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\begin{document}

\title{Honeybee visitation to shared flowers increases Vairimorpha ceranae
prevalence in bumblebees}



\author[1]{Maryellen Zbrozek}%
\author[1]{Michelle Fearon}%
\author[1]{Chloe Weise}%
\author[1]{Elizabeth Tibbetts}%
\affil[1]{University of Michigan}%


\vspace{-1em}



  
  \date{May 23, 2024}


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\selectlanguage{english}
\begin{abstract}
Vairimorpha (=Nosema) ceranae is a widespread pollinator parasite that
commonly infects honeybees and wild pollinators, including bumblebees.
Honeybees are highly competent V. ceranae hosts and previous work in
experimental flight cages suggests V. ceranae can be transmitted during
visitation to shared flowers. However, the relationship between floral
visitation in the natural environment and the prevalence of V. ceranae
among multiple bee species has not been explored. Here, we analyzed the
number and duration of pollinator visits to particular components of
squash flowers---including the petals, stamen, and nectary---at six
farms in southeastern Michigan, USA. We also determined the prevalence
of V. ceranae in honeybees and bumblebees at each site. Our results
showed that more honeybee flower contacts and longer duration of
contacts with pollen and nectar was linked with greater V. ceranae
prevalence in bumblebees. Honeybee visitation patterns appear to have a
disproportionately large impact on V. ceranae prevalence in bumblebees
even though honeybees are not the most frequent flower visitors. Floral
visitation by other pollinators was not linked with V. ceranae
prevalence in bumblebees. Further, V. ceranae prevalence in honeybees
was unaffected by floral visitation behaviors by any pollinator species.
These results suggest that honeybee visitation behaviors on shared
floral resources may be an important contributor to increased V. ceranae
spillover to bumblebees in the field. Understanding how V. ceranae
infection risk is influenced by pollinator behavior in the shared floral
landscape is critical for reducing parasite spillover into declining
native bee populations.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Recent declines in native and managed bee populations threaten the
stability of pollination services that are vital for maintaining natural
and agricultural ecosystems (Beismeijer et al. 2006, Potts et al. 2010).
Several factors contribute to these declines, including the spread of
multi-host pathogens, habitat loss, and climate change (Ricketts et al.
2008, Burkle et al. 2013, Furst et al. 2014). Losses in pollinator
community biodiversity and abundance lead to changes in flower
visitation patterns (Beismeijer et al. 2006, Albrecht et al. 2012,
Burkle et al. 2013), as well as changes in the risk of infectious
disease within reduced pollinator communities (Figueroa et al. 2020,
Graystock et al. 2020, Fearon and Tibbetts 2021). Yet, it remains
unclear how differences in floral visitation behaviors within pollinator
communities affected by these declines may in turn affect the spread of
pathogens.

Many pollinator pathogens and parasites (hereafter, `parasites') are
transmitted within and among species by visitation to flowers that were
previously visited by infected bees (Durrer and Schmid-Hempel 1994,
Graystock et al. 2015, M\selectlanguage{ngerman}üller et al. 2019, Purkiss and Lach 2019). The
likelihood of parasite deposition and subsequent transmission on flowers
depends on multiple factors, including flower traits, flower morphology,
pollinator behavior, and the environment (Durrer and Schmid-Hempel 1994,
Alger et al. 2019, Figueroa et al. 2019, Russell et al. 2019). Depending
on the parasite, different plant components, including the floral
tissue, pollen, and nectar, are implicated in transmission among
pollinators (reviewed by McArt et al. 2014). In particular, differences
in the rates of parasite deposition and acquisition of microorganisms on
various flower parts may depend on how bees interact with the flowers
during foraging visits. For example, bees foraging for pollen had
greater rates of microbe deposition and acquisition on flowers than did
bees foraging for nectar (Russell et al. 2019). However, pollinator
visitation behaviors have been shown to have a complex relationship with
the prevalence of bee parasites on flowers. In a study on pollinator
viruses, flowers receiving longer visits were more likely to host
viruses, but those with high visitation rates were less likely to host
viruses (Alger et al. 2019). In a different study, \emph{Crithidia
bombii} survived longer when deposited inside the corolla rather than on
the bract, but infection occurring from an encounter with the bract
resulted in more intense infection (Figueroa et al. 2019). Therefore,
the ways in which infected bees interact with specific flower features
and the duration and frequency of their visits will alter the likelihood
of parasite deposition on floral surfaces and influence the probability
of infection for later visitors. However, most studies on this topic
have been conducted in the laboratory and have not fully considered the
potential for parasite transmission via shared floral resources in
natural settings.

Agricultural fields and the surrounding hedgerows may represent
potential `hot spots' for parasite transmission within and among bee
species on shared floral resources. Managed honeybees (\emph{Apis
mellifera)} are frequently brought to agricultural fields to provide
pollination services, where they have ample opportunity to interact with
wild pollinators that are also attracted to plentiful crop flowers or
nearby hedgerows with wildflowers (Goulson and Hughes 2015). The
worldwide dispersal of \emph{A. mellifera} (hereafter honeybees) and its
many parasites has consequently led to spillover (i.e., parasite
transmission from reservoir populations to sympatric wildlife) to many
naïve wild pollinators (Daszak et al. 2000, Keesing et al. 2006, Goulson
and Hughes 2015, Purkiss and Lach 2019). Since honeybee colonies tend to
send generalist foragers to a few flower patches at a time (Visscher and
Seeley 1982), it is possible that an infected colony may create
localized floral hot-spots where wild bees may acquire parasites.
Increasingly, parasites previously thought to only infect honeybees are
found in diverse populations of wild pollinators and seem to be
contributing to their decline (Furst et al. 2014, Arbulo et al. 2015,
Goulson and Hughes 2015, Porrini et al. 2017, Müller et al. 2019,
Purkiss and Lach 2019).

One parasite of particular concern is the widely-dispersed
microsporidian parasite \emph{Vairimorpha} (= \emph{Nosema}
)\emph{ceranae} (Tokarev et al. 2020)\emph{,} which has been rapidly
infecting honeybees and spilling over into wild bee populations over the
past three decades (Paxton et al. 2007, Chen et al. 2008, Fries
2010)\emph{.} Although \emph{V. ceranae} is transmitted within honeybee
hives through contaminated feces and pollen stores, transmission may
also occur when bees encounter spores on contaminated flowers (Higes et
al. 2008b, 2010). Graystock et al. (2015) demonstrated that multiple
pollinator parasites, including \emph{V. ceranae} , can be effectively
dispersed onto flowers by competent hosts and then vectored from flowers
back to colonies by other pollinator species. Additionally, \emph{V.
ceranae} spores have been detected on the flowers of at least 14 plant
genera in the field (Graystock et al. 2020). Therefore, contamination of
shared flower resources is a likely mode of transmission for \emph{V.
ceranae} between different pollinator species, with dispersal
potentially occurring through defecation on floral surfaces or through
the rubbing off of spores that were attached to the bee cuticle
(Graystock et al. 2015, Bodden et al. 2019, Piot et al. 2020).
Furthermore, Graystock et al. (2015) found that \emph{V.
ceranae}transmission was very rapid in small experimental flight cages,
but they recognized that whether parasite dispersal is similar in nature
will depend on the characteristics of pollinator communities and
environmental conditions. Despite clear experimental evidence
for\emph{V. ceranae} transmission on flowers, the relationship between
specific pollinator visitation patterns and \emph{V. ceranae} prevalence
across managed and wild pollinator species in the field has remained
understudied.

Here, we examine whether the prevalence of \emph{V. ceranae} in managed
and wild bee populations is influenced by the floral visitation
behaviors of bees in the natural environment. We conducted an
observational study of \emph{V. ceranae} in honeybee (\emph{A.
mellifera} ) and bumblebee (\emph{Bombus} spp.) populations among
different pollinator communities to understand how floral visitation
patterns differ among pollinator species and whether the visitation
patterns are linked with \emph{V. ceranae} prevalence in both host
species. Specifically, we investigated how \emph{V. ceranae} prevalence
is linked with the number of honeybee, bumblebee, and other pollinator
visits to flowers and the time each bee species spent interacting with
different parts of the flowers during each visit. We hypothesized that
higher numbers of visits and longer visits by potentially infected bees
would increase the likelihood of \emph{V. ceranae} transmission and
correlate with higher \emph{V. ceranae} prevalence. These findings will
be important for determining the pollinator visitation behaviors that
contribute the most to \emph{V. ceranae} exposure and subsequent
infection in honeybees and bumblebees as well as helping to establish
whether \emph{V. ceranae} transmission on flowers occurs under field
conditions.

\textbf{METHODS}

\emph{Study System}

\emph{V. ceranae} is a microsporidian parasite with a nearly global
distribution. It was initially discovered in \emph{Apis ceranae} and
later spilled over into \emph{A. mellifera} honeybees, where it appears
to be more virulent than closely related parasites such as \emph{V.
apis} (Paxton et al. 2007). Recent studies have shown that wild native
bees are also infected with \emph{V. ceranae} , including many wild
bumblebees (\emph{Bombus} spp.), stingless bees (\emph{Tetragonula
hockingsi} , \emph{Tetragonisca} spp., \emph{Scaptotrigona}
spp.,\emph{Melipona} spp.), and solitary bees (\emph{Osmia bicornis} )
(Plischuk et al. 2009, Graystock et al. 2013, Furst et al. 2014, Müller
et al. 2019, Purkiss and Lach 2019, Salvarrey et al. 2021, Cilia et al.
2022). Transmission of \emph{V. ceranae} between individuals is
primarily fecal--oral or oral--oral, as it is spread through ingestion
of contaminated food or contact with the feces of diseased hosts (Chen
et al. 2008, Smith 2012). \emph{V. ceranae} germinates in the midgut of
the bee, where the spore count can reach over 30 million, and it is then
excreted as feces (Paxton et al. 2007, Chen et al. 2008, Higes et al.
2008a), potentially contributing very large numbers of spores to the
environment (e.g., on floral surfaces). Symptoms of infection in
honeybees include digestive disorders, shortened life spans, atypical
breeding behavior, reduced sucrose sensitivity, and diminished honey
production; however, colony infection is often asymptomatic until sharp
depopulation occurs, often in autumn and winter (Chen et al. 2008, Higes
et al. 2008a, 2010, Graystock et al. 2013). Symptoms are generally
assumed to be the same for wild bees, but data on this is limited aside
from a few reports that \emph{V. ceranae} may cause reduced survival,
learning impairment, sucrose sensitivity, and cellular immunosuppression
in bumblebees or stingless bees (Graystock et al. 2013, Piiroinen and
Goulson 2016, Macías-Macías et al. 2020). Furthermore, \emph{V.
ceranae}infections suppress the pollinator immune response, which can
lead to coinfection with other pathogens or parasites and an increased
likelihood of mortality (Antúnez et al. 2009). The drastic effects
of\emph{V. ceranae} on pollinator health have been linked to the sudden
collapse of honeybee colonies (Higes et al. 2008a) and may be an
important factor in the recent declines of some wild bees (Graystock et
al. 2013, Furst et al. 2014, Goulson and Hughes 2015).

