi of honey bees, honey, and flowers

Mikko Tiusanen, Antoine Becker-Scarpitta, Helena Wirta

► To cite this version:

Mikko Tiusanen, Antoine Becker-Scarpitta, Helena Wirta. Distinct communities and differing dispersal routes in bacteria and fungi of honey bees, honey, and flowers. Microbial ecology, 2024, 87, pp.100. 10.1007/s00248-024-02413-z. http://dx.doi.org/10.1007/s00248-024-02413-z

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<figure><img src="image_1.png" /></figure>

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# Distinct Communities and Differing Dispersal Routes in Bacteria and Fungi of Honey Bees, Honey, and Flowers

Mikko Tiusanen<fnref n="1" />,<fnref n="2" /> · Antoine Becker-Scarpitta<fnref n="1" />,<fnref n="3" /> · Helena Wirta<fnref n="1" />,<fnref n="4" />

Received: 17 January 2024 / Accepted: 16 July 2024

© The Author(s) 2024

## Abstract

Microbiota, the communities of microbes on and in organisms or organic matter, are essential for the functioning of ecosystems. How microbes are shared and transmitted delineates the formation of a microbiota. As pollinators forage, they offer a route to transfer microbes among the flowering plants, themselves, and their nests. To assess how the two components of the microbiota, bacteria and fungi, in pollination communities are shared and transferred, we focused on the honey bee *Apis mellifera* and collected honey bee, honey (representing the hive microbiota), and flower samples three times during the summer in Finland. We identified the bacteria and fungi by DNA metabarcoding. To determine the impact of honey bees' flower choices on the honey bee and hive microbiota, we identified also plant DNA in honey. The bacterial communities of honey bees, honey, and flowers all differ greatly from each other, while the fungal communities of honey bees and honey are very similar, yet different from flowers. The time of the summer and the sampling area influence all these microbiota. For flowers, the plant identity impacts both bacterial and fungal communities' composition the most. For the dispersal pathways of bacteria to honey bees, they are acquired directly from the honey and indirectly from flowers through the honey, while fungi are directly transmitted to honey bees from flowers. Overall, the distinctiveness of the microbiota of honey bees, honey, and the surrounding flowers suggests the sharing of microbes among them occurs but plays a minor role for the established microbiota.

**Keywords** Pollination · *Apis mellifera* · Microbe · DNA metabarcoding

## Introduction

We are surrounded by communities of microbes living on and in organisms or organic matter. This microbiota is needed for the functioning of ecosystems and the health of organisms [1]. How microbes are transmitted among organisms is crucial in the formation of an individual's microbiota [2]. Overall, organisms acquire microbiota in different

ways. As an example, as a honey bee emerges, it has no gut microbes, but within a few days, they are obtained through contact with nurse bees and nurse bees' feces [3]. In general, the microbiota of an individual is acquired through three modes: maternal inheritance, social transmission, and environmental acquisition [4]. All of these modes impact the microbiota of insects [2, 5]. In addition, suitability of the host as a habitat for the microbe, the host filtering of microbes, and the microbe-microbe interactions within the host determine the composition of a microbiota [6].

Pollinators play an irreplaceable ecological role, as the vast majority of flowering plants need animal-mediated pollination to ensure the quantity and quality of fruits and seeds [7, 8]. In regard to many important crop species, honey bees contribute significantly to their pollination [9]. While foraging and pollinating, pollinators contribute to the dispersal of microbes in and on plants [10], and microbes can be seen as the third partners of bee-angiosperm mutualism [4]. Microbes may inhabit flower tissues, such as pollen, ovules, and seeds, or reside on the surfaces of the flower organs or

<figure><img src="image_2.png" /></figure>

<footnote>Helena Wirta
helaena.wirta@helsinki.fi</footnote>

<footnote n="1">¹ Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland</footnote>

<footnote n="2">² Department of Environmental Systems Science, Eidgenössische Technische Hochschule Zürich (ETH Zürich), Zurich, Switzerland</footnote>

<footnote n="3">³ UMR PVBMT, CIRAD, Saint Pierre, 97410 La Réunion, France</footnote>

<footnote n="4">⁴ Department of Ecology and Environmental Science, Umeå University, Umeå, Sweden</footnote>
in nectar. For instance, the microbes in nectar are dispersal-
limited, making pollinators crucial vectors for them [11].
Bacteria and fungi living on flowers and in nectar can alter
a flower's attractiveness to pollinators [7, 12]. Conversely,
the identity of the pollinator and its nectar-foraging behavior
can influence the nectar's microbial community [13]. Over-
all, microbes and their chemical effects can influence the
interactions between pollinators and flowers and thus affect
pollination [7, 14]. However, understanding the structures
of the microbiota within pollination communities remains
fragmented [15].

