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\DOI{10.5802/crchim.231}
\datereceived{2023-01-28}
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\dateposted{2023-06-08}
\begin{document}

%\copyeditor{RK}

\begin{noXML}

%\TopicEF{Chemical Ecology -- Chemical Mediation in the
%Environment}{\'Ecologie chimique -- M\'ediation chimique dans
%l'environnement}

\title{Tree-dimensional resources: hedgerow and orchard pollen for
bumble bees}

\author{\firstname{Antoine} \lastname{Geki\`{e}re}\IsCorresp\CDRorcid{0000-0001-5337-1305}}
\address{Laboratory of Zoology, Research Institute for Biosciences,
University of Mons, Mons, Belgium}
\email[A. Geki\`{e}re]{antoine.gekiere@umons.ac.be}

\author{\firstname{Apolline} \lastname{Michel}}
\addressSameAs{1}{Laboratory of Zoology, Research institute for
Biosciences, University of Mons, Mons, Belgium}
\email[A. Michel]{apolline.michel@student.umons.ac.be}

\author{\firstname{Ir\`{e}ne} \lastname{Semay}\CDRorcid{0000-0002-4197-0316}}
\address{Organic Synthesis and Mass Spectrometry Laboratory, Research
Institute for Biosciences, University of Mons, Mons, Belgium}
\email[I. Semay]{irene.semay@umons.ac.be}

\author{\firstname{Cl\'{e}ment} \lastname{Tourbez}\CDRorcid{0000-0003-1783-5824}}
\addressSameAs{1}{Laboratory of Zoology, Research institute for Biosciences,
University of Mons, Mons, Belgium}
\email[C. Tourbez]{clement.tourbez@umons.ac.be}

\author{\firstname{Martin} \lastname{Begou}}
\addressSameAs{1}{Laboratory of Zoology, Research institute for Biosciences,
University of Mons, Mons, Belgium}
\email[M. Begou]{martin.begou@student.umons.ac.be}

\author{\firstname{Denis} \lastname{Michez}\CDRorcid{0000-0001-8880-1838}}
\addressSameAs{1}{Laboratory of Zoology, Research institute for Biosciences,
University of Mons, Mons, Belgium}
\email[D. Michez]{denis.michez@umons.ac.be}

\author{\firstname{Pascal} \lastname{Gerbaux}\CDRorcid{0000-0001-5114-4352}}
\addressSameAs{2}{Organic Synthesis and Mass Spectrometry Laboratory, Research
Institute for Biosciences, University of Mons, Mons, Belgium}
\email[P. Gerbaux]{pascal.gerbaux@umons.ac.be}

\author{\firstname{Maryse} \lastname{Vanderplanck}\CDRorcid{0000-0002-0110-8019}}
\address{CEFE, Univ Montpellier, CNRS, EPHE, IRD, Montpellier, France}
\email[M. Vanderplanck]{maryse.vanderplanck@cefe.cnrs.fr}

\shortrunauthors

\keywords{\kwd{Tree pollen}
\kwd{Willow}
\kwd{Bumble bee}
\kwd{Parasite}
\kwd{Orchard}
\kwd{Hedgerow}
\kwd{Flavonoids}}

\begin{abstract}
Intensive landscape modifications have led to the loss of floral
resources, partly in early spring when bumble bee queens need suitable
pollen to establish their brood. Adequate floral resources are also
crucial to mitigate parasite infection, a stress compromising
reproductive success. Among early blooming trees, willows represent an
important and highly suitable pollen resource. Alas, riparian areas and
their associated willows have been declining. In this study, we found
that hedgerow and orchard pollen were at least as suitable as willow
pollen for bumble bee survival and microcolony development. Moreover,
orchard pollen seemed an interesting candidate to help reduce parasite
infection, but unlikely due to its flavonoids. Such non-willow trees
could then be favoured in agri-environmental schemes implemented in bee
conservation strategies, but pollen chemicals underlying beneficial
effects remain to be determined.
\end{abstract}

\maketitle

\vspace*{.5pc}

\twocolumngrid

\end{noXML}

\section{Introduction}\label{sec1}

In the current Anthropocene epoch, insect populations face drastic
decline (``insectageddon'';~\cite{1}) and bees (Hymenoptera: Anthophila)
are no exception~\cite{2}. Bees are among the most important groups of
pollinators for wild plants and agricultural crops. They support the
seed set of more than 85\% of wildflower species~\cite{3} and benefit
the production of roughly 75\% of the 115 most important crops
 worldwide~\cite{4,5}. In Europe, although biotic
pollination is crucial for only 12\% of the cropland area, it enables
more than 31\% of the EU income from crop production~\cite{6}, reaching
more than \$17 billion annually~\cite{7}. Moreover, bees provide
nutrient-rich food for human populations~\cite{8}. They therefore play
a compelling role in natural ecosystems as well as in human
societies~\cite{9,10,11}, making their persistence a matter of both
scientific and societal importance. 

\break
The consensus is that bee population decline is the product of multiple
factors that can act singly or in combination, the most known being
diseases and parasites, invasive species, intensive agriculture,
climate change, and pesticides (i.e., insecticides, herbicides and
fungicides)~\cite{12,13}. Among these factors, land use (i.e.,
agricultural intensification and its associated landscape
simplification) is considered an important driver of pollinator
decline~\cite{14,15,16}. Indeed, in the ongoing human-driven landscape
homogenisation (i.e., diminishing resource quality) and natural habitat
destruction (i.e., diminishing resource quantity~\cite{17}), bee
populations face serious challenges to find suitable feeding and
nesting resources~\cite{18,19,20}. Besides, human activities also
influence natural bee-pathogen dynamics, either increasing or
decreasing pathogen prevalence, typically through the global trade of
commercial pollinators~\cite{21} and landscape modification~\cite{22}.
Such human-disturbed host-pathogen dynamics also contribute to bee population
decline~\cite{23}, since bees suffer from taxonomically diverse
pathogens and parasites~\cite{24} {that play crucial roles in shaping
their communities~\cite{25}. A dearth of suitable resources may then be
even more worrying as bees depend on specific flower species not only
to meet their nutritional demand, but also to deal with parasite
infection, most likely relying on some beneficial specialised
metabolites in pollen and nectar~\cite{26,27}. For example, flavonoids,
a broad class of specialised metabolites widely found in
pollen~\cite{28}, have been shown to display antioxidant and
antimicrobial properties~\cite{29}. The flavonoid kaempferol and the
isoflavonoid biochanin A have been found to reduce}
\textit{Vairimorpha} spp. (formerly \textit{Nosema} spp.;
Microsporidia: Nosematidae) load in honey bees~\cite{30} and bumble
bees~\cite{31}, respectively. Flavonoids have even been shown to help
bees face other stressors, such as pesticide exposure
(e.g.,~\cite{32,33}).\looseness=-1