\emph{Sampling pollinators in the field}

\emph{A. mellifera} and \emph{Bombus} spp. (hereafter honeybee and
bumblebee, respectively) samples were collected from six winter squash
farms in southeastern Michigan, USA (Appendix S1: Table S1) during two
visits to each site between 26 July and 30 August 2016 during the peak
squash bloom. The pollinator sampling described here includes a subset
of the sites that were previously sampled in Fearon and Tibbetts (2021)
and Fearon et al. (2022). In this study, we focus on \emph{V.
ceranae}infection in honeybees and bumblebees, while the prior studies
examined links between the pollinator community composition and bee
viral prevalence. Sites were at least 10 km apart to ensure that the
pollinator communities were isolated from each other (Greenleaf et al.
2007). We only sampled on sunny days with windspeeds less than 2 m/s. To
collect the bees, four 50 m transects were randomly placed at each field
site. Three transects were placed in the field along the crop rows,
while the fourth transect was placed along a field edge to sample bees
foraging near native flowers and invasive weeds. All honeybees and
bumblebees observed along the transect lines were collected using
handheld nets or pan traps. Details on the trapping methods are included
in Appendix S2 and Fearon and Tibbetts (2021).

All pollinator samples were stored on dry ice in the field, and later
placed in a -80 \selectlanguage{english}@C freezer to maintain the integrity of the DNA for
detection of \emph{V. ceranae} infection. All bees were identified to
species using the Discover Life key (Ascher and Pickering 2013). The
collected bumblebee species were primarily \emph{Bombus impatiens} , but
also included \emph{Bombus auricomus} , \emph{Bombus bimaculatus}
,\emph{Bombus griseocollis} , \emph{Bombus fervidus, Bombus
pensylvanicus} , \emph{Bombus sandersoni} , and \emph{Bombus vagans} at
very low densities (\textless{} 8 individuals total). \emph{A.
mellifera} and \emph{B. impatiens} were common at all six field sites.

\emph{Collecting Flower Visitation Data}

During each visit to the six farms, we took 30-min video recordings of
pollinators visiting eight randomly selected male squash flowers per
site (N = 112, mean video length: 30.87 min {[}sd: 3.75 min{]}). Each
video was recorded between 07:30 AM and 12:00 PM on sunny, non-windy
days. Video recordings were watched to record data on the identity and
frequency of pollinator visitors to the flowers. Pollinators captured on
video were identified to genus where possible (e.g., \emph{Apis}
,\emph{Bombus} , \emph{Eucera} ), or to morphospecies for species that
require close inspection and/or a key for accurate identification
(Appendix S1: Table S2). Honeybees and bumblebees were easy to identify
in the video recordings due to their relatively large body size and
distinctive coloration. The behaviors of all other pollinators observed,
including small green and olive halictids (e.g., \emph{Augochlora}
,\emph{Augochlorella} , \emph{Augochloropsis} , \emph{Halictus} ,
and\emph{Lasioglossum} genera), \emph{Melissodes} spp., \emph{Eucera}
spp.,\emph{Triepeolus} spp., \emph{Vespula} wasp spp., and hover flies,
were grouped together into an `other pollinators' category to compare to
honeybee and bumblebee behaviors in later analyses (see Statistics
section).

During each individual pollinator's visit to the observed flower, we
recorded the duration (seconds) of each visitor's interactions with
specific flower parts, including petals (petal-only), nectar
(nectar-only), pollen (pollen-only), and both pollen and nectar
simultaneously (pollen+nectar). Typically, large-bodied bees, including
honeybees and bumblebees, could not avoid contacting the stamen while
drinking nectar (pollen+nectar) and led to relatively few observations
of nectar-only interactions with flowers (Appendix 1: Table S3). For
this reason, the nectar-only interactions were not considered as a
substantial interaction type and were not included as a response
variable in our main analyses. For each flower observed, the total
duration of all types of interactions were summed for each pollinator
group (honeybees, bumblebees, or all other pollinators) and then divided
by the number of flower visits for the respective pollinator group to
generate the duration spent per visit by each pollinator group to each
flower. Finally, to test how each pollinator group's visitation behavior
impacted \emph{V. ceranae} prevalence, we averaged the calculated
visitation metrics for all flowers observed during the same site visit
for each pollinator group. We followed the same process to calculate the
average duration per visit of time spent on petal-only, pollen-only, and
pollen+nectar interactions for each pollinator group. The number of
visits for each pollinator group was the raw count of each type of
pollinator that visited each observed flower within the 30-min
observation period, which was then averaged for each of the two visits
to each site.

Evaluating the average duration bees spent per floral visit ensured that
the duration metrics accurately reflected the time bees spent
interacting with flowers without being skewed by the number of bee
visitors. Each additional bee visitor inherently increased the total
duration of time bees spent on flowers (\emph{r} = 0.76, t = 11.52, df =
95, p \textless{} 0.001) but did not necessarily increase the duration
per visit time (\emph{r} = 0.02, t = 0.21, df = 95, p = 0.84). We
predicted that bees that spent a greater amount of time per visit
interacting with flowers would have a greater likelihood of either
depositing or picking up \emph{V. ceranae} spores on flowers and would
be correlated with higher \emph{V. ceranae} prevalence.

\emph{Detecting V. ceranae Infection Presence}

Approximately eight honeybee and eight bumblebee individuals per visit
to each field site were randomly selected to test for the presence or
absence of \emph{V. ceranae} infection (target N per species per site =
16; total honeybee, n = 75; bumblebee, n = 86; Appendix S1: Table S4).
Only sites with a minimum of eight bees total were included in the
analysis. When less than eight individuals of each species were
collected during one of the two visits to a site, infection was tested
in all individuals collected (Appendix S1: Table S4). The selected
bumblebees were predominantly \emph{Bombus impatiens} , but also
included single individuals from \emph{Bombus fervidus, Bombus
bimaculatus,} and\emph{Bombus pensylvanicus} species that were all
collected from a single field site visit that had relatively low
\emph{Bombus impatiens}abundance (Site E, Visit 1). Ultimately, we
modeled the binary presence or absence of \emph{V. ceranae} in
individual honeybees and bumblebees and all sites had a minimum of eight
samples tested for \emph{V. ceranae} presence per host species.

Abdominal contents were dissected from each sample using sterilized
forceps and immediately placed on dry ice. Half of the abdomen was
placed in a microcentrifuge tube for DNA analysis, and the other half
was stored for reference. DNA was extracted using the DNeasy Blood \&
Tissue Kit (Qiagen, Germantown, MD, USA) following the manufacturer's
instructions for tissue samples. Following extraction, DNA purity and
concentration were quantified using Nanodrop 2000 software (Thermo
Fisher Scientific, Waltham, MA, USA). One sample with a nucleic acid
concentration less than 10 ng/\selectlanguage{greek}µ\selectlanguage{english}L was removed from the study due to
insufficient DNA extraction (a honeybee from Site E, Visit 1).

To ensure adequate extraction of bee DNA, polymerase chain reactions
(PCR) were conducted on all samples using \emph{A. mellifera} 18S rRNA
gene primers, which produced bands at 784 bp (Cardinal et al. 2010).
Sequences for these bands were confirmed via Sanger sequencing. To
determine presence or absence of \emph{V. ceranae} infection in each
sample, PCR was conducted with \emph{V. ceranae} -positive and
H\textsubscript{2}O negative controls using the primers Nosema-F
(5'-CGGATAAAAGAGTCCGTTACC-3') and Nosema-R (5'-TGAGCAGGGTTCTAGGGAT-3')
for the \emph{V. ceranae} large subunit ribosomal RNA gene (GenBank
Accession No: DQ486027; Chen et al. 2008). Details on the PCR procedure
can be found in Appendix S2. A subset of samples was selected for Sanger
sequencing to confirm the identification of \emph{V. ceranae} (GenBank
Accession Numbers: bee 18S rRNA, OQ545564--OQ545565 and \emph{V.
ceranae} large subunit rRNA, OQ550096--OQ550100).