Honey bees, *Apis mellifera*, are the most abundant pol-
linators of the world [8, 16]. Honey bees harbor a distinct,
well-studied gut microbiota, mainly consisting of bacteria
[17, 18], and being mainly transmitted by social interactions
among colony members, and to a lesser extent by hive sur-
faces [19, 20]. Also, the environment shapes the gut bacter-
rial community of honey bees [21, 22]. On the other hand,
honey bees influence the microbiota of flowers [21], and
the environment influences the overall microbiota of honey
bees [22]. On top of interacting with the microbes in their
guts, honey bees interact with microbes in their food stores,
hive surfaces, floral resources, and other parts of their envi-
ronment. There, the importance of microbial dispersal from
different sources to honey bees remains unexplored [23] yet
would be important because of their interplay [24]. Honey
bees have several well-known pathogens [25–27]. Some of
them are known to be spread to other pollinators too, pos-
sibly through flowers [28–30]. Therefore, it is important to
understand the pathway of transmission. The hives and food
stores of honey bees also host a diversity of microbes, with
different environmental factors shaping these microbial com-
munities [31, 32]. Flowers share microbes with the visiting
bees and the food stores of honey bees [23, 33, 34], yet host
physiology and priority effects affect which microbes can
establish in and on a honey bee [7]. Most research on the
microbes of honey bees, of their nests, and of the flowers
they visit has focused on bacteria (for an exception, see a
comparison of bacteria and fungi in bee bread, the stored
pollen [35]), although fungi also play an important role in
pollination communities by changing the attractiveness of
nectar differently from bacteria [36, 37], for example, patho-
genic fungi, species of *Vairimorpha* (*Nosema*), are known
to be distributed through flowers to honey bees and bumble
bees [38]. For another managed bee, *Megachile rotundata*,
bacteria and fungi from floral resources are brought to their
nests, and their nests are likely to serve as reservoirs of both
bacterial and fungal pathogens of flowers and the bees them-
selves to act as transferring agents [39].

Given the vast role in pollination attributed to the honey
bee, we examine how bacteria and fungi are shared and
transferred by honey bees, among the pollinating species
itself, its nest (i.e., the hive), and its food resources (i.e.,

flowers in the surroundings). For assessing bacteria and
fungi in the hives, we sample honey, which stores DNA
well. The processing of nectar into honey includes spread-
ing it into a high number of open combs in small amounts,
for excess water to evaporate [40], and this allows DNA
present in any form within the hive to enter honey. The time
taken for nectar to ripen into honey tends to be 3 to 7 days
after which honey bees collect the nectar, now turned into
honey, from multiple combs into few to fill the combs up
and cover these with wax [40, 41]. Thus, the bacterial and
fungal DNA in newly covered honey would offer a sample of
the bacteria and fungi honeybees have encountered in their
hive during approximately the previous week. While the
structure of the pollination network has been pin-pointed as
important for the dispersal of the microbes to flowers [23],
here, we aim at identifying the movement of microbes and
their dispersal routes to honey bees, through their hives and
through flowers. We identify and compare the bacterial and
fungal communities of honey bees, honey (representing their
hives), and flowers in the surroundings of the hives by DNA
metabarcoding. We focus our analyses onto the strain level
(genetic variants of microbe species), as this is essential to
detect which exact microbes are shared [2]. For this, we use
zero-radius operational taxonomic units (ZOTUs) [42], the
smallest taxonomic units based on sequence data.