Issues could arise when plant species with medicinal properties
decline, as Koch \etal~\cite{34} {raised {\unskip\break}concerns regarding
worldwide heathland decline since the norisoprenoid callunene found in
\textit{Calluna vulgaris} (Ericales: Ericaceae) nectar helps bumble bee
wipe out the obligate intestinal parasite \textit{Crithidia} sp.
(Euglenozoa: Trypanosomatidae). This parasite is known to cause
premature death in food-stressed bumble bees~\cite{35} as well as 
impair foraging efficiency~\cite{36}, decrease likelihood of
reproduction~\cite{37}, decrease queen bee's survival to
diapause~\cite{38} and reduce successful colony
initiation~\cite{39}. In addition, bees' immunocompetence is a
cornerstone in the nutrition-infection interplay and this parameter has
often been assessed through fat body content, a crucial tissue involved
in energy metabolism and antimicrobial peptide production in
insects~\cite{40}. Both nutrition and infection, including specialised
metabolites (e.g.,~\cite{41,42}) and \textit{Crithidia} sp.~\cite{43},
have been shown to impact this organ. As a consequence of the
nutritional and parasite stress undergone by bees, there has been an
unanimous concern over the loss of bee populations and an urge for
conservation strategies ~\cite{44,45}, which have led to an increasing
number of research around bees (Supplementary Figure~S1) to promote
suitable floral resources.

Most studies focusing on bee resources have principally considered
herbaceous plants (e.g.,~\cite{46}) but recently, 
Donkersley~\cite{47} {underlined the importance of neglected woody species
for pollinators, notably because (i) they provide wooden nesting
materials and sites, (ii) they offer refuge during wind and rain, (iii)
they produce a great amount of floral resources (i.e., in three
dimensions) and (iv) they show an extended flowering time. 
Wood \etal~\cite{48} even showed that woody species are as suitable (e.g.,
Fabaceae species) or even more suitable than some herbaceous species
(e.g., Asteraceae species) for bumble bee colony development.
Considering the phenology is also crucial since bumble bee queens
establish their colony in early spring and hence rely on early
flowering plants for brood development. One remarkable tree species is
the willow (\textit{Salix} spp.; Salicales: Salicaceae) that represents
an important resource for spring bee species, particularly in wetlands
and wet heathlands, blooming in early spring and offering a huge amount
of nutritive and easily accessible pollen (e.g.,~\cite{49,50,51}).
However, riparian zones and their associated willows are declining,
notably as a consequence of stream diversions, groundwater pumping,
extended drought, and replacement with less\unskip\break desirable shrubs and
trees~\cite{52,53,54}, thereby threatening bumble bee colony
establishment. Hence, bumble bee queens must rely on other tree
resources in early spring to initiate their brood with abundant and
nutritive pollen, but also to deal with parasites such as
\textit{Crithidia} {sp}.

Among spring-blooming tree species, bumble bees can forage on fruit
trees in orchards. For instance, mass-flowering orchard species such as
sweet cherry (\textit{Prunus avium}; Rosales: Rosaceae), apple
(\textit{Malus domestica}; Rosales: Rosaceae) and pear (\textit{Pyrus
communis}; Rosales: Rosaceae) trees represent important resources for
early emerging bee pollinators~\cite{55,56,57}. {Indeed, human
populations rely increasingly on mass-flowering crops, including fruit
crops~\cite{17}, but their consequences for pollinator-parasite
dynamics remain unclear~\cite{58,59}. Besides, bumble bees can also
benefit from non-crop trees used in hedgerows such as the hawthorn
(\textit{Crataegus monogyna}; Rosales: Rosaceae) known as a dominant
woody species in hedgerows~\cite{60}, benefiting insect
pollinators~\cite{61,62} and especially bumble bees~\cite{63}. Indeed,
to address pollinator decline in Europe, agri-environment schemes have
been introduced, including the implementation of hedgerows along field
boundaries to provide additional food resources and nesting sites for
bees~\cite{64,65}. However, despite the well-founded importance of such
trees for pollinators, the suitability of their pollen---including
their specialised metabolites---for social bee species development as
well as their potential benefits to deal with parasites remain poorly
explored~\cite{48}.