\emph{Statistical Analysis}

Analyses were performed using the statistical program R (version 4.2.1;
R Core Team 2020). First, we evaluated how pollinator visitation
behavior varied among honeybees, bumblebees, and other pollinators with
a separate model for the following response variables: 1) number of
visits to flowers per 30 min video observation period, 2) total duration
per visit of pollinator visits to flowers (seconds/visit), 3) duration
pollinators spent on petals only per visit (seconds/visit), 4) duration
pollinators spent on pollen only per visit (seconds/visit), and 5)
duration pollinators spent simultaneously on pollen + nectar per visit
(seconds/visit; model output in Appendix S1: Table S5). Each model was a
zero-inflated generalized linear mixed effects model (GLMM) using a
negative binomial distribution with a log link function and pollinator
group (honeybees, bumblebees, and other pollinators) as the main
predictor for both the zero-inflated and GLMM portions of the model
(\emph{glmmTMB} package; Brooks et al. 2017). The pollinator visitation
data was aggregated by each flower observed for each pollinator group;
therefore, a nested random effect of Flower ID (8 flowers/visit/site)
within visit to a site (2 visits/site) within site (6 sites) was used in
each model. To model the duration per visit, we used the duration of
behaviors in seconds as the response variable with an offset of the log
of the number of pollinator visits + 1 to correct for flowers with zero
visits. We removed one outlier point from the number of visits per 30
min data, where other pollinators visited a single flower over twice as
many times as the next most visited flower in our study. Each model was
checked for overdispersion, zero-inflation, and spatial autocorrelation;
none of these tests were significant (\emph{DHARMa} package; Hartig
2020). Then we followed up each model with a post-hoc test to evaluate
significant differences among honeybees, bumblebees, and other
pollinators' visitation behaviors (Appendix S1: Table S6;
\emph{emmeans}package; Lenth et al. 2020).

To evaluate \emph{V. ceranae} prevalence in honeybees and bumblebees, we
initially calculated the total apparent \emph{V. ceranae} prevalence in
each host species (\emph{epiR} package; Stevenson et al. 2021) and used
a Chi-squared test of two proportions to determine if there was a
significant difference among the two host species\emph{.} Then we ran
two sets of models: the first to evaluate how the number of visits to
flowers (per 30 min) at each site influenced \emph{V. ceranae}prevalence
in honeybees and bumblebees\emph{,} and the second to test how the
duration per visit of specific behaviors on the flowers was correlated
with \emph{V. ceranae} prevalence in each host species\emph{.} All
models were generalized linear mixed effects models with \emph{V.
ceranae} prevalence in either honeybees or bumblebees as the response
variable, a binomial distribution, and a logit link function (package
\emph{lme4} ; Bates et al. 2015). For all models, we used a random
effect of each visit to a site nested within site to account for both
the variation between the two different dates on which each site was
visited and the variation among different sites, though the random
effects were singular for many models (model outputs in Appendix S1:
Table S7 and S8). In the first set, models included the average number
of honeybee visits, bumblebee visits, and all other pollinator species
visits to flowers during the 30-min observation period as main effects.
In the second set, we ran a series of models to evaluate the average
total duration per visit (seconds/visit) and the durations per visit of
petal-only, pollen-only, and pollen+nectar interactions of honeybees,
bumblebees\emph{,} and all other pollinators that visited the flowers.
To deal with zeros in the data, all main effects had `1' added to the
value before log transforming the variable, and then they were scaled
and centered to generate standardized estimates from the models. The
variance inflation factor (VIF) for all main effects in all models was
\textless{} 2.2, indicating that there was no multicollinearity in our
models. Additionally, none of the models were over-dispersed. There was
no evidence of spatial autocorrelation in the model residuals,
indicating that \emph{V. ceranae} prevalence was not correlated among
sites based on their spatial proximity (\emph{DHARMa} package; Hartig
2020). Finally, we used a Bonferroni Correction of four comparisons to
adjust our alpha significance threshold from 0.05 to 0.0125 to account
for four separate analyses, one for each of four different pollinator
duration behavioral parameters (total duration per visit, duration on
petals per visit, duration on pollen per visit, duration on pollen +
nectar per visit). The number of visits model was not included in the
Bonferroni Correction and was evaluated with the usual 0.05 alpha
threshold.

\textbf{RESULTS}

The number of pollinator visits by the three pollinator groups
(honeybee, bumblebee, and other pollinators) varied considerably among
observed flowers (ranges: honeybees = 0--7, bumblebees = 0--38, other =
0--54; \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 16.12, df = 2, p = 0.0003). Honeybees had
fewer visits compared to bumblebees (p = 0.0003) but did not differ from
combined other pollinator visits (p = 0.15; Figure 1a; Appendix S1:
Table S3, S5, and S6). On the other hand, the time bees spent on flowers
per visit (seconds) did not differ among honeybees, bumblebees, and
other pollinators (Figure 1b; \selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2} = 4.95, df = 2, p =
0.08), despite substantial variation in total duration per visit among
flowers observed (ranges: honeybees = 0--219.25, bumblebees = 0--222.75,
other = 0--484 seconds). We further explored how bee species may differ
in how much time per visit they spend interacting with different aspects
of the flower, including the petals, pollen, and simultaneously
contacting the pollen and nectar (pollen+nectar). Bumblebees spent less
time per visit on petals compared to honeybees (p \textless{} 0.0001) or
other pollinators (p \textless{} 0.0001; Figure 1c). Other pollinators
spent more time per visit on pollen-only visits relative to honeybees (p
= 0.0025) and bumblebees (p \textless{} 0.0001; Appendix S1: Fig. S1).
On average, all three pollinator groups spent similar amounts of time
per visit in contact with pollen+nectar (Figure 1d;
\selectlanguage{greek}χ\selectlanguage{english}\textsuperscript{2}= 3.60, df = 2, p = 0.17), though there was a wide
range of visit times for pollen+nectar (ranges: honeybees = 0--197.75,
bumblebees = 0--218.75, other = 0--295 seconds). Overall, each
pollinator group differed in the number of visits and duration of time
spent per visit interacting with different aspects of the flowers, which
could contribute to variation in the likelihood of bees depositing or
picking up parasite spores during floral visits.

\emph{V. ceranae} was highly prevalent in both honeybees and bumblebees
at all six field sites. In total, 68.0\% (95\% CI: 56.7--77.9\%) of
honeybees and 64.0\% (95\% CI: 52.9--73.6\%) of bumblebees had \emph{V.
ceranae} detected in their midguts. \emph{V. ceranae} prevalence did not
significantly differ between host species (\selectlanguage{greek}\emph{χ} \selectlanguage{english}\textsuperscript{2}
= 0.14, df = 1, p = 0.71). Among different sites, \emph{V.
ceranae}prevalence ranged from 57.1\% to 81.3\% in honeybees and from
40.0\% to 93.8\% in bumblebees (Appendix S1: Fig. S2, Table S9).

To determine whether floral visitation behaviors were linked
with\emph{V. ceranae} prevalence, we explored how the number of
pollinator visits and the duration of time per visit spent interacting
with certain parts of the flower correlated with \emph{V. ceranae}
prevalence in honeybees and bumblebees. Despite a lower number of
honeybee visits compared to bumblebee visits (Figure 1a), the number of
honeybee visits was the only factor that had a significant impact on
\emph{V. ceranae}prevalence. \emph{V. ceranae} prevalence in bumblebees
was positively linked with honeybee flower visits (p = 0.005; Table 1,
Figure 2a) but not bumblebee flower visits (p = 0.98, Figure 3a). In
contrast, \emph{V. ceranae} prevalence in honeybees was not linked with
honeybee flower visitation (p = 0.57, Table 1, Figure 2a) or bumblebee
flower visitation (p = 0.47, Figure 3a). \emph{V. ceranae} prevalence in
honeybees and bumblebees was also not linked with flower visitation by
other bee genera (both p \textgreater{} 0.54, Figure 3b; Table 1,
Appendix S1: Table S7 and S8).

We also expected that greater amounts of time bees spent per visit on
flowers and interacting with different aspects of the flower (e.g.,
petals, pollen, and pollen+nectar) would increase the \emph{V.
ceranae}prevalence by increasing the chances of parasite transmission.
For the duration per visit models, we used a Bonferroni-corrected
significance threshold of 0.0125 because the durations per pollinator
visit to each part of the flower were analyzed separately. \emph{V.
ceranae} in honeybees and bumblebees was not associated with the total
duration per visit of honeybees, bumblebees, or other pollinators (Table
1). However,\emph{V. ceranae} prevalence in bumblebees was marginally,
but not significantly, higher the longer that honeybees spent
interacting with flowers per visit (p = 0.014, Figure 2b). We further
explored this result by breaking down the total floral visit duration by
the duration of time that bees spent interacting with different flower
parts, including petals, pollen, and pollen+nectar to determine which
specific behaviors contributed most to \emph{V. ceranae} prevalence in
each host species (Table 1; Appendix S1: Table S7 and S8). \emph{V.
ceranae}prevalence in bumblebees was higher the longer honeybees
interacted with pollen+nectar (p = 0.009; Figure 2c), despite no overall
differences in time spent on pollen+nectar per visit among honeybees,
bumblebees, and other pollinators (Figure 1d). \emph{V. ceranae} in both
host species was not impacted by bumblebee or other pollinator duration
spent per visit on pollen+nectar (Figure 3c,d; Table 1). \emph{V.
ceranae} in honeybees and bumblebees was not correlated with the time
per visit that bees spent on petals or pollen, regardless of bee species
(Table 1).

\textbf{DISCUSSION}

We observed that honeybees, bumblebees, and other pollinators differed
in the number of visits to flowers and the duration per visit to petals
and pollen but did not vary in the total length of time they spent on
flowers. Honeybees had fewer flower visits than bumblebees, and all
visiting pollinators spent similar amounts of time per visit interacting
with the pollen and nectar simultaneously (Figure 1). Yet, the sites
with more and longer honeybee visits to shared flowers had
higher\emph{V. ceranae} prevalence in bumblebees. Therefore, honeybee
visitation to flowers appears to have a disproportionate impact
on\emph{V. ceranae} prevalence in local bumblebee populations.
Visitation by bumblebees or other pollinators, in terms of the number of
visits or time spent on flowers, was not associated with \emph{V.
ceranae}prevalence in either host species. These findings suggest
honeybees may play an important role in the spread of \emph{V. ceranae}
to bumblebees through indirect contact via shared flowers in the natural
environment. Such pathogen spillover from honeybees to bumblebees is
likely to have negative consequences for bumblebee populations (Colla et
al. 2006, Furst et al. 2014).