We examine the following questions:

1. The honey bees, their honey, and surrounding flowers share bacteria and fungi?

2. What is the spatiotemporal structure of the bacterial and fungal communities of honey bees, honey, and flowers?

3. How do the bacteria and fungi from flowers and the honey transfer to the microbiota of the honey bees?

Methods

Sampling, Sample Preprocessing, and Dna
Metabarcoding

To study the microbiota of honey bees, honey, and flowers,
we studied 36 honey bee colonies (29 colonies at the end of
the summer), placed in six apiaries [as in 43] and the sur-
rounding flowering plants in South-Finland.

We collected honey bee, honey, and flower samples in
mid-June, mid-July, and mid-August in 2021. For microbiota
of honey bees, we collected one deciliter of a mixture of
nurse and forager bees, and a subsample of this was homog-
enized to determine microbiota in and on honey bees. We
used whole individuals, as the whole-body microbiota rep-
resent the gut microbiome and also capture the whole-body
microbial exposure [44]. For the honey microbiota, we col-
lected newly covered honey. For microbiota of flowers, we

<figure><img src="image_3.png" /></figure>
sampled inflorescences of the abundantly flowering species
into 99% EtOH in different habitat types close to the apiar-
ies (herbaceous plants and shrubs were sampled, as listed in
Text S1). All samples were stored frozen.

Before extracting the DNA, the samples were preproc-
essed. For honey bees, to extract DNA of microbes from
on and in the body of honey bees, homogenized honey bee
tissue was used. For honey, 10 g of honey was diluted to
30 ml of DNA clean water, the sample was centrifuged, and
the supernatant was discarded. For flowers, a water-bath
sonicator and vortexing were used to detach the microbes.
After removing most of the flower tissue, the sample was
centrifuged and the supernatant discarded (Text S1).

The DNA was extracted in the same way from all sam-
ples with the DNeasy Plant Mini Kit (Qiagen, Germany).
To identify bacteria, a part of the gene region 16S was
amplified and for fungi a part of ITS2, with taxon-specific
primers (16S_515FB and 16S_806RB [45, 46] and ITS2-F
and ITS2-R [47], respectively). To identify plants in honey
samples, a part of ITS2 was amplified with plant-specific
primers ITS2-F and ITS2-R [48, 49]. After the initial ampli-
fication with tagged primers, the samples we labeled with
unique dual-index combinations in a second PCR and pooled
libraries of the three gene regions were formed. The librar-
ies were sequenced with Illumina MiSeq V3 (Text S1). The
bioinformatics processing of reads followed Kaunisto et al.
[50] (see details of the bioinformatic processing in Text S1).
For the bioinformatic processing, the reads of all samples
were combined per gene region. The reads were truncated,
merged, and quality controlled. Primers were removed, and
the reads were dereplicated and singletons were removed.
The reads were denoised to ZOTUs [42]. The taxonomic
assignation of ZOTUs was done by comparison against a
specific reference database for each gene region (Text S1).

## Statistical Analyses

Comparison of Bacterial and Fungal Communities in Honey
Bee, Honey, and Flower Samples

Statistical analyses were performed in R v.4.3.1 [51]. All the analyses were run separately for both bacterial and fungal communities, at the taxonomic level of ZOTUs. Additionally, the main analyses were run with families to assess the robustness of patterns in regard to the taxonomic level used. All the analyses were based on presence–absence data, unless separately indicated that relative read abundances were used. Thus, this does put emphasis on rare taxa even though the rarest ones were omitted from the analyses (see below).

To answer how much of the bacterial and fungal taxa are
shared among the honey bee, honey, and flower samples, in
comparison to being found uniquely in each, we drew Euler

diagrams with the *eulerr* package [52]. To determine the
difference in the bacterial and fungal community compo-
sition between honey bee, honey, and flower samples, we
applied the multivariate homogeneity of group dispersion
of Bray–Curtis dissimilarity using “betadisper” in package
vegan 2.6–4 [53]. A significant difference in the multivariate
distance between the plots and the sample type-specific cen-
troid indicates heterogeneity in variance in species composi-
tion among communities, i.e., difference in beta-diversity.
For this and the following analyses assessing factors affect-
ing the community compositions, we omitted samples with
less than 2000 reads of the targeted taxonomic group (bacte-
ria or fungi), to avoid including samples with few sequences
due to amplification or sequencing issues. Taxa occurring in
less than three samples were omitted from the community
analyses.