To address this gap, we evaluated how tree pollen found either in
mass-flowering orchards (i.e., sweet cherry, apple and pear) or 
in hedgerows (i.e., hawthorn) and their respective flavonoids impact
healthy and parasite-challenged buff-tailed bumble bees \textit{Bombus
terrestris} (Hymenoptera: Apidae), the second-most economically
important bee pollinator worldwide~\cite{66}. For the parasite
challenge, we used the trypanosomatid gut parasite} \textit{Crithidia}
sp. (Euglenozoa: Trypanosomatidae), the most prevalent parasite species
in bumble bee populations~\cite{67,68}  on which non-flavonoid
specialised metabolites were found to have an effect in previous
studies (e.g.,~\cite{27,69}). Specifically, we provided \textit{B.
terrestris} microcolonies with tree pollen diets or flavonoid
extract-laced control diets, and observed the effects at the
microcolony and individual levels. We also\unskip\break assessed whether impacts
varied between healthy and infected bumble bees, and the
differences in parasite load.
\vspace*{60pt}


\section{Materials and methods}\label{sec2}

\subsection{Experimental design}\label{sec21}

The ``hedgerow'' experiment (i.e., hawthorn pollen) was conducted from
April to June 2021 while the ``orchard'' experiment (i.e., mix of sweet
cherry, apple, and pear pollen; hereafter ``orchard mix'') was conducted
from April to June 2022. In both experiments, \textit{B. terrestris} 
microcolonies were distributed among a $2\times 3$ factorial design (i.e., six
treatments), with infection (uninfected or infected) crossed with diet
(control, natural or flavonoid). The control diet consisted of willow
\textit{Salix} sp. pollen, the natural diet consisted of hawthorn
pollen or orchard mix, and the flavonoid diet consisted of willow
\textit{Salix} sp. pollen laced with flavonoid extract from hawthorn
pollen or orchard mix. Honey bee-collected willow \textit{Salix} sp.
pollen was purchased from the company ``Ruchers de Lorraine'' (Nancy,
France) while hawthorn pollen and the orchard mix were purchased from
the company ``Pollenergie'' (Saint-Hilaire-de-Lusignan, France). Pollen
was ground and mixed with sugar syrup (water:sucrose 35:65 w/w) to be
provided to microcolonies as candies (see Supplementary Section~1 for
flavonoid extraction and details about diet preparation).

For both experiments, a set of five colonies was ordered from
\textit{Biobest bvba} (Westerlo, Belgium; the five colonies in the two
experiments were different}). Fifteen and ten queenless \textit{B.
terrestris} microcolonies were established for each treatment in the
hedgerow and orchard experiments, respectively (i.e., three and two
microcolonies per colony, respectively). Each microcolony was made of
five workers (inoculated or not) placed in distinct plastic boxes 
(10~cm ${\times}$ 16~cm ${\times}$ 16~cm). For the hedgerow experiment,
microcolonies ($n = 90$) were initiated with 1 g of willow pollen for
three days before being fed with their respective diet while in the
orchard experiment, microcolonies ($n = 60$) were directly provided
with their respective diet upon the onset of the bioassay. Every
microcolony was fed \textit{ad libitum} with its respective diet for 35
days using pollen candies (1--3 g depending on the size of the
microcolony) and sugar syrup (water:sucrose 35:65~w/w). Pollen candies
were freshly prepared and renewed every other day while weighing pollen
left and syrup container to determine resource collection. Collection
data were corrected for evaporation using controls placed in bee-free
boxes. Dead workers and ejected larvae were checked every other day.
Workers that died during the experiment were removed, weighed and
replaced by new tagged workers originating from the same colony.
Microcolonies were reared in a dark room ($27 \pm 1$~\textdegree C;  60
${\pm}$ 10\% humidity) and manipulated under red light to minimise
disturbance. Upon the end of the experiment, the masses of adult
individuals were noted, the brood was carefully dissected, and the
masses of individuals in each developmental stage were recorded. No
mortality among emerged males was observed during the experiments.

\subsection{Parasite inoculation and monitoring}\label{sec22}

Three {\textit{B. terrestris}} {queens were collected in March 2021 and
determined to be infected by} {\textit{Crithidia}} sp. through faecal
inspection under a microscope (it is impossible to differentiate the 
cryptic species \textit{C. bombi} and \textit{C. expoeki} under a
microscope~\cite{70}). Contaminated faeces
were mixed with sugar syrup (water:sucrose 50:50 w/w) and the resulting
solution was provided in caps to two commercial \textit{B.
terrestris} colonies continuously fed with willow pollen. These two
colonies were used as parasite reservoirs for the hedgerow experiment.
For a year, infection was transmitted seven times through commercial
colonies and thus, in March 2022, two other infected colonies were used
as parasite reservoirs for the orchard experiment. For microcolony
inoculation, faeces were collected from 30 workers from the two
colonies used as parasite reservoirs. In the hedgerow experiment,
parasite load was measured every three days after inoculation (i.e., 11
measurements) while in the orchard experiment, parasite load was
measured at days 3, 5, 7, 9, 11, 15, 19 and 34 after inoculation (i.e.,
eight measurements). The inoculum preparation, the inoculation protocol
and the parasite load monitoring are fully described in Geki\`{e}re
\etal~\cite{42}. Briefly, faeces from workers from the
parasite reservoirs were collected and purified following a modified
``triangulation'' method~\cite{71}. Then, workers used in the
microcolonies were starved for five hours and inoculated with 25,000}
{\textit{Crithidia}} {sp. cells placed in 10~${{\upmu}}$L of sugar
syrup (water:sucrose 60:40 w/w) in the tip of a 10 mL syringe. Finally,
workers that consumed the inoculum were placed in their respective
microcolonies. To avoid any confounding effect due to handling and
starvation stress, uninoculated workers were also isolated and starved
for five hours before being placed in their microcolonies. Parasite
load was monitored by counting} {\textit{Crithidia}} sp. cells found
in faeces under a phase contrast microscope. In the hedgerow
experiment, faeces were collected from a randomly chosen worker in
every infected microcolony whereas in the orchard experiment, faeces
were collected from the exact same worker in every microcolony over the
experiment. Uninfected microcolonies were checked to be free of
parasite at the end of the experiment through faecal examination under
a microscope.  

\subsection{Microcolony parameters}\label{sec23}

To estimate the performance and development of the
microcolonies~\cite{72}, we analysed (i) pollen and syrup collection as
well as (ii) colony growth (i.e., the total mass of non-isolated
larvae, isolated and pre-defecating larvae, isolated and
post-defecating larvae, pupae, non-emerged and emerged males). We also
analysed stress parameters based on (iii) larval ejection (i.e., the
number of larvae removed from the brood by workers over the experiment
divided by the number of hatched offspring), (iv) pollen efficacy
(i.e., the mass of alive hatched offspring divided by the total mass of
collected pollen), (v) pollen dilution (i.e., the total mass of
collected syrup divided by the total mass of collected pollen) and (vi)
worker mortality (i.e., the number of dead workers over the
experiment).