The spillover from honeybees to bumblebees may occur differently
compared to transmission among honeybees. \emph{V. ceranae} is easily
transmitted within honeybee hives when bees clean up fecal material, eat
contaminated food, or perform trophallaxis (Chen et al. 2008, Higes et
al. 2010). Further, drifting of honeybees among hives is known to occur
and is thought to play a role in the transmission of parasites,
including \emph{V. ceranae} (Higes et al. 2010, Eberl and Muhammad
2022). As \emph{V. ceranae} is a well-established concern for managed
honeybee populations (Higes et al. 2013) and is thought to spill over
from managed honeybee populations to native bumblebee populations (Furst
et al. 2014, Goulson and Hughes 2015, Alger et al. 2018), high \emph{V.
ceranae} prevalence in honeybees may be driven by intraspecific
transmission occurring among and within honeybee hives. In
contrast,\emph{V. ceranae} prevalence in bumblebees may be driven in
part by parasite spillover from shared flowers with honeybees. Thus,
spillover from honeybees to bumblebees could explain why honeybee
visitation behavior was strongly correlated with \emph{V. ceranae}
prevalence in bumblebees, but not with prevalence in honeybees.

Our results are consistent with prior small-scale lab experiments which
demonstrated that pollinator parasites, including \emph{V. ceranae} ,
are transmitted via contact with flowers (Durrer and Schmid-Hempel 1994,
Graystock et al. 2015, Purkiss and Lach 2019). Several recent studies
have further shown that pollinator parasites are commonly found on
flowers in the field, but their abundance varies based on flower
morphology, the environment, and pollinator visitation patterns (Alger
et al. 2019, Figueroa et al. 2019, Russell et al. 2019, Graystock et al.
2020). Furthermore, Graystock et al. (2015) experimentally showed that
23\% of uninfected bumblebees that foraged on flowers recently visited
by infected honeybees became infected with \emph{V. ceranae} . This
suggests that flowers can become hotspots for parasite dispersal once
contaminated (Graystock et al. 2015). However, few studies have examined
how differences in the pollinator community's floral visitation
behaviors may impact parasite prevalence across multiple host species in
nature (but see Graystock et al. 2020), and \emph{V. ceranae} in
particular has been neglected. As pathogens are a key driver of
pollinator population decline (Potts et al. 2010), it is crucial to
understand patterns of their transmission within and among pollinator
species in the natural environment. Our findings corroborate prior
experimental work and add that honeybee visitation to shared
flowers---especially in areas with generally high \emph{V.
ceranae}infection levels in honeybees---facilitates greater \emph{V.
ceranae}spillover from managed honeybees to wild bumblebees in the
natural environment.

While \emph{V. ceranae} spillover via contaminated flowers seems likely,
little is known about how pollinator interactions with different parts
of inflorescences may affect the likelihood of \emph{V.
ceranae}transmission. We examined the association between \emph{V.
ceranae}prevalence and the duration per visit by honeybees, bumblebees,
and other pollinators to flower petals, nectaries, and pollen to explore
which parts of inflorescences may have the greatest impact on \emph{V.
ceranae} spread. We found that higher \emph{V. ceranae} prevalence in
bumblebees was associated with longer durations of honeybee interactions
per visit spent simultaneously contacting both the pollen and nectar of
inflorescences (pollen+nectar). These visits were characterized by
active foraging behavior for nectar and/or pollen while deeply embedded
within the corolla of the large squash flowers. Additionally, since
there was no difference among honeybees, bumblebees, and other
pollinators in time spent per visit on the pollen+nectar (Figure 1d),
our results suggest that time spent by honeybees on flowers
disproportionately increases the likelihood of parasite spillover to
bumblebees relative to time spend on flowers by bumblebees or other
pollinators.

The length of time that infected honeybees spend closely interacting
with both pollen and nectar---food resources that are consumed by many
pollinator species---likely contributes to \emph{V. ceranae} spore
deposition on flower surfaces, which may be picked up and consumed by
subsequent floral visitors. \emph{V. ceranae} is a fecal-orally
transmitted parasite (Chen et al. 2008, Smith 2012) and bees commonly
defecate on floral surfaces while foraging, with longer visits
increasing the likelihood of defecation (Bodden et al. 2019). \emph{V.
ceranae} has been detected in honeybee salivary glands (Chen et al.
2009) and viable and infectious \emph{V. ceranae} spores have been found
in the corbicular pollen of honeybees (Higes et al. 2008b), suggesting
that pollen can become contaminated during pollen collection. Therefore,
it is possible that the pollen on the stamen may be a key hot spot for
the deposition of \emph{V. ceranae} by infected bees and the acquisition
of this contaminated pollen by susceptible bees. In contrast, nectar may
be a poor location for pathogen transmission because high sugar
concentrations can inhibit microbial growth and pathogen survival (Adler
et al. 2021). We observed that honeybees and bumblebees seemed to spend
more time on pollen+nectar interactions compared to pollen-only or
nectar-only interactions (Appendix S1: Table S3), likely owing to their
large size making it difficult to only contact one food source at a
time. Therefore, the long visits with high floral contact during which
honeybees and bumblebees foraged for pollen and nectar may have
increased the chances for transmission to occur.

We did not observe any relationships between \emph{V. ceranae}prevalence
and the length of time pollinators spent interacting with only the
petals or pollen. Though many bees spent time on the petals, bees were
typically observed either resting or crawling on the petals for very
short periods of time. Though other pollinator parasites are transmitted
via floral petals (Figueroa et al. 2019), in our study petal-only
interactions were not linked with \emph{V. ceranae}prevalence.
Pathogenic spores can often survive well on floral surfaces (McArt et
al. 2014), but their survival likely varies among different plant
species, flower parts, and the centrality of the plant in the
plant--pollinator network (Palmer-Young et al. 2016, Naughton et al.
2017, Adler et al. 2018, Figueroa et al. 2019, Piot et al. 2020). Since
this study only considered a single plant species and did not consider
the plant--pollinator network, future studies are needed to empirically
test how floral traits among different plant species affect
pollinator--flower interactions, explore the distribution of spores on
different floral surfaces in the natural environment, and determine the
consequences for \emph{V. ceranae} parasite dispersal via different
parts of the inflorescences.

In contrast to \emph{V. ceranae} prevalence in bumblebees, we
consistently found that \emph{V. ceranae} prevalence in honeybees was
not correlated with flower visitation by any species\emph{. V.
ceranae}prevalence in honeybees was high at all sites (57.1\% to 81.3\%;
Appendix S1 Figure S1, Table S9), indicating that honeybees experience
consistently high \emph{V. ceranae} infection levels across the
landscape. The spillover of \emph{V. ceranae} from managed honeybee
hosts to wild bumblebee populations would suggest that honeybees are a
highly competent host for \emph{V. ceranae} that could be facilitating
transmission to other native bee species in pollinator communities
through indirect interactions on shared flowers.

\textbf{Conclusions}

We found that \emph{V. ceranae} prevalence in bumblebees was strongly
associated with the floral visitation behaviors of honeybees. More
honeybee visits and time spent interacting with both the pollen and
nectar contributed to higher \emph{V. ceranae} prevalence in bumblebees,
despite honeybees visiting flowers less than bumblebees. These results
suggest that even a few visits by honeybees to shared crop flowers may
be having a disproportionately large effect on \emph{V.
ceranae}spillover from managed honeybee populations to wild bumblebee
populations in the agricultural landscapes. Our study provides a first
look at how specific pollinator visitation behaviors on flowers impact
the likelihood of parasite spillover among native pollinators in nature.
Understanding how the risk of \emph{V. ceranae} infection for different
bee species changes with regard to their shared floral landscape with
honeybees is critical for reducing parasite spillover into declining
native bee populations. This knowledge may be particularly important in
agricultural settings where managed honeybees and wild pollinators from
the surrounding environment may frequently interact on crop flowers and
nearby hedgerows, creating potential hotspots for parasite transmission
on flowers.

\textbf{Acknowledgements}

We thank the many people that helped with field collection, pollinator
sample processing, and recording pollinator behaviors from video
recordings on this project: M. Frank, H. Zonnevylle, C. Fleming, J.
Graham, M. Scales, K. Lam, Y. Oden-Plants, G. Flint, E. Collins-Carey,
L. Davis, N. Gratsch, J. Martin-Johnston, and J. Peterson. All molecular
work was conducted in the University of Michigan (UM) Biodiversity
Laboratory. Sanger sequencing was done by the UM Advanced Genomics Core.
This research was funded by NSF IOS-1557564 and NSF IOS-2134910, the
North American Pollinator Protection Campaign, Pollinator Partnership,
The Garden Club of America, the UM Rackham Graduate School, the UM
Department of EEB, and the Noreen and Kenneth Buckfire Scholarship Fund
through the UM LSA Honors Program. MLF was supported in part by The Dow
Chemical Company Foundation through the UM Dow Sustainability Fellows
Program.

\textbf{Author contributions} : M. Zbrozek and M. Fearon should be
considered joint first author. MZ and MLF developed manuscript ideas,
MLF carried out field data collection, and MZ and CW conducted the
molecular data collection. MLF and MZ performed all analyses with advice
from EAT. MZ and MLF wrote the first draft of the manuscript, and all
authors contributed substantially to manuscript revisions.

\textbf{Data and Code Accessibility:} All data used for the analyses and
figures in this manuscript are available on Dryad at the following DOI
url upon publication: https://doi.org/10.5061/dryad.vt4b8gtxt. Data is
available at this private-for-peer-review
link:~https://datadryad.org/stash/share/rqYo0GHtWdNnQimIpJRViRZiHpQuuN9i20hpiQxKhGE.
All code used in calculations and analyses will be available at Zenodo
with a DOI upon publication and has been submitted with our manuscript
documents for review. All videos associated with the data in this
manuscript will be available upon request from M.L. Fearon
(mlfearon@umich.edu). All sequences generated in this study are
available on GenBank Accession numbers OQ545564--OQ545565 and
OQ550096--OQ550100.

\textbf{Competing Interests:} The authors declare that they have no
conflict of interest.