To explore the factors influencing bacterial and fungal
communities, we built three separate models: (1) to test the
spatial and temporal effects on the microbial communities
of honey bees, honey, and flowers; (2) to test the spatial
and temporal effects and flower identity on flower microbial
communities; and (3) to test the effects of the most-visited
flowering plants present in honey samples on microbial
communities of honey bees and honey (for details, see Text
S2). The effects of different variables on community com-
position were estimated with redundancy analyses (RDAs)
using “rda” in vegan 2.6–4 [53]. Last, to examine how the
bacterial and fungal communities in honey and flower sam-
ples explain the respective communities in honey bees and
potential transmission pathways, we constructed structural
equation models (SEMs). We modeled direct pathways
from flowers to honey, and from both honey and flowers to
honey bees, and an indirect path from flowers to honey bees
through honey. We built separate models for bacterial and
fungal communities. For the modeling, we used the propor-
tions of each sample type (honey bee, honey, flower) where
a certain ZOTU was present (567 and 1072 ZOTUs for the
bacterial and fungi, respectively). The models were fitted
using package lavaan [54].

Results

We collected 98 honey bees, 99 honey, and 143 flower sam-
ples in 2021 (Text S3 and Table S1). After the bioinform-
matic processing, 98 and 97 honey bee samples had filtered
bacterial and fungal reads, respectively. All honey samples
had bacterial, fungal, and plant reads. Of the flower samples
110 and 133 had bacterial and fungal reads, respectively,
due to majority of the flower samples’ reads being of plant
origin instead of the targeted bacteria or fungi (only 8.1%
and 40.3% of the filtered reads, respectively; Tables S2 and
S3). Of all the filtered reads assigned to bacteria, fungi
and plants, 99.2%, 70.8%, and 100.0%, respectively, were
assigned to a family (Table S2).

Bacterial and Fungal Communities' Composition
of Honey Bee, Honey, and Flower Samples

The frequency of occurrence of bacterial and fungal families and genera among samples varied largely between the different sample types (Tables S4 and S5). As an example, the bacterial genera, which the five core honey bee gut microbial species clusters belong to (*Lactobacillus* Firm-4 and Firm-5, *Snodgrassella alvi*, *Gilliamella apicola*, and *Bifidobacterium* spp. [55]), were found in all honey bee samples, as expected. They were found in a far smaller proportion of honey samples and were nearly absent in the flower samples.

The proportions of shared ZOTUs and families between honey bee, honey, and flower samples varied between bacteria and fungi (Figs. 1 and S1). The portion of taxa shared between honey bee and honey samples for bacteria is clearly smaller than that of fungi (56.2% and 75.5% of honey bee ZOTUs detected in honey samples, respectively). Vice versa, there were more bacterial than fungal sharing between flowers and honey (34% and 19.9% of flower ZOTUs detected in honey, respectively). All these patterns are consistent also at the family level (Fig. S1). Meanwhile, honey bees shared less bacteria than fungi with flowers (22.5% and 36.1% of honey bee ZOTUs detected in flowers, respectively). Only 3.8% of bacterial ZOTUs and 6.8% of fungal ZOTUs were detected in all three sample types.

While assessing the similarity of bacterial and fungal
community composition between sample types, bacterial
communities are clearly more clustered than the fungal com-
munities, for which the compositional overlap is stronger
(Fig. 2). The group dispersion (β-diversity) of the bacte-
rial communities of honey bee samples is lower than that of
honey or flower samples, with the average distance to cen-
troids being 0.17, 0.59, and 0.47, respectively, while it is of

similar magnitude for fungal communities, ranging between
0.50 and 0.57 (Table S6, Fig. 2). The community composi-
tion of different sample types is more similar when con-
sidering families (Fig. S2). Yet, the overall patterns remain
despite the taxonomic level as well as when considering also
the relative read abundances, not only presence-absence data
(Fig. S2).