\subsection{Individual parameters}\label{sec24}

To estimate the immunocompetence of bumble bee individuals~\cite{73},
we measured abdominal fat body content of two workers and two males per
microcolony (hedgerow: $n = 360$; orchard: $n = 280$) at the end of the
experiment following Ellers~\cite{74}. The abdomens were first
removed from the thorax and dehydrated in an incubator at
70~\textdegree C for three days and weighed. They were then placed for
one day in 2 mL of diethyl ether in order to solubilise the lipids
before being washed twice and incubated at 70~\textdegree C for seven
days and then weighed again. The mass difference before and after lipid
solubilisation corresponds to the mass of the fat body. Fat body
content is calculated by dividing the fat body mass by the dry abdomen
mass prior to solubilisation.

\subsection{Statistical analyses}\label{sec25}
We built distinct models for the hedgerow and orchard experiments since
these two experiments were conducted with a one-year interval and
included their respective controls. 

To test for differences in total mass of hatched alive offspring and in
pollen dilution, we built linear mixed-effect models (LMMs) and checked
for linearity, normality and homoscedasticity of residuals. Pollen
dilution was log-transformed in the orchard experiment to meet the
previous assumptions. To test for differences in pollen efficacy,
larval ejection and fat body content, we built generalised linear
mixed-effect models (GLMMs) and checked the deviance residual
diagnostics to ensure goodness of fit. Pollen efficacy and fat body
content were assessed using a beta error structure with a logit link,
since they are continuous variables restricted to the interval [0, 1].
For pollen efficacy, we discarded two outliers in the orchard
experiments. Larval ejection was assessed using a binomial error
structure with a logit link with the number of ejected larvae
considered as the number of ``successes'' and the total number of hatched
alive offspring considered as the number of ``failures'' (cbind()
argument). LMMs and GLMMs included diet, parasite and their interaction
as fixed effects and colony of origin as random effect. For larval
ejection, microcolony nested within colony was used as a random effect
to deal with overdispersion (i.e., observation-level random effect).
Besides, for fat body content, we included caste and microcolony nested
within colony as random effects to avoid pseudo-replication. Cumulative
pollen and syrup collection, as well as parasite load were assessed
using generalised additive mixed-effect models (GAMMs), to relax the
GLMM restriction that relationships must be a weighted sum, by allowing
covariates to vary according to non-linear spline functions~\cite{75}.
Cumulative syrup collection and log-transformed cumulative pollen
collection were fitted using a Gamma error structure with a log link.
Square root-transformed parasite load was fitted using a Gaussian
location-scale error structure with an identity link for the mean and a
logb link for the standard deviation. Pollen collection GAMM included
diet, parasite and their interaction as fixed effects and microcolony
nested within colony as random effect to account for repeated measures,
while parasite load GAMM only considered diet as fixed effect (since
infection was only assessed in infected treatments). The
goodness-of-fit was checked using diagnostic information and plots.
Survival analyses were conducted using mixed-effect proportional
hazards regression models (Cox models) after checking for proportional
hazard assumptions. Cox models included diet, parasite and their
interaction as fixed effects and colony as random effect. When models
highlighted a significant effect of diet (\textit{p-value}  $&lt;$ 0.05), 
we ran between-diet contrasts conditioned on the
infection status because we were not interested by the impacts of the
parasite itself in this study (i.e., uninfected treatments were never
contrasted to infected treatments). Error due to multiple comparisons
was controlled using the one stage false discovery rate (FDR) method
and the corrected \textit{p-values} are presented. All analyses
and graphs were run in R version~4.1.3~\cite{84a} using the packages
\textit{mgcv}~\cite{76}, \textit{glmmTMB}~\cite{77},
\textit{emmeans}~\cite{78}, \textit{DHARMa}~\cite{79},
\textit{survival}~\cite{80}, \textit{survminer}~\cite{81},
\textit{coxme}~\cite{82}, \textit{ggpubr}~\cite{83} and
\textit{ggplot2}~\cite{84}. Detailed statistical outputs from the
models can be found in Supplementary Table~S3.

\section{Results}\label{sec3}

\subsection{Hedgerow experiment}\label{sec31}

\subsubsection{Resource collection}\label{sec311}
\noindent
Irrespective of the infection, we found a lower cumulative pollen
collection in microcolonies fed the natural diet than in microcolonies
fed either the control (Uninfected:  $t = -3.278$, $p = 0.003$;
Infected: $t= -4.334$,  $p &lt; 0.001$) or supplemented diet (Uninfected:
$t = -2.165$, $p = 0.046$; Infected: $t = -3.217$, $p = 0.002$), while
the cumulative pollen collection in microcolonies fed one of the
two latter diets did not differ from each other (Supplementary Figure~S2A,B).
Regarding syrup, there was no significant impact of diet on cumulative
collection (Supplementary \unskip\break Figure~S3A,B).

\subsubsection{Reproduction}\label{sec312}
\noindent
The only difference in mass of alive hatched offspring was detected in
uninfected microcolonies, whereby we found a lower mass of alive
hatched offspring in microcolonies fed the supplemented diet when
compared to microcolonies fed either the control ($t = -4.324$, $p
&lt;0.001$) or natural diet ($t = -3.547$,  $p = 0.001$). In these
uninfected microcolonies, the mass of alive hatched offspring did not
differ between microcolonies fed the control diet and microcolonies fed
the natural diet (Supplementary Figure~S4A,B).