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2008. Landscape effects on crop pollination services: are there general
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Roulston, T., S. Smith, and A. Brewster. 2007. A Comparison of Pan Trap
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Russell, A. L., M. Rebolleda-Gómez, T. M. Shaible, and T. L. Ashman.
2019. Movers and shakers: Bumble bee foraging behavior shapes the
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Salvarrey, S., K. Antúnez, D. Arredondo, S. Plischuk, P. Revainera, M.
Maggi, and C. Invernizzi. 2021. Parasites and RNA viruses in wild and
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Smith, M. L. 2012. The honey bee parasite Nosema ceranae: Transmissible
via food exchange? PLoS ONE 7:1--6.

Stevenson, M., E. Sergeant, T. Nunes, C. Heuer, J. Marshall, J. Sanchez,
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\textbf{TABLES}

\textbf{Table 1.} Scale standardized model estimates for the effects of
the number of visits, total duration per visit, duration per visit of
interactions with petals only, pollen only, and pollen and nectary
simultaneously by honeybees, bumblebees, and all other pollinators
on\emph{V. ceranae} prevalence in honeybees and bumblebees. Standardized
estimates with a larger magnitude indicate a stronger relationship
with\emph{V. ceranae} prevalence. Full model output in Appendix S1:
Table S7 and S8.\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\textbf{Response variable} & \textbf{Visitation by} & \textbf{Visit
Number Estimates} & \textbf{Total Duration Per Visit Estimates+} &
\textbf{Petal Duration Per Visit Estimates+} & \textbf{Pollen Duration
Per Visit Estimates+} & \textbf{Pollen \& Nectary Duration Per Visit
Estimates+} & \textbf{Pollen \& Nectary Duration Per Visit
Estimates+}\tabularnewline
\midrule
\endhead
\emph{V. ceranae} \textbf{prevalence in honeybees} & \emph{Apis} & 0.171
& 0.260 & 0.092 & 0.043 & 0.490 &\tabularnewline
& \emph{Bombus} & 0.262 & -0.312 & 0.714* & 0.044 & -0.408
&\tabularnewline
& Other & 0.190 & -0.109 & -0.504 & 0.150 & -0.230 &\tabularnewline
\emph{V. ceranae} \textbf{prevalence in bumblebees} & \emph{Apis} &
\textbf{1.144**} & 0.825* & 0.573* & 0.790* & \textbf{1.878**}
&\tabularnewline
& \emph{Bombus} & -0.008 & -0.016 & 0.680* & -0.094 & -0.017
&\tabularnewline
& Other & -0.118 & -0.306 & -0.389 & -0.376 & -0.426 &\tabularnewline
\bottomrule
\end{longtable}

Significant estimates are bolded.

\textbf{+ Bonferroni-corrected alpha threshold of 0.0125 for four
comparisons applied to models with duration per visit main factors.}

Significant: ** p \textless{} 0.01; trending: * p \textless{} 0.05

\textbf{FIGURE CAPTIONS}

\textbf{Figure 1.} Honeybees had fewer visits to flowers compared to
bumblebees and other pollinators, and honeybees and other pollinators
spent more time on petals than bumblebees. Total duration per visit and
duration on pollen + nectar per visit did not differ among pollinator
species. (a) Number of visits observed per 30 min by pollinator species,
(b) total duration per visit (seconds/visit) by pollinator species, (c)
duration on petals per visit (seconds/visit), (d) duration on pollen +
nectar per visit (seconds/visit). Y-axes are on a log scale, where zero
values are on the x-axis. Colored points are the raw data per flower
observed, and the black points are the model-predicted marginal means
with 95\% confidence intervals. Significant differences are indicated by
the number of stars for each pair (Appendix S1: Table S6).

\textbf{Figure 2.} (a) The average number of honeybee visits to flowers
was correlated with greater \emph{V. ceranae} prevalence in bumblebees
(p = 0.005), but not in honeybees (p = 0.58). (b) The average total
duration of time honeybees spent on flowers per visit did not correlate
with \emph{V. ceranae} prevalence in honeybees (p = 0.40) or bumblebees
(p = 0.014). (c) The average duration of time honeybees spent per visit
interacting with pollen and nectar simultaneously correlated with
greater \emph{V. ceranae} prevalence in bumblebees (p = 0.009), but
not\emph{V. ceranae} prevalence in honeybees (p = 0.12). The number and
duration of visits by honeybees were converted to their original scales
for figure clarity. Significant slopes are indicated by solid lines,
while insignificant slopes are indicated by dotted lines. Honeybee
visits (per 30 min) were evaluated based on a 0.05 alpha threshold,
while the visit duration (seconds/visit) were evaluated according to the
Bonferroni-corrected alpha threshold of 0.0125.

\textbf{Figure 3.} Neither the number of bumblebee and other pollinator
visits per 30 min nor the duration per visit to pollen + nectar
impacted\emph{V. ceranae} prevalence in honeybees or bumblebees. There
was no change in \emph{V. ceranae} prevalence in honeybees or bumblebees
based on (a) average number of bumblebee visits (per 30 min), (b)
average number of other pollinator visits (per 30 min), (c) average
duration on pollen + nectar per bumblebee visit (second/visit), and (d)
average duration on pollen + nectar per other pollinator visit
(second/visit). Significant slopes are indicated by solid lines, while
insignificant slopes are indicated by dotted lines.

\textbf{FIGURES}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} Honeybees had fewer visits to flowers compared to
bumblebees and other pollinators, and honeybees and other pollinators
spent more time on petals than bumblebees. Total duration per visit and
duration on pollen + nectar per visit did not differ among pollinator
species. (a) Number of visits observed per 30 min by pollinator species,
(b) total duration per visit (seconds/visit) by pollinator species, (c)
duration on petals per visit (seconds/visit), (d) duration on pollen +
nectar per visit (seconds/visit). Y-axes are on a log scale, where zero
values are on the x-axis. Colored points are the raw data per flower
observed, and the black points are the model-predicted marginal means
with 95\% confidence intervals. Significant differences are indicated by
the number of stars for each pair (Appendix S1: Table S6).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2.} (a) The average number of honeybee visits to flowers
was correlated with greater \emph{V. ceranae} prevalence in bumblebees
(p = 0.005), but not in honeybees (p = 0.58). (b) The average total
duration of time honeybees spent on flowers per visit did not correlate
with \emph{V. ceranae} prevalence in honeybees (p = 0.40) or bumblebees
(p = 0.014). (c) The average duration of time honeybees spent per visit
interacting with pollen and nectar simultaneously correlated with
greater \emph{V. ceranae} prevalence in bumblebees (p = 0.009), but
not\emph{V. ceranae} prevalence in honeybees (p = 0.12). The number and
duration of visits by honeybees were converted to their original scales
for figure clarity. Significant slopes are indicated by solid lines,
while insignificant slopes are indicated by dotted lines. Honeybee
visits (per 30 min) were evaluated based on a 0.05 alpha threshold,
while the visit duration (seconds/visit) were evaluated according to the
Bonferroni-corrected alpha threshold of 0.0125.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} Neither the number of bumblebee and other pollinator
visits per 30 min nor the duration per visit to pollen + nectar
impacted\emph{V. ceranae} prevalence in honeybees or bumblebees. There
was no change in \emph{V. ceranae} prevalence in honeybees or bumblebees
based on (a) average number of bumblebee visits (per 30 min), (b)
average number of other pollinator visits (per 30 min), (c) average
duration on pollen + nectar per bumblebee visit (second/visit), and (d)
average duration on pollen + nectar per other pollinator visit
(second/visit). Significant slopes are indicated by solid lines, while
insignificant slopes are indicated by dotted lines.