Different Spatiotemporal Structure
of the Microbiota of Honey Bees, Honey,
and Flowers

All the models exploring the factors influencing bacterial
and fungal communities (Text S2) were statistically sup-
ported, while the adjusted *R*² values varied from 2.8% for
bacterial communities of flowers to 18.3% for fungal com-
munities of honey bees (Table S7). Based on the variance
partitioning, the variance accounted for by the spatiotem-
poral variables, by region and month together, vary largely
among the bacterial and fungal communities, ranging from
2.8% (month: 1.0% + region: 1.8%) for the bacterial commu-
nities of flowers to 18.6% (month: 13.8% + region: 4.8%) for
the fungal communities of honey bees (Table 1). The meth-
odological bias quantified by the number of reads accounted
for a minor proportion of the total variation, ranging from
0.5 to 2.6% (Table 1).

Of all the sample types and communities tested, only the
bacterial communities in flowers were not affected by the
time of the summer (Table S7). The two types of microbial
communities in the honey bee samples are not structured in
the same way by the spatiotemporal variables, with a greater
effect of the time of the summer and the region on fungal
than on bacterial communities (Tables 1 and S8). The effect
of time is either of the same order of magnitude or much
greater than the effect of space (Table 1). The results based
on the ZOTUs are supported also by models run at the fam-
ily level (Table S8).

<figure><img src="image_4.png" /><figcaption>**Fig. 1** Euler diagrams show-
ing the number of shared and
non-shared bacterial and fungal
ZOTUs between honey bee,
honey and flower samples,
shown with the symbols, with
all samples and all taxa included</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2** Similarity of bacterial and fungal communities in honey bee, honey, and flower samples based on principal coordinates analyses, with presence-absence data, for the ZOTUs. Samples with less than 2000 reads and taxa with less than three occurrences are omitted from</figcaption></figure>

<figure><img src="image_6.png" /></figure>

the analyses. The first and second PCoA axes explained 33.3 and 9.6% and 21.9 and 11.3% of the variation of the bacterial and fungal communities, respectively (Table S6)

<table><caption>**Table 1** Results of the redundancy analyses for the microbial communities of the different sample types, considering the communities of ZOTUs and using presence-absence data. Below are shown the results of the variance partitioning between the fractions. Detailed results of the model and the ANOVA partitioning are given in Table S7</caption><thead><tr><th rowspan="2">Fractions</th><th colspan="2">Bacteria</th><th colspan="2">Fungi</th></tr><tr><th>Df</th><th>adjR<sup>2</sup></th><th>Df</th><th>adjR<sup>2</sup></th></tr></thead><tbody><tr><td colspan="5"><strong>Honey bees</strong></td></tr><tr><td>Month</td><td>2</td><td>7.6%</td><td>2</td><td>13.8%</td></tr><tr><td>Region</td><td>2</td><td>1.8%</td><td>2</td><td>4.8%</td></tr><tr><td>Reads</td><td>1</td><td>2.6%</td><td>1</td><td>0.5%</td></tr><tr><td>Residuals</td><td></td><td>90.0%</td><td></td><td>81.1%</td></tr><tr><td colspan="5"><strong>Honey</strong></td></tr><tr><td>Month</td><td>2</td><td>8.8%</td><td>2</td><td>6.9%</td></tr><tr><td>Region</td><td>2</td><td>9.0%</td><td>2</td><td>9.3%</td></tr><tr><td>Reads</td><td>1</td><td>3.3%</td><td>1</td><td>2.3%</td></tr><tr><td>Residuals</td><td></td><td>81.7%</td><td></td><td>81.5%</td></tr><tr><td colspan="5"><strong>Flowers</strong></td></tr><tr><td>Month</td><td>2</td><td>1.0%</td><td>2</td><td>8.1%</td></tr><tr><td>Region</td><td>2</td><td>1.8%</td><td>2</td><td>0.6%</td></tr><tr><td>Reads</td><td>1</td><td>1.9%</td><td>1</td><td>2.5%</td></tr><tr><td>Residuals</td><td></td><td>95.3%</td><td></td><td>89.4%</td></tr></tbody></table>

Including the identity of the focal plant, as the plant family it belongs to, in the model for flower samples (Text S3), increased the proportion of the variance explained (Table S9). In the model, the contribution of the plant family is clearly higher than the one of spatiotemporal variables, yet for bacteria, the plant family was not statistically significant

for the ANOVA partitioning ($p=0.066$; Table S9). The impact of the flower families used by the honey bees is smaller on the honey bees' bacterial community but of similar magnitude for that of honey and the fungal community in both honey bee and honey samples (Text S3, Table S10).