\subsubsection{Stress response}\label{sec313}
\noindent
In the uninfected treatments, we observed a higher larval ejection in
microcolonies fed the supplemented diet than in microcolonies fed the
control ($t = 2.300, p = 0.036$) or natural diet ($t = 2.464$, $p =
0.036$), while the larval ejection in microcolonies fed the one of the
two latter diets did not differ between each other
(Supplementary Figure~S5A). By contrast, in the infected treatments, we
found a higher larval ejection in microcolonies fed the supplemented
diet (supplemented vs. control: $t = 5.594$, $p &lt; 0.001$;
supplemented vs. natural: $t = 2.567$, $p = 0.012$), an intermediate
larval ejection in microcolonies fed the natural diet (natural vs.
control: $t = 3.219$, $p = 0.003$), and a lower larval ejection in
microcolonies fed the control diet (Supplementary Figure~S5B). Regarding the pollen
efficacy, irrespective of the infection, it was higher in microcolonies
fed the natural diet than in microcolonies fed the control diet
(Uninfected: $t = 7.313$, $p &lt; 0.001$; Infected: $t = 10.771$, $p &lt;
0.001$) or supplemented diet (Uninfected: $t = 7.771$, $p &lt; 0.001$;
Infected: $t = 8.523$, $p &lt;0.001$), while the pollen efficacy in
microcolonies fed one of the two latter diets did not differ
between each other (Figure~\ref{fig1}A,B). Regarding the pollen
dilution, irrespective of the infection, it was lower in microcolonies
fed the control diet when compared to microcolonies fed either the
natural (Uninfected: $t = -11.544$, $p &lt; 0.001$; Infected: $t =
-4.684$, $p &lt; 0.001$) or supplemented diet (Uninfected: $t = -12.735$,
$p &lt; 0.001$; Infected: $t = -3.750$, $p = 0.001$), while the pollen
dilution in microcolonies fed one of the two latter diets did not
differ between each other (Supplementary Figure~S6A,B).

\begin{figure*}[p]
\includegraphics{fig01}
\caption{\label{fig1}Parameters recorded in the hedgerow experiment
from uninfected (A,C,E) and infected (B,D,F) microcolonies.
Control diet $=$ \textit{Salix} sp. pollen. Natural diet $=$
\textit{Crataegus monogyna} pollen. Flavo diet $=$ Control pollen laced
with flavonoids from natural pollen. (A,B)~Pollen efficacy,
defined as the mass of alive hatched offspring divided by total mass of
collected pollen per microcolony. (C,D) Fat body content in
bumble bee individuals. \entmath{25B4}{$\blacktriangle$}: Female individuals.
$\bullet$: Male individuals. (E,F)~Kaplan--Meier survival
curves for microcolonies across diets. Note that $y$-axis have been
truncated for easier interpretation. Two treatments sharing a letter
are not significantly different (GLMM). {n.s.} Not significant.}
\end{figure*}

\subsubsection{Immunity and health}\label{sec314}
\noindent
In the uninfected treatments, we found a higher fat body content in
individuals housed in microcolonies fed the control diet (control vs.
natural: $t = 9.112$, $p &lt; 0.001$; control vs. supplemented: $t =
2.117$,  $p = 0.035$), an intermediate fat body content in individuals
housed in microcolonies fed the supplemented diet (supplemented vs.
natural: $t = 7.065$, $p &lt; 0.001$), and a lower fat body content in
individuals housed in microcolonies fed the natural diet
(Figure~\ref{fig1}C). By contrast, in the infected treatments, we
observed a lower fat body content in individuals housed in
microcolonies fed the natural diet than in individuals housed in
microcolonies fed the control ($t =-5.027$,  $p &lt; 0.001$) or
supplemented diet ($t = -4.634$,  $p &lt; 0.001$), while fat body content
in individuals housed in microcolonies fed one of the two latter
diets did not differ between each other (Figure~\ref{fig1}D). Regarding
the other parameters, we did not find any significant impact of diets
on worker mortality (Figure~\ref{fig1}E,F) or on parasite load
(Figure~\ref{fig2}A).\vspace*{10pt}

\begin{figure}[t!]
{\vspace*{4pt}}
\includegraphics{fig02}
{\vspace*{6pt}}
\caption{\label{fig2}Parasite load in \textit{Crithidia}-infected
\textit{Bombus terrestris} individuals. Control diet $=$ \textit{Salix}
sp. pollen. Flavonoid diet $=$ Control pollen laced with flavonoids from
natural pollen. ({A}) Hedgerow experiment (i.e., natural diet $=$
\textit{Crataegus monogyna} pollen). (B)~Orchard experiment
(i.e., natural diet $=$ mix of \textit{Prunus avium}, 
\textit{Malus domestica} and \textit{Pyrus communis} pollen). Two
treatments sharing a letter are not significantly different (GAMM).
{n.s.} Not significant.}
\end{figure}

\subsection{Orchard experiment}\label{sec32}
\vspace*{5pt}
\subsubsection{Resource collection}\label{sec321}
\noindent
We found that uninfected microcolonies fed the supplemented diet had a
lower cumulative pollen collection when compared to uninfected
microcolonies fed either the control ($t = -9.272$, $p &lt; 0.001$) or
natural diet ($t = -9.352$, $p &lt; 0.001$), while the cumulative pollen
collection in microcolonies fed one of the two latter diets did not
differ between each other (Supplementary Figure~S2C). On the contrary,
there was no difference in the cumulative pollen collection across
diets in infected microcolonies (Supplementary Figure~S2D). Regarding
syrup, there was no significant impact of diet on cumulative collection
(Supplementary Figure~S3C,D).\vspace*{10pt}

\subsubsection{Reproduction}\label{sec322}
\noindent
In the uninfected treatments, we found a higher mass of alive hatched
offspring in microcolonies fed the natural diet (natural vs. control:
$t = 5.086$, $p &lt; 0.001$; natural vs. supplemented: $t = 9.853$,  $p &lt;
0.001$), an intermediate total mass of alive hatched offspring in
microcolonies fed the control diet \unskip\break (control vs. supplemented: $t =
4.767$, $p &lt; 0.001$), and a lower total mass of alive hatched
offspring in microcolonies fed the supplemented diet (Supplementary 
Figure~S4C). By contrast, in the infected treatments, we found a higher 
mass of alive hatched offspring in microcolonies fed the natural when 
compared to microcolonies fed the control ($t = 3.036$,  $p = 0.006$) or
supplemented diet ($t = 4.824$, $p &lt; 0.001$), while the mass of alive
hatched offspring in microcolonies fed one of the two latter diets
did not differ between each other (Supplementary Figure~S4D).