\textbf{APPENDIX S1}

\textbf{Table S1.} Field site abbreviation, farm name, dates of each
visit to the field site (mm/dd/yyyy), zone, and Easting and Northing
coordinates in the UTM GPS system. All field sites are located in the
southeastern region of the Lower Peninsula of Michigan, USA. Permission
from landowners was granted for all pollinator collection.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
Site Code & Farm Name & First Visit & Second visit & Zone & Easting &
Northing\tabularnewline
\midrule
\endhead
BP & Brimley's Pumpkin Patch & 8/10/2016 & 8/26/2016 & 16T & 714474 &
4716740\tabularnewline
K & Kapnick Orchards & 8/21/2016 & 8/28/2016 & 17T & 257729 &
4648607\tabularnewline
PR & Peacock Road Farms & 7/26/2016 & 8/23/2016 & 16T & 714244 &
4746884\tabularnewline
GT & Green Things Farm & 8/17/2016 & 8/24/2016 & 17T & 276741 &
4689607\tabularnewline
E & Erwin Orchards & 7/27/2016 & 8/22/2016 & 17T & 280997 &
4708908\tabularnewline
PL & Plymouth Orchards & 8/11/2016 & 8/30/2016 & 17T & 289557 &
4690343\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S2.} Morphospecies classifications used to identify
individuals visiting squash flowers in the visitation videos. For
analyses, visitations by all other pollinator groups excluding\emph{Apis
mellifera} and \emph{Bombus} spp. were combined into the `Other'
category.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Code} & \textbf{Possible included species} & \textbf{Total Num.
Visits} & \textbf{Characteristics}\tabularnewline
\midrule
\endhead
APIS & \emph{Apis mellifera} & 50 &\tabularnewline
AUGO & \emph{Augochlora}, \emph{Augochlorella}, \emph{Augochloropsis} &
232 & Small green halictid\tabularnewline
BOMB & \emph{Bombus} & 477 &\tabularnewline
HALI & \emph{Halictus}, \emph{Lasioglossum} & 128 & Small non-green
halictid\tabularnewline
HFLY & Hover fly & 13 &\tabularnewline
MELI & \emph{Melissodes} & 2 &\tabularnewline
PEPO & \emph{Eucera (=Peponapis)} & 180 &\tabularnewline
TRIE & \emph{Triepeolus} & 1 & Cuckoo bee, parasitizes \emph{Eucera}
spp.\tabularnewline
VESP & \emph{Vespula} (wasp) & 1 &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S3.} Means and ranges of each visitation variable at each
site. Durations per visit are calculated as the number of seconds that a
bee species visited the flower per visit (i.e., number of seconds
honeybees spent doing a given behavior divided by the number of honeybee
visits to the flower).\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& \textbf{SITE} & \textbf{Honeybees} & \textbf{Bumblebees} &
\textbf{Other Pollinators}\tabularnewline
\midrule
\endhead
\textbf{VISIT NUMBER PER 30 MIN} & \textbf{BP} & 1.75 (0, 7) & 9.25 (0,
25) & 1.63 (0, 7)\tabularnewline
& \textbf{E} & 0 (0,0) & 1.63 (0, 8) & 7.31 (0, 43)\tabularnewline
& \textbf{GT} & 0.24 (0, 3) & 4.47 (0, 15) & 1.41 (0, 8)\tabularnewline
& \textbf{K} & 0 (0,0) & 1.31 (0, 6) & 1.31 (0, 4)\tabularnewline
& \textbf{PL} & 1 (0, 7) & 4.44 (0, 15) & 3.25 (0, 12)\tabularnewline
& \textbf{PR} & 0.13(0, 2) & 8.44 (0, 38) & 19 (0, 136)\tabularnewline
\textbf{TOTAL DURATION PER VISIT} & \textbf{BP} & 34.33 (0, 219.3) &
19.57 (0, 70) & 12.94 (0, 58.6)\tabularnewline
& \textbf{E} & 0 (0,0) & 20.39 (0, 222.8) & 19.01 (0,
113.7)\tabularnewline
& \textbf{GT} & 5.39 (0, 90.7) & 18.90 (0, 42.3) & 13.26 (0,
85)\tabularnewline
& \textbf{K} & 0 (0,0) & 15.41 (0, 65) & 31.08 (0, 94)\tabularnewline
& \textbf{PL} & 8.78 (0. 64.3) & 20.63 (0, 107) & 14.44 (0,
117.3)\tabularnewline
& \textbf{PR} & 5.28 (0, 84.5) & 11.99 (0, 40.3) & 63.71 (0,
484)\tabularnewline
\textbf{PETAL-ONLY DURATION PER VISIT} & \textbf{BP} & 4.03 (0, 23) &
0.83 (0, 3.1) & 6.35 (0, 33.7)\tabularnewline
& \textbf{E} & 0 (0,0) & 0.29 (0, 4) & 1.00 (0, 12)\tabularnewline
& \textbf{GT} & 5.10 (0, 86.7) & 0.98 (0, 6) & 5.54 (0,
32.8)\tabularnewline
& \textbf{K} & 0 (0,0) & 0.85 (0, 4.3) & 10.13 (0, 69.3)\tabularnewline
& \textbf{PL} & 2.31 (0, 11.7) & 1.60 (0, 4) & 5.70 (0,
23.4)\tabularnewline
& \textbf{PR} & 3.94 (0, 63) & 0.55 (0, 4) & 17.29 (0,
75.7)\tabularnewline
\textbf{NECTAR-ONLY DURATION PER VISIT} & \textbf{BP} & 0.29 (0, 2.5) &
0.03 (0, 0.55) & 1.40 (0, 14.3)\tabularnewline
& \textbf{E} & 0 (0,0) & 0 (0,0) & 0.02 (0, 0.4)\tabularnewline
& \textbf{GT} & 0 (0,0) & 0 (0,0) & 5.12 (0, 46.4)\tabularnewline
& \textbf{K} & 0 (0,0) & 0.03 (0, 0.5) & 18.15 (0, 80)\tabularnewline
& \textbf{PL} & 4.66 (0, 52.7) & 0.67 (0, 8.4) & 6.23 (0,
99.8)\tabularnewline
& \textbf{PR} & 0.66 (0, 10.5) & 0 (0,0) & 15.17 (0, 116)\tabularnewline
\textbf{POLLEN-ONLY DURATION PER VISIT} & \textbf{BP} & 2.00 (0, 11) &
2.50 (0, 6.4) & 1 (0, 6.3)\tabularnewline
& \textbf{E} & 0 (0,0) & 1.30 (0, 7) & 5.63 (0, 50.7)\tabularnewline
& \textbf{GT} & 0.29 (0, 4) & 2.63 (0, 9) & 1.49 (0, 12)\tabularnewline
& \textbf{K} & 0 (0,0) & 0.80 (0, 3) & 2.65 (0, 14)\tabularnewline
& \textbf{PL} & 0.22 (0, 3) & 2.10 (0, 14.3) & 1.21 (0,
9.1)\tabularnewline
& \textbf{PR} & 0.22 (0, 3.5) & 1.26 (0, 4.6) & 8.69 (0,
26.3)\tabularnewline
\textbf{POLLEN+NECTAR DURATION PER VISIT} & \textbf{BP} & 28.01 (0,
197.8) & 16.17 (0, 67) & 4.20 (0, 45.4)\tabularnewline
& \textbf{E} & 0 (0,0) & 17.84 (0, 218.8) & 12.36 (0, 60)\tabularnewline
& \textbf{GT} & 0 (0,0) & 15.29 (0, 39.7) & 1.10 (0,
14.8)\tabularnewline
& \textbf{K} & 0 (0,0) & 13.66 (0, 60.5) & 0.13 (0, 2)\tabularnewline
& \textbf{PL} & 1.59 (0, 17.4) & 14.20 (0, 103) & 1.29 (0,
9)\tabularnewline
& \textbf{PR} & 0.47 (0, 7.5) & 9.93 (0, 37.8) & 22.56 (0,
295)\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S4.} Numbers of honeybee (\emph{Apis mellifera} ) and
bumblebee (\emph{Bombus} spp.) individuals tested and positive
for\emph{V. ceranae} presence across six sites and two visits per site.
We aimed to sample eight individuals per species per visit to each site
where possible (target N = 16 per species per site), but there was some
variation in the abundance of honeybees and bumblebees across visits to
each field site.\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
Species & Species & honeybees & honeybees & Bumblebees & Bumblebees &
Combined & Combined\tabularnewline
\midrule
\endhead
Site & \textbf{VISIT} & \textbf{Tested} & \textbf{Positive} &
\textbf{Tested} & \textbf{Positive} & \textbf{Tested} &
\textbf{Positive}\tabularnewline
BP & 1 & 8 & \emph{4} & 8 & 7 & 16 & 11\tabularnewline
BP & 2 & 8 & \emph{7} & 8 & 8 & 16 & 15\tabularnewline
E & 1 & 8 & \emph{6} & 7 & 2 & 15 & 8\tabularnewline
E & 2 & 8 & \emph{4} & 8 & 4 & 16 & 8\tabularnewline
GT & 1 & 7 & \emph{3} & 8 & 4 & 15 & 7\tabularnewline
GT & 2 & 5 & \emph{5} & 7 & 4 & 12 & 9\tabularnewline
K & 1 & 3 & \emph{2} & 7 & 4 & 10 & 6\tabularnewline
K & 2 & 5 & \emph{3} & 8 & 4 & 13 & 7\tabularnewline
PL & 1 & 8 & \emph{5} & 7 & 5 & 15 & 10\tabularnewline
PL & 2 & 8 & \emph{8} & 8 & 8 & 16 & 16\tabularnewline
PR & 1 & 0 & \emph{0} & 2 & 1 & 2 & 1\tabularnewline
PR & 2 & 7 & \emph{4} & 8 & 4 & 15 & 8\tabularnewline
\emph{TOTAL} & \emph{TOTAL} & \textbf{75} & 51 & \textbf{86} & 55 &
\textbf{161} & 106\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S5.} Full model output for each zero-inflated hurdle GLMM
for pollinator species (honeybees, bumblebees, or other pollinators)
effects on the number of visits per 30 min and total, petal, pollen, and
pollen+nectar durations per visit. The table includes the model
estimate, standard error, z value, and p-value for each main effect in
the conditional and zero-inflated portions of the models, as well as the
variance and standard deviation for the nested random effects of each
observed flower within site visit (i.e., first or second visit to each
site) within site. All models used a negative binomial distribution with
a log link function. Some models have singular random effects,
indicating no variation in all or some of the nested random effects.
Significant p-values are bolded.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
Response variable & Main Effect & Estimate & Std Error & z value &
P-value & Random Effect & Variance & Std Dev.\tabularnewline
\midrule
\endhead
\emph{Number of visits per 30 min} & \textbf{Conditional model:} & & & &
& & &\tabularnewline
& Intercept & 0.25 & 0.48 & 0.52 & 0.602 & FlowerID:Visit:Site & 0.00 &
0.00\tabularnewline
& Genus-Bumblebees & 1.48 & 0.38 & 3.91 & \textbf{\textless{}0.0001} &
Visit:Site & 0.42 & 0.65\tabularnewline
& Genus-Other Pollinators & 1.08 & 0.57 & 1.87 & 0.061 & Site & 0.19 &
0.44\tabularnewline
& \textbf{Zero-inflation model:} & & & & & & &\tabularnewline
& Intercept & 0.58 & 0.43 & 1.34 & 0.179 & & &\tabularnewline
& Genus-Bumblebees & -2.06 & 0.54 & -3.79 & \textbf{0.00015} & &
&\tabularnewline
& Genus-Other Pollinators & -1.67 & 0.68 & -2.47 & \textbf{0.014} & &
&\tabularnewline
\emph{Total duration per visit} & \textbf{Conditional model:} & & & & &
& &\tabularnewline
& Intercept & 3.25 & 0.32 & 10.07 & \textbf{\textless{}0.0001} &
FlowerID:Visit:Site & 0.10 & 0.32\tabularnewline
& Genus-Bumblebees & -0.36 & 0.34 & -1.04 & 0.3 & Visit:Site & 0.04 &
0.19\tabularnewline
& Genus-Other Pollinators & 0.18 & 0.36 & 0.49 & 0.624 & Site & 0.00 &
0.00\tabularnewline
& \textbf{Zero-inflation model:} & & & & & & &\tabularnewline
& Intercept & 1.30 & 0.27 & 4.88 & \textbf{\textless{}0.0001} & &
&\tabularnewline
& Genus-Bumblebees & -2.47 & 0.42 & -5.90 & \textbf{\textless{}0.0001} &
& &\tabularnewline
& Genus-Other Pollinators & -1.71 & 0.35 & -4.88 &
\textbf{\textless{}0.0001} & & &\tabularnewline
\emph{Duration on Petals per visit} & \textbf{Conditional model:} & & &
& & & &\tabularnewline
& Intercept & 1.92 & 0.39 & 5.0 & \textbf{\textless{}0.0001} &
FlowerID:Visit:Site & 0.00 & 0.00\tabularnewline
& Genus-Bumblebees & -2.29 & 0.36 & -6.4 & \textbf{\textless{}0.0001} &
Visit:Site & 0.00 & 0.00\tabularnewline
& Genus-Other Pollinators & 0.18 & 0.37 & 0.5 & 0.618 & Site & 0.24 &
0.49\tabularnewline
& \textbf{Zero-inflation model:} & & & & & & &\tabularnewline
& Intercept & 1.22 & 0.30 & 4.07 & \textbf{\textless{}0.0001} & &
&\tabularnewline
& Genus-Bumblebees & -20.12 & 3615.1 & -0.01 & 0.996 & &
&\tabularnewline
& Genus-Other Pollinators & -1.69 & 0.40 & -4.20 &
\textbf{\textless{}0.0001} & & &\tabularnewline
\emph{Duration on Pollen per visit} & \textbf{Conditional model:} & & &
& & & &\tabularnewline
& Intercept & 0.26 & 0.42 & 0.64 & 0.525 & FlowerID:Visit:Site & 0.02 &
0.13\tabularnewline
& Genus-Bumblebees & 0.19 & 0.41 & 0.46 & 0.643 & Visit:Site & 0.00 &
0.00\tabularnewline
& Genus-Other Pollinators & 1.45 & 0.44 & 3.33 & \textbf{0.0009} & Site
& 0.02 & 0.15\tabularnewline
& \textbf{Zero-inflation model:} & & & & & & &\tabularnewline
& Intercept & 1.12 & 0.41 & 2.71 & \textbf{0.007} & & &\tabularnewline
& Genus-Bumblebees & -18.04 & 4156.2 & 0.00 & 0.997 & & &\tabularnewline
& Genus-Other Pollinators & -1.12 & 0.48 & -2.36 & \textbf{0.018} & &
&\tabularnewline
\emph{Duration on Pollen + Nectar per visit} & \textbf{Conditional
model:} & & & & & & &\tabularnewline
& Intercept & 2.64 & 0.46 & 5.76 & \textbf{\textless{}0.0001} &
FlowerID:Visit:Site & 0.58 & 0.76\tabularnewline
& Genus-Bumblebees & 0.10 & 0.44 & 0.23 & 0.821 & Visit:Site & 0.13 &
0.37\tabularnewline
& Genus-Other Pollinators & -0.56 & 0.49 & -1.15 & 0.25 & Site & 0.00 &
0.00\tabularnewline
& \textbf{Zero-inflation model:} & & & & & & &\tabularnewline
& Intercept & 1.94 & 0.33 & 5.90 & \textbf{\textless{}0.0001} & &
&\tabularnewline
& Genus-Bumblebees & -2.47 & 0.41 & -6.08 & \textbf{\textless{}0.0001} &
& &\tabularnewline
& Genus-Other Pollinators & -1.27 & 0.41 & -3.14 & 0.0017 & &
&\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S6.} Pairwise contrasts between each pollinator species
group (honeybees, bumblebees, and other pollinators) for each pollinator
visitation behavior metric, including number of visits per 30 min, total