## Bacteria and Fungi have Different Dispersal Pathways between the Microbiota

Bacterial communities transfer from flowers to hive, represented by honey, more than fungal ones (Fig. 3, Table S11). The direct pathway from hive to honey bees is of similar importance for both bacteria and fungi, but the indirect pathway from flowers to honey bees through hive is much stronger for bacteria than for fungi. Meanwhile, bacterial taxa are not transferred directly from flowers to honey bees, while for fungi, this also happens. These patterns prevail also when analyzing the data at the taxonomic level of families and, in addition, the pathways are stronger except for the direct pathway from hive to honey bees in fungi (Fig. S3).

## Discussion

We found the bacterial and fungal microbiota of honey bees, their honey, and the surrounding flowers to be partially shared even at the level of individual DNA-strains, indicating microbial dispersal between them. However, the dispersal routes differed between fungi and bacteria. While most bacterial ZOTUs in or on the honey bees are not shared with or obtained from their environment, nearly one fourth
**Fig. 3** Pathways of the bacterial and fungal ZOTUs from flowers to honey and to honey bees based on the SEM analyses. The direct lines show the direct pathways, while the circle shows the interaction of the pathways flower-to-honey and honey-to-honey bees. All the pathways shown are statistically supported

<figure><img src="image_7.png" /></figure>

of them are, as we observed, 22.5% of the honey bees' bacte-
rial ZOTUs also in the flower samples.

Considering the bacteria found in honey samples, repre-
senting microbes living in the hive and brought into the hive
by the honey bees from their environment, only few of the
bacterial taxa were also found in and on honey bees. This
is in line with previous research showing that gut bacteria
contribute the vast majority of microbial DNA sequences
of honey bees, while only a little microbial DNA originates
from the honey bees outside surfaces in a DNA metabarcod-
ing-based assessment when comparing samples of whole
honey bees, bee guts, and bee individuals from which the
guts were removed [44]. The highly specialized bacterial
community of honey bee guts [56, 57] is known to be mainly
obtained through the interactions with colony members, but
also from hive materials [57]. The gut as an environment for
microbes is very different from hive surfaces, highlighting
how exposure to microbes is only one step of the microbiota
formation [58]. In contrast to the gut bacteria, the bacte-
ria on the honey bees could be expected to be more shared
with their hive, their food sources, and other parts of their
environment, and thus, the bacteria found shared among
honey bees, honey, and flowers in our study, as an exam-
ple of ZOTUs from the genera *Pseudomonas* (*Pseudomon-
aceae*), and *Spiroplasma* (*Spiroplasmataceae*), are likely to
be mainly from the surfaces of the bees. Members of both
these genera have been associated both with honey bees and
flowers and with members of *Pseudomonas* likely to prevent
diseases while members of *Spiroplasma* are likely to cause
such [25, 59, 60].

Interestingly, the fungal communities of the honey bees
and their honey show a totally different pattern from that
of bacteria, as the majority of the taxa are shared, and the
communities are highly similar. As flowers provide the main

source of food for bees, the fungi found in nectar and pollen
can directly contribute to microbiota in bee nests and guts,
and thus, the likely source for them has been proposed to be
the plants [34]. On top of being exposed to fungi arbitrarily
while foraging, honey bees have been observed to collect
fungal spores from plant surfaces, to complement their nutri-
tion with these [34, 61]. Fungi in honey bees' food stores
also changes in time, initially resembling those of flowers
but decreasing in diversity and abundance over time. This
has been found to happen in fungi associated with stored
pollen, i.e., bee bread [62, 63]. We here examined DNA
traces in freshly covered honey, presenting a sample of any
DNA present in the hive as the honey would have been pre-
pared from nectar spread to a large number of open combs,
allowing any DNA traces to enter and be stored in it. Yet,
sampling bee bread and hive surfaces directly for microbes
would possibly complement the list of microbes encountered
in and brought to the hive and lead to discovering a greater
overlap between microbial communities [64] than we dis-
covered by sampling only honey.