\subsubsection{Stress response}\label{sec323}
\noindent
Regarding the larval ejection, there was no significant difference
among diets, irrespective of the infection (Supplementary Figure~S5C,D).
Regarding the pollen efficacy, irrespective of the infection, it was
lower in microcolonies fed the supplemented diet compared to
microcolonies fed either the control (Uninfected: $t = -3.472$,  $p =
0.002$; Infected: $t = -3.725$, $p = 0.002$)  or natural diet
(Uninfected: $t = -4.436$, $p &lt; 0.001$; Infected: $t = -2.839$, $p =
0.010$), while the pollen efficacy in microcolonies fed the one of the
two latter diets did not differ between each other
(Figure~\ref{fig3}A,B). Regarding pollen dilution, in
uninfected microcolonies, we observed a higher pollen dilution in
microcolonies fed the supplemented diet (supplemented vs. control: $t =
2.893$, $p = 0.006$; supplemented vs. natural: $t = 6.114$, $p &lt;
0.001$), an intermediate pollen dilution in microcolonies fed the
control diet (control vs. natural: $t = 3.221$,  $p = 0.003$), and a
lower pollen dilution in microcolonies fed the natural diet
(Supplementary Figure~S6C). By contrast, in infected microcolonies, we observed a
higher pollen dilution in microcolonies fed the control diet (control
vs. natural: $t = 4.973$, $p &lt; 0.001$; control vs. supplemented: $t =
2.770$, $p = 0.012$), an intermediate pollen dilution in microcolonies
fed the supplemented diet (supplemented vs. natural: $t = 2.203$, $p =
0.032$), and a lower pollen dilution in microcolonies fed the natural
diet (Supplementary Figure~S6D).

\begin{figure*}[p]
{\vspace*{-2pt}}
\includegraphics{fig03}
{\vspace*{-2pt}}
\caption{\label{fig3}\fontsize{9.6}{12}\selectfont
Parameters recorded in the orchard experiment from
uninfected (A,C,E) and infected (B,D,F) microcolonies. Control diet
$=$ \textit{Salix} sp. pollen. Natural diet $=$ mix of \textit{Prunus
avium}, \textit{Malus} \textit{domestica} and \textit{Pyrus communis}
pollen. Flavo diet $=$ Control pollen laced with flavonoids from
natural pollen. (A,B) Pollen efficacy, defined as the mass of
alive hatched offspring divided by total mass of collected pollen per
microcolony. (C,D)~Fat body content in bumble bee individuals.  
\entmath{25B4}{$\blacktriangle$}: Female individuals. ${\bullet}$:  Male
individuals. (E,F)~Kaplan--Meier survival curves for
microcolonies across diets. Note that $y$-axis have been truncated for
easier interpretation. Two treatments sharing a letter are not
significantly different (GLMM). n.s. Not significant.}
\end{figure*}


\subsubsection{Immunity and health}\label{sec324}
\noindent
The only difference in fat body content was detected in uninfected
microcolonies, whereby individuals housed in microcolonies fed the
supplemented diet showed higher fat body content than individuals
housed in microcolonies fed the control diet ($t = 2.979$, $p =
0.010$;  Figure~\ref{fig3}C,D). Regarding mortality, it was higher in
microcolonies fed the supplemented diet than in microcolonies fed the
natural diet, irrespective of the infection (Uninfected: HR $=$ 7.012,
$z = -2.564$, $p = 0.031$; Infected: HR $=$ 3.067, $z = -2.676$, $p =
0.022$; Figure~\ref{fig3}E,F). We also observed a higher mortality in
uninfected microcolonies fed the control diet than in uninfected
microcolonies fed the natural diet (HR $=$ 5.230, $z = 2.136$, $p =
0.049$; Figure~\ref{fig3}E). Regarding the parasite load, it was lower
in individuals housed in microcolonies fed the natural diets over
individuals housed in microcolonies fed either the control ($t =
-2.188$, $p = 0.045$) or supplement diet ($t = -3.145$, $p = 0.006$),
while parasite load in individuals housed in microcolonies fed one
of the two latter diets did not differ between each other
(Figure~\ref{fig2}B).
\vspace*{50pt}

%\pagebreak
\section{Discussion}\label{sec4}
\subsection{Hedgerow and orchard pollen as suitable diets}\label{sec41}

Based on the mass of alive hatched offspring, hedgerow (i.e., hawthorn)
and orchard (i.e., sweet cherry, apple and pear) pollen were as
adequate or even better than willow pollen, respectively, for the
microcolony development of the buff-tailed bumble bee. For the hedgerow
experiment, these results are in line with Wood \etal~\cite{48} 
who found no beneficial effect of hawthorn pollen over willow pollen
for bumble bee microcolony development, despite a higher
protein-to-lipid ratio and higher amino acid content in the former. For
the orchard experiment, these results are in line with Aupinel
\etal~\cite{85} and Genissel \etal~\cite{86} who
highlighted a higher offspring production and a reduced oophagy in
microcolonies fed sweet cherry pollen when compared to microcolonies
fed willow pollen, likely due to higher protein, amino acid and sterol
content in sweet cherry pollen~\cite{87,88}. Especially, the latter
contains a 20-times higher concentration of 24-methylenecholesterol
than willow pollen~\cite{87,88}, a crucial sterol for bee moulting and
ovary development~\cite{89}. To our knowledge, no study has ever
assessed the impact of pure apple or pear tree pollen on bumble bee
microcolony development. Barraud \etal~\cite{90} showed that a
mix of apple, pear and willow pollen did not lead to significant
differences in microcolony development when compared to pure willow
pollen, suggesting that apple or pear tree pollen should not have
drastic negative consequences for bumble bee development (e.g., toxic
phytochemicals). Quinet \etal~\cite{57} suggested that apple and
pear trees are complementary for bumble bees as they produce a huge
quantity of pollen and nectar, respectively. Hedgerow and orchard
pollen suitability for bumble bees was further confirmed by a low
mortality rate in microcolonies. 