duration per visit, and duration per visit on petals, pollen, and
pollen+nectar. Odds ratios are shown for the pairwise difference between
the two host species compared in each row and are calculated on the log
scale. P values are Tukey adjusted for comparing a family of three, and
significant p-values are bolded. Data for these tests are shown in
Figure 1 and Figure S1.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\textbf{Response variable} & \textbf{Contrast} & \textbf{Ratio} &
\textbf{Std Error} & \textbf{DF} & \textbf{z value} &
\textbf{P-value}\tabularnewline
\midrule
\endhead
Number of visits per 30 min & Honeybees / Bumblebees & 0.227 & 0.086 &
Inf & -3.911 & \textbf{0.0003}\tabularnewline
& Honeybees / Other Pollinators & 0.341 & 0.196 & Inf & -1.873 &
0.1467\tabularnewline
& Bumblebees / Other Pollinators & 1.504 & 0.653 & Inf & 0.94 &
0.6152\tabularnewline
Total duration per visit & Honeybees / Bumblebees & 1.427 & 0.489 & Inf
& 1.036 & 0.5539\tabularnewline
& Honeybees / Other Pollinators & 0.839 & 0.301 & Inf & -0.49 &
0.876\tabularnewline
& Bumblebees / Other Pollinators & 0.588 & 0.143 & Inf & -2.184 &
0.0738\tabularnewline
Duration on Petals per visit & Honeybees / Bumblebees & 9.902 & 3.5253 &
Inf & 6.44 & \textbf{\textless{}0.0001}\tabularnewline
& Honeybees / Other Pollinators & 0.8326 & 0.3063 & Inf & -0.498 &
0.8722\tabularnewline
& Bumblebees / Other Pollinators & 0.0841 & 0.0214 & Inf & -9.739 &
\textbf{\textless{}0.0001}\tabularnewline
Duration on Pollen per visit & Honeybees / Bumblebees & 0.827 & 0.3382 &
Inf & -0.464 & 0.8882\tabularnewline
& Honeybees / Other Pollinators & 0.235 & 0.1024 & Inf & -3.326 &
\textbf{0.0025}\tabularnewline
& Bumblebees / Other Pollinators & 0.284 & 0.0653 & Inf & -5.477 &
\textbf{\textless{}0.0001}\tabularnewline
Duration on Pollen + Nectar per visit & Honeybees / Bumblebees & 0.904 &
0.402 & Inf & -0.226 & 0.9722\tabularnewline
& Honeybees / Other Pollinators & 1.754 & 0.856 & Inf & 1.152 &
0.4824\tabularnewline
& Bumblebees / Other Pollinators & 1.94 & 0.685 & Inf & 1.876 &
0.1458\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S7.} Full model output for each GLMM for \emph{V.
ceranae}prevalence in honeybees, including the model estimate, standard
error, z value, and p-value for each main effect in the models, as well
as the variance and standard deviation for the nested random effects of
each site visit (i.e., first or second visit to each site) within site.
There are separate models for each pollinator behavior variable. Some
models have singular random effects, indicating no variation in all or
some of the nested random effects. Significant p-values are bolded, and
all duration per visit models use the Bonferroni-corrected alpha
threshold of 0.0125 instead of 0.05. Note that data on the nectar-only
interactions are only included in the Appendix and were not part of the
analyses in the main text and are not included as a group in the
Bonferroni correction for four comparisons (Table 1).\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
Response variable & Main Effect & Estimate & Std Error & z value &
P-value & Random Effect & Variance & Std Dev.\tabularnewline
\midrule
\endhead
\emph{V. ceranae} in honeybees & Intercept & 0.783 & 0.273 & 2.865 &
\textbf{0.004} & Site:Visit & 0.041 & 0.202\tabularnewline
& Number of honeybee visits per 30 min & 0.171 & 0.307 & 0.558 & 0.577 &
Site & 0 & 0\tabularnewline
& Number of bumblebee visits per 30 min & 0.262 & 0.365 & 0.717 & 0.473
& & &\tabularnewline
& Number of other visits per 30 min & 0.190 & 0.311 & 0.610 & 0.542 & &
&\tabularnewline
\emph{V. ceranae} in honeybees & Intercept & 0.783 & 0.276 & 2.842 &
\textbf{0.004} & Site:Visit & 0.063 & 0.251\tabularnewline
& Total duration per visit of honeybee visits & 0.260 & 0.310 & 0.840 &
0.401 & Site & 9.61E-10 & 3.1E-5\tabularnewline
& Total duration per visit of bumblebee visits & -0.312 & 0.355 & -0.878
& 0.380 & & &\tabularnewline
& Total duration per visit of other visits & -0.109 & 0.324 & -0.336 &
0.737 & & &\tabularnewline
\emph{V. ceranae} in honeybees & Intercept & 0.818 & 0.263 & 3.109 &
\textbf{0.002} & Site:Visit & 0 & 0\tabularnewline
& Duration per visit of honeybee petal-only interactions & 0.092 & 0.290
& 0.316 & 0.752 & Site & 0 & 0\tabularnewline
& Duration per visit of bumblebee petal-only interactions & 0.714 &
0.348 & 2.051 & 0.040 & & &\tabularnewline
& Duration per visit of other petal-only interactions & -0.504 & 0.341 &
-1.479 & 0.139 & & &\tabularnewline
\emph{V. ceranae} in honeybees & Intercept & 2.025 & 1.191 & 1.700 &
0.089 & Site:Visit & 1.07E-16 & 1.04E-08\tabularnewline
& Duration per visit of honeybee nectar-only interactions & 0.270 &
0.776 & 0.347 & 0.728 & Site & 1.05E-16 & 1.03E-08\tabularnewline
& Duration per visit of bumblebee nectar-only interactions & 4.006 &
3.119 & 1.284 & 0.199 & & &\tabularnewline
& Duration per visit of other nectar-only interactions & -0.392 & 0.302
& -1.300 & 0.194 & & &\tabularnewline
\emph{V. ceranae} in honeybees & Intercept & 0.783 & 0.286 & 2.742 &
\textbf{0.006} & Site:Visit & 0.139 & 0.372\tabularnewline
& Duration per visit of honeybee pollen-only interactions & 0.043 &
0.300 & 0.143 & 0.886 & Site & 0 & 0\tabularnewline
& Duration per visit of bumblebee pollen-only interactions & 0.044 &
0.398 & 0.109 & 0.913 & & &\tabularnewline
& Duration per visit of other pollen-only interactions & 0.150 & 0.387 &
0.388 & 0.698 & & &\tabularnewline
\emph{V. ceranae} in honeybees & Intercept & 0.798 & 0.258 & 3.087 &
\textbf{0.002} & Site:Visit & 0 & 0\tabularnewline
& Duration per visit of honeybee pollen+nectar interactions & 0.490 &
0.311 & 1.574 & 0.116 & Site & 0 & 0\tabularnewline
& Duration per visit of bumblebee pollen+nectar interactions & -0.408 &
0.315 & -1.297 & 0.195 & & &\tabularnewline
& Duration per visit of other pollen+nectar interactions & -0.230 &
0.294 & -0.784 & 0.433 & & &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S8.} Full model output for each GLMM for \emph{V.
ceranae}prevalence in \emph{Bombus} spp\emph{.} (bumblebees), including
the model estimate, standard error, z value, and p-value for each main
effect in the models, as well as the variance and standard deviation for
the nested random effects of each site visit within site. Some models
have singular random effects, indicating no variation in all or some of
the nested random effects. Significant p-values are bolded, and all
duration per visit models use the Bonferroni-corrected alpha threshold
of 0.0125 instead of 0.05. Note that data on the nectar-only
interactions are only included in the Appendix and were not part of the
analyses in the main text and are not included as a group in the
Bonferroni correction for four comparisons (Table 1).\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
Response variable & Main Effect & Estimate & Std Error & z value &
P-value & Random Effect & Variance & Std Dev.\tabularnewline
\midrule
\endhead
\emph{V. ceranae} in bumblebees & Intercept & 0.807 & 0.288 & 2.803 &
\textbf{0.005} & Site:Visit & 0 & 0\tabularnewline
& Number of honeybee visits per 30 min & 1.144 & 0.410 & 2.789 &
\textbf{0.005} & Site & 0 & 0\tabularnewline
& Number of bumblebee visits per 30 min & -0.008 & 0.333 & -0.023 &
0.982 & & &\tabularnewline
& Number of other visits per 30 min & -0.118 & 0.267 & -0.443 & 0.658 &
& &\tabularnewline
\emph{V. ceranae} in bumblebees & Intercept & 0.683 & 0.284 & 2.407 &
0.016 & Site:Visit & 0 & 0\tabularnewline
& Total duration per visit of honeybee visits & 0.825 & 0.335 & 2.465 &
0.014 & Site & 0.089 & 0.299\tabularnewline
& Total duration per visit of bumblebee visits & -0.016 & 0.342 & -0.046
& 0.963 & & &\tabularnewline
& Total duration per visit of other visits & -0.306 & 0.352 & -0.868 &
0.386 & & &\tabularnewline
\emph{V. ceranae} in bumblebees & Intercept & 0.648 & 0.271 & 2.387 &
0.017 & Site:Visit & 0 & 0\tabularnewline
& Duration per visit of honeybee petal-only interactions & 0.573 & 0.274
& 2.088 & 0.037 & Site & 0.078 & 0.280\tabularnewline
& Duration per visit of bumblebee petal-only interactions & 0.680 &
0.312 & 2.180 & 0.029 & & &\tabularnewline
& Duration per visit of Other petal-only interactions & -0.389 & 0.305 &
-1.277 & 0.201 & & &\tabularnewline
\emph{V. ceranae} in bumblebees & Intercept & 1.873 & 1.096 & 1.708 &
0.088 & Site:Visit & 7.76E-10 & 2.79E-05\tabularnewline
& Duration per visit of honeybee nectar-only interactions & 0.616 &
0.762 & 0.808 & 0.419 & Site & 0.129 & 0.360\tabularnewline
& Duration per visit of bumblebee nectar-only interactions & 3.948 &
3.048 & 1.295 & 0.195 & & &\tabularnewline
& Duration per visit of other nectar-only interactions & -0.360 & 0.300
& -1.200 & 0.230 & & &\tabularnewline
\emph{V. ceranae} in bumblebees & Intercept & 0.708 & 0.263 & 2.692 &
\textbf{0.007} & Site:Visit & 0 & 0\tabularnewline
& Duration per visit of honeybee pollen-only interactions & 0.790 &
0.352 & 2.246 & 0.025 & Site & 0.007 & 0.086\tabularnewline
& Duration per visit of bumblebee pollen-only interactions & -0.094 &
0.356 & -0.265 & 0.791 & & &\tabularnewline
& Duration per visit of other pollen-only interactions & -0.376 & 0.333
& -1.130 & 0.258 & & &\tabularnewline
\emph{V. ceranae} in bumblebee & Intercept & 1.069 & 0.401 & 2.666 &
\textbf{0.008} & Site:Visit & 3.12E-16 & 1.77E-08\tabularnewline
& Duration per visit of honeybee pollen+nectar interactions & 1.878 &
0.722 & 2.601 & \textbf{0.009} & Site & 0 & 0\tabularnewline
& Duration per visit of bumblebee pollen+nectar interactions & -0.017 &
0.261 & -0.064 & 0.949 & & &\tabularnewline
& Duration per visit of other pollen+nectar interactions & -0.426 &
0.270 & -1.578 & 0.114 & & &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table S9.} The \emph{V. ceranae} prevalence for honeybees and
bumblebees at each site, including the standard error (SE), degrees of
freedom (df), and the lower and upper asymptotic confidence intervals.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
Genus & Site & Estimated Prevalence & SE & df & Lower CL & Upper
CL\tabularnewline
\midrule
\endhead
Honeybees & BP & 0.688 & 0.116 & 146 & 0.431 & 0.865\tabularnewline
Honeybees & E & 0.625 & 0.121 & 146 & 0.375 & 0.822\tabularnewline
Honeybees & GT & 0.667 & 0.136 & 146 & 0.374 & 0.870\tabularnewline
Honeybees & K & 0.625 & 0.171 & 146 & 0.282 & 0.876\tabularnewline
Honeybees & PL & 0.813 & 0.098 & 146 & 0.550 & 0.939\tabularnewline
Honeybees & PR & 0.571 & 0.187 & 146 & 0.228 & 0.858\tabularnewline
Bumblebees & BP & 0.938 & 0.061 & 146 & 0.661 & 0.991\tabularnewline
Bumblebees & E & 0.400 & 0.127 & 146 & 0.190 & 0.654\tabularnewline
Bumblebees & GT & 0.533 & 0.129 & 146 & 0.291 & 0.761\tabularnewline
Bumblebees & K & 0.533 & 0.129 & 146 & 0.291 & 0.761\tabularnewline
Bumblebees & PL & 0.867 & 0.088 & 146 & 0.592 & 0.967\tabularnewline
Bumblebees & PR & 0.500 & 0.158 & 146 & 0.223 & 0.777\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure S1.} Other pollinators spent more time per visit
(seconds/visit) on pollen than both honeybees and bumblebees. This
finding may be driven by the time spent by \emph{Eucera pruinosa} , a
squash pollen specialist, that was included in the `other pollinator'
category. Significant differences are indicated by the number of stars
for each pair (Appendix S1: Table S6). The y-axis is log scaled, and
zero values are shown along the x-axis. Colored points are the raw data
per flower observed, and the black points are the model predicted
marginal means with 95\% confidence intervals.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure S2.} \emph{V. ceranae} prevalence in honeybees and
bumblebees at each of the six field sites. (Appendix: Tables S9). The
error bars signify a 95\% confidence interval.