Overall, to our knowledge, other studies have not exam-
ined the difference in sharing of fungi and bacteria of honey
bees with their honey and environment. Most research in
regard to microbes of bees has focused on bacteria, yet fungi
may be pathogenic, living in food storages causing spoilage,
as well as commensal or even mutualistic [34]. Especially,
the non-pathogenic fungal members of honey bee gut micro-
biota are mainly unstudied [24]. Yet, ecologically interesting
fungal taxa are common members of the fungal communi-
ties of both honey and honey bees also in our study, for
example, the fungal honey bee pathogens, belonging to the
genera *Ascosphaera* and *Aspergillus* [27, 65], and yeasts like
*Metschnikowia* living in nectar and moist honey [66]. Most
microbiota studies typically focus on a single host type [23]

<figure><img src="image_8.png" /></figure>
and thus cannot explore the relationships between the differ-
ent microbiota. Here, through studying the microbiota within
a connected system, we were able to address the multitrophic
dispersal pathways of the microbes. While many of the fun-
gal or bacterial taxa observed here in or on bees, honey, and
flowers may not have an ecologically meaningful role on
the specific substrate or host on which they were observed,
their presence or absence tells us about the relationships of
bees, honey, and flowers and where different microbes may
be encountered. Our examination of the patterns based on
presences and absences does delimit the applicability of our
results as the abundances of fungi and bacteria are likely
to vary greatly in different parts of the pollination system,
with as an example bacteria being highly more abundant in
honey bee guts than fungi [3, 24]. Yet, with the occurrences
of taxa, most of the bacterial and fungal taxa of the flower
microbiota were not detected as members of the microbiota
of either honey or honey bees, and both the bacterial and
fungal community compositions of flowers were totally dis-
tinct from the communities of honey bees and only over-
lapping a little with the communities of the honey. These
results contradict recent findings proposing that the dispersal
of microbes between plants, mediated by pollinators, is an
important factor shaping microbial communities of plants
and the pollinators [23]. This could be due to a lesser role of
dispersal per se, in comparison to the characteristics of the
habitat and the interspecific interactions with the microbes
already inhabiting the habitat, in the formation of a micro-
biota, in other words, the filtering of microbial taxa [23].

The identity of the flower visitor plays a central role in
this dispersal [10], yet based on our results, *Apis mellifera*
does not play a substantial role in shaping the bacterial or
fungal communities of flowers, although they might have a
large impact on the transfer of certain microbes. In support
of this minor role, Ushio et al. [67] found that the impact of
a visit of a pollinator is small on the diversity of bacteria on
flower surfaces, although different insects do leave differ-
ing microbial footprints. Yet, for the honey bee, it has been
shown their visits increase the diversity of bacteria on grape-
fruit, *Citrus paradisi*, flowers [21] as well as the diversity
of both bacteria and fungi on *Salix* inflorescences [68]. As
microbes may change nectar properties, as an example by
changing the sugar composition and decreasing the sugar
concentration [69], a change in a flower's microbiota might
impact pollinator visits, and this may change the sharing of
microbes among the flowers and pollinators [7]; thus, even
small contributions to the microbiota of flowers by the honey
bee may be impactful.

Spatiotemporal Structure of Microbiota

We found that both the sampling region and time of the sea-
son influenced the studied bacterial and fungal communities.

Still, the bacterial communities of honey bees of all the
sampled colonies in the different regions, and in different
months, were very similar. The same has been found in
another study, where the bacterial communities of whole
honey bee individuals varied only slightly over the course
of a honey production season and not at all between differ-
ent hive types [70]. This is consistent with the honey bee
microbiota being strongly dominated with the restricted set
of gut bacteria [56, 71], yet the bacteria on the body surfaces
could be more exposed to temporal changes.

Our results show that the relative abundance of the honey
bees' most abundantly used plant families affect both the
bacterial and fungal communities of the honey bees and the
honey. This is in line with previous studies showing varying
diversity in microbial composition across different flower
species and flower characteristics, such as volatile organic
compounds, defining which microbes inhabit them [72, 73].
Sampling time and region largely define the community of
flowers available to honey bees to select from, but there, the
relative proportions of different plant families honey bees
have used, have a strong impact on the microbiota of honey
and honey bees.

Honey Bees Acquire Bacteria and Fungi Differently
from Honey and Flowers

When considering the pathways of microbial taxa from the
surrounding microbe pool of flowers to the honey bees and
the honey, fungal taxa are introduced into the honey bee
microbiota directly from flowers and the hive, and from the
flowers through the hive. For bacteria, taxa are transferred
from flowers to honey, but not directly to honey bees at all.