By contrast, immunocompetence was slightly impacted by the natural
pollen treatments, as they were as adequate or less adequate than
willow pollen for fat body content of the buff-tailed bumble bee. It
could be due to quantitative or qualitative reasons: (i)
bumble bees collected slightly less natural than control pollen, or
(ii) natural pollen differ in their metabolite content when compared to
control pollen. Differences in nutrient intakes are likely to influence
fat body development~\cite{91,92,93,94}, as in Geki\`{e}re 
\etal~\cite{42} wherein sunflower pollen reduced fat body
content when compared to willow pollen. By contrast, another study
conducted by our team showed that bumble bees fed natural heather
pollen (\textit{Calluna vulgaris}; Ericales: Ericaceae) had higher fat
body content than bumble bees fed willow pollen 
(Tourbez \etal~\cite{new95}).
However, many studies did not find any influence of the pollen
diet on fat body content~\cite{77,95,96,97,98}. Overall,
nutritional compounds underlying fat body development remain obscure
and definitely warrant further attention. Likewise, we must stress that
there is an increasing uncertainty regarding the adequacy of fat body
content as a proxy for immunocompetence, as results are inconsistent
and hard to interpret. Although fat bodies have been shown as the main
sites of antimicrobial peptide production~\cite{99} and the main
organs for lipid metabolization~\cite{100}---two crucial steps in
immune responses---no study has ever properly demonstrated a
correlation between fat body content and immunocompetence in insects
(Geki\`{e}re, Dewaele, \etal~\cite{new101}). 

\subsection{Natural pollen diets shape resistance towards parasite
infection}\label{sec42}

Regarding pollen medicinal effects, we found that orchard pollen
slightly increased bumble bee resistance towards infection (i.e.,
decreased load), which could be due to the poor adaptation of our
parasite strains for orchard pollen. Indeed, {\textit{Crithidia}} sp.
require pollen to thrive in the gut lumen and specific strains are
likely selected when bumble bees are chronically fed the same pollen
diet~\cite{101}. Because our strains were sampled from stock bumble
bees fed willow pollen for more than a year (2021--2022), it is likely
that these strains thrived better on willow pollen than orchard pollen.
This interpretation is in line with the hedgerow experiment wherein
bumble bees fed willow pollen did not differ in their parasite load
from bumble bees fed hawthorn pollen, likely because parasite strains
were freshly acquired from the wild (spring 2021), and did not develop
for more than two weeks in stock colonies fed willow pollen. 

Although we focussed on the chemical roles of natural pollen on
\textit{Crithidia}-infected bumble bees in this study, we would like
to remind that the impacts of natural pollen on gut parasites could be
purely mechanical. In 2018, Adler and her team showed that
sunflower pollen (Asterids: Asteraceae) reduced \textit{Crithidia}
sp. load in \textit{Bombus impatiens}~\cite{102} but further
research failed to identify any specialised metabolites accountable for
such effects~\cite{103}. Recently, they found that sunflower medicinal
effects were due to its spiny exine, rather than to its chemical
profile~\cite{104}. Here, the pollen species we used belong to the
Rosaceae family which harbours smooth exines~\cite{105}. Hedgerow and
orchard pollen were consequently unlikely to display any medicinal
effects through their morphological features.

\subsection{Slight detrimental impacts of flavonoid
extracts}\label{sec43}

An intriguing observation to underline is the different results
obtained in microcolonies fed flavonoid diets when compared to
microcolonies fed natural diets, whereas both diets harbour the same
flavonoid profiles. We propose that although pollen grains containing
these phytochemicals are constantly ingested by bumble bees, it could
be that these phytochemicals are not readily bioavailable in the bumble
bee gut (e.g., because of the pollen wall). For instance, Omar 
\etal~\cite{106} demonstrated that honey bees digested 60\% of the
proteins found in corn pollen when fed raw pollen, while honey bees fed
with crushed pollen digested {$&gt;$}70\% of the proteins. Further, some
phytochemicals are confined in the pollen wall and are therefore likely
to never be digested (e.g., the biopolymer sporopollenin)~\cite{107}.
By treating pollen with chemical solvents, we presumably rendered
bioavailable phytochemicals that would not be naturally digested in the
bee gut, or at lower concentrations. Besides, as we used willow pollen
in the mix for flavonoid diets, we exposed bumble bees not only to
flavonoid extracts but also to flavonoids naturally occurring in willow
pollen. Using willow pollen in the mix for flavonoid diets also
provided bumble bees with central metabolites (e.g., lipids) different
from the ones in the natural diets. This limitation prevents us from
drawing clear conclusions on hedgerow and orchard flavonoid
consequences on bumble bees. As already stressed in previous studies
(e.g.,~\cite{94,97}), this research further calls for the development
of controlled pollen substitutes for laboratory experiments on bees.