\textbf{APPENDIX S2}

\textbf{Trapping methodology}

To catch bees using the netting method, each transect was walked once
for 30 minutes at approximately 08:00, 10:00, 11:00, and 12:00 before
the squash flowers closed around midday. Any observed honeybees and
bumblebees that were visiting squash flowers within 1.5 m of the
transect were captured. To allow for maximum sample sizes, pan traps
were used alongside netting. Brightly colored pan traps attract bees,
who are subsequently trapped by soapy liquid in the pans and drown
(Roulston et al. 2007). In our study, fluorescent blue, yellow, and
white pan traps were placed in an alternating color pattern 5 m apart
along each transect between the crop rows. Each trap was filled with a
mixture of water and clear dish soap. Traps were set up by 07:00 am,
checked every three hours for captured pollinators, and collected after
the squash flowers closed around midday, for a total average duration of
six hours.

\textbf{PCR procedure}

The PCR master mix contained 12.5 \selectlanguage{greek}µ\selectlanguage{english}L dH\textsubscript{2}O, 2 \selectlanguage{greek}µ\selectlanguage{english}L 10x
buffer, 0.4 \selectlanguage{greek}µ\selectlanguage{english}L 10 mM dNTPs, 1 \selectlanguage{greek}µ\selectlanguage{english}L of each primer (10 mM), 2 \selectlanguage{greek}µ\selectlanguage{english}L 25 mM
MgCl\textsubscript{2}, and 0.1 \selectlanguage{greek}µ\selectlanguage{english}L 5 U/\selectlanguage{greek}µ\selectlanguage{english}L Taq polymerase (Invitrogen,
Carlsbad, CA, USA) per reaction. Reactions were run with an initial
denaturation step at 94 @\selectlanguage{english}C for 2 min, 40 cycles containing denaturation
at 94 @\selectlanguage{english}C for 30 s, annealing at 61 @\selectlanguage{english}C for 45 s, and extension at 72 @\selectlanguage{english}C
for 2 min, followed by a final extension at 72 @\selectlanguage{english}C for 7 min and a
cooling period at 10 @\selectlanguage{english}C for 2 min.

The PCR product was visualized on a 2\% agarose gel by observing a 250
bp band. We extracted the 250 bp band with a High Pure PCR Product
Purification and Gel Extraction kit (Roche, Basel, Switzerland) to clean
the product for sequencing.

\selectlanguage{english}
\FloatBarrier
\end{document}