Honey bees spend the first weeks of their lives in the
hives and have very similar bacterial communities with each
other, with progressive changes as they age [74, 75]. Our
study further suggests that only a minority of the bacteria
occurring in the hive or in the surrounding environment of
hives becomes a part of the honey bee microbiota, reinforc-
ing the hypothesis of an ecological filter through selection
due to biotic interactions [6].

Meanwhile, the larger variability in their fungal micro-
biomes shows that there is more room for environmental
acquisition, and honey bees would acquire fungal taxa both
from the honey and the flowers based on our analyses.
Here, the role that bacteria versus fungi play in honey
bees is likely to be of importance, as fungi are only rarely
part of the gut microbiota of honey bees [76]. Thus, fungi
may inhabit other structures of honey bees and especially
the surfaces, allowing for a proportionally larger sharing
of them. We found that flowers, hive, and flowers through
hive can act as dispersal routes of microbes to honey bees.
This supports the view of hive as an extended organism
to honey bees in terms of their microbiota [19] and offers
greater understanding how the different communities of microbiota are shared and interact, yet, taking into account all the above, more research on especially the sharing and transfer of fungi by honey bees is needed, to understand the formation and functioning of the full microbiota of this system.

## Conclusions

Honey bees' movements between flowers and the hive make the hives potential hotspots from where the microbes might be dispersed further directly (e.g., to flowers) and indirectly (e.g., to other pollinators through flowers) by the honey bees. While several studies have shown microbe sharing to take place between flowers and pollinators, based on our results, the encounters of honey bees and flowers shape the others' microbiota relatively little, this being especially revealed when looking at the level of individual strains of microbes, as here with ZOTUs. While the microbiota of the honey bee, the hive (as represented by DNA of microbes in honey), and the surrounding flowers are all impacted by the time and region in a relatively similar magnitude, it appears that their bacterial and fungal communities are fairly distinct. Considering how different honey bees, honey, and flowers are as habitats for bacteria and fungi to live in, in regard to nutrient composition, environmental conditions, and longevity, this result is not contradictory despite the sharing of microbes [77]. While the gut microbiota of honey bee is known to be specialized, so are those of many other bee species, especially social ones, which have a route to share the microbes to offspring, and thus, provide the microbiota an opportunity to evolve within the host species [5, 57]. Consequently, our results of horizontal microbe sharing in the world's most dominant pollinator are likely to some extent be generalizable to other closely related social bees. However, many other pollinator groups acquire their gut microbiota more opportunistically, leaving the role of different bees, flies, butterflies, and moths in microbe sharing of pollination networks to be further examined.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s00248-024-02413-z.

**Acknowledgements** We would like to thank Tristan Ubaldi and Matti Leponiemi for the help with fieldwork and Marjo Kilpinen and Eija Hakala for assisting in the laboratory work. We also thank the two beekeepers for maintaining the hives during the study. We would also like to thank the anonymous reviewers for providing thorough and helpful advices. The Biomedicum Functional Genomics Unit is acknowledged for the sequencing services and CSC - IT Center for Science, Finland, for computational resources. Open access of the article is funded by Helsinki University Library. The study was funded by a grant to HW from Kone foundation. ABS is supported by the European Union: Agricultural Fund for Rural Development (EAFRD).

**Author Contribution** HW designed the study. HW and MT collected the samples. HW did the DNA laboratory and the bioinformatic analyses. ABS, MT and HW did the statistical analyses. HW wrote the first draft of the manuscript. All authors contributed to and approved the manuscript.

**Funding** Open Access funding provided by University of Helsinki (including Helsinki University Central Hospital). Open access of the article is funded by Helsinki University Library. The study was funded by a grant to HW from Kone foundation. ABS is supported by the European Union: Agricultural Fund for Rural Development (EAFRD).

**Data Availability** The sequence datasets generated during the current study are available in the Sequence Read Archive repository, in the BioProject PRJNA930592 (https://www.ncbi.nlm.nih.gov/sra/PRJNA930592).

## Declarations

**Competing Interests** The authors declare no competing interests.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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