In our experiment, flavonoid extracts mostly had detrimental impacts
for the microcolony development and the survival of the buff-tailed
bumble bee. We are not aware of any studies tackling the influence of
these specialised metabolites on bumble bee microcolony development,
but flavonoids did not increase mortality rate in honey 
bees~\cite{108}. Given that flavonoids are ubiquitous in pollen
species~\cite{109}, we expect that they should have only minor effects
on bumble bee colonies, which somewhat contrasts with our results. This
is however in accordance with a previous research conducted in our lab,
wherein we found that phenolamide extracts---a major class of
phenylpropanoid metabolites evolutionarily conserved across 
angiosperms---had detrimental impacts on bumble bee microcolony
development~\cite{42}. In bees, larval development is highly shaped by
pollen properties, whether it is due to pollen metabolite content or
harmful morphological features, because bee species differ in their
abilities to deal with specific pollen species~\cite{110,111}. Our
experiment does not enable us to disentangle if the impeded offspring
production in microcolonies fed flavonoid diets is caused by either
altered worker behaviours or phytochemical unsuitability for the
larvae. Yet, one may suggest that it is due to phytochemical
unsuitability because our results showed that pollen collection did not
seem to explain offspring production. Further experiments using larval
rearing \textit{in vitro}~\cite{112} and isolating pollen
wall~\cite{113} are required to directly assess diet phytochemical and
morphological suitability for bumble bee larval development.\looseness=-1 

\vspace*{4pt}
Strikingly, flavonoid extracts mostly had beneficial consequences on
fat body content, although this parameter must be interpreted with
caution as previously discussed. Based on Ellers~\cite{74}, we
hypothesise that it could be explained by a trade-off between
immunocompetence and reproduction, whereby egg-laying workers allocated
their fat resources in their ovaries instead of their body fat.
Indeed, higher body fat content was found in microcolonies fed
flavonoid diets which had a lower reproductive success. We could argue
that we should not observe this trade-off in our results since it is
assumed that only one worker lays egg in microcolonies~\cite{114}, and
that we did not target these pseudo-queens for fat body analyses.
However, personal observations and previous studies showed that several
workers could have well-developed ovaries in queen-less
microcolonies~\cite{115}, which is consistent with our results. 

Surprisingly, flavonoids did not reduce parasite load, whereas
flavonoids were shown to have antimicrobial properties (e.g.,
\textit{Vairimorpha} spp. load reduction in honey bees~\cite{30}
{and bumble bees~\cite{31}). As far as we know, there is no report of
flavonoid impacts on \textit{Crithidia} sp. in bumble bees, but
Palmer-Young \etal~\cite{116} studied flavonoid impacts
\textit{in vitro} on \textit{C.~mellificae} (host: honey bees)
and \textit{C. fasciculata} (host: mosquitoes). They found no
effect on \textit{Crithidia} spp. growth except for the flavonoid
chrysin, which hampered \textit{C. fasciculata} but not
\textit{C. mellificae}. They suggested a flavonoid resistance for
\textit{C. mellificae} since this parasite is recurrently exposed to
flavonoids in pollen and propolis~\cite{117}, while} 
\textit{C. fasciculata} is only exposed to flavonoids found in
nectar~\cite{109}. Despite the flavonoid antimicrobial
properties~\cite{29}, it is hence perfectly reasonable to assume that
\textit{Crithidia} sp. is able to deal with flavonoid exposure.


\subsection{Bee conservation strategies and perspectives}\label{sec44}

Overall, our results are supporting the development of orchards and
hedgerows as mitigation strategies to face decline of wetlands, wet
heathlands and their associated willow trees due to human activities
and extreme climate events~\cite{52,53,54}, since bumble bees greatly
rely on willows for colony initiation ~\cite{49,50,51}. Given
the phenology of the investigated tree species (Supplementary Figure~S7),
our results suggest that bumble bees could lean on sweet cherry, pear
and apple tree pollen for colony initiation in early spring, while
hawthorn pollen could rather be used by older bumble bee colonies later
in the season. However, although sweet cherry, apple and pear orchards
have been increasingly harvested worldwide since 1960, a worrying
decline has been observed for the last 20 years
(Supplementary Figure~S8~\cite{new17}). Besides, there is no data around
hedgerow management, and it is therefore impossible to tell whether
hedgerows will significantly contribute to bumble bee diets in future
springs. Moreover, even though flavonoids did not help bumble bees
reduce infection, they did not show any harmful effects and remain
important dietary phytochemicals to deal with environmental stress,
such as agrochemical exposure~\cite{33,118}. In conclusion, trees
represent nutritional resources of tremendous importance for bumble
bees, especially early flowering species that are crucial to sustain
colony initiation. Sweet cherry, apple and pear trees bloom in early
spring, and are good candidates to offset the ongoing decline of early
flowering willows, while hawthorn is a suitable resource for the late
season. In addition to preserving early blooming tree species,
conservation strategies should further consider specialised metabolite
profiles, especially flavonoids, when designing agri-environmental
schemes to support wild bee health.

\section*{Conflicts of interest}
Authors have no conflict of interest to declare.

\section*{Author contributions} 

Conceptualisation, AG and MV; formal analysis, AG, AM and MV;
funding acquisition, DM, PG and MV; investigation, AG, AM,
CT and MB; methodology, AG, IS and MV; resources, DM and
PG; supervision, DM and MV; visualisation, AG;
writing---original draft, AG with the help of MV;
writing---review and editing, AG, AM, IS, CT, MB,
DM, PG and MV.

\section*{Funding}

This work was part of the ARC ``Actions de Recherche Concert\'{e}es''
project ``METAFLORE'', 2019--2023. AG is supported by a
F.R.S.-FNRS PhD grant ``Aspirant''. The PhD grant of IS is supported by
the ARC project METAFLORE. The PhD grant of CT is supported by the
University of Mons (UMons).

\section*{Acknowledgements}
We would like to thank D. Evrard and L. Verdy for their help in
logistics as well as the numerous people that helped during microcolony
dissection. Many thanks to L. Marin for her help in daily monitoring.
We are very grateful to the Laboratory of Cellular Biology (UMons) for
their help in microscopic analyses.

\back{}

\section*{Supplementary data}

Supporting information for this article is available on the journal's
website under \printDOI\ or from the author.  

\CDRsupplementaryTwotypes{supplementary-material}{\cdrattach{crchim-231-suppl.pdf}}

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