Dimitry
Wintermantel1,*, Maria-Helena
Pereira-Peixoto1, Nadja Warth1,,
Kristin Melcher1, Michael Faller1,
Joachim Feuer1, Matthew J. Allan2,
Robin Dean3, Giovanni Tamburini4,
Anina C. Knauer5, Janine Melanie
Schwarz5, Matthias Albrecht5 and
Alexandra‑Maria Klein1
1 University of Freiburg, Nature Conservation and
Landscape Ecology, Freiburg, Germany;
2 Atlantic Pollination Ltd, Eastleigh, United Kingdom;
3 Red Beehive Company, Bishops Waltham, United
Kingdom;
4 University of Bari, Department of Soil, Plant and
Food Sciences (DiSSPA - Entomology), Bari, Italy;
5Agroscope, Agroecology and Environment, Zurich,
Switzerland.
Email addresses : dimitry.wintermantel@nature.uni-freiburg.de
(DW), helenametal@yahoo.com.br
(MHPP), nadja.warth123@web.de (NW), kristin_melcher@web.de (KM),
michi.faller@gmx.de (MF), joachim_feurer@web.de (JF),
matt@atlanticpollination.com (MJA), redbeehive@btopenworld.com (RD),
giovanni.tamburini@uniba.it (GT), anina.knauer@agroscope.admin.ch,
janine.schwarz@agroscope.admin.ch, matthias.albrecht@agroscope.admin.ch
(MA), alexandra.klein@nature.uni-freiburg.de (AMK).
*Corresponding author : Dimitry Wintermantel, University of
Freiburg, Nature Conservation and Landscape Ecology, Freiburg, Germany.
E-mail: dimitry.wintermantel@nature.uni-freiburg.de. Phone: +49 (0) 761
203 -9484.
Running title : Flowering resources alter fungicide impacts
Keywords: Bombus terrestris , azoxystrobin, nutrition,
pesticide sensitivity, pollen quality, floral diversity, flowering
resource quality, agrochemical, plant protection product, semi-field
study
Type of article: Letter
Number of
- words in the abstract: 150
- words in the main text: 5000
- references: 86
- figures: 4
- tables: 1
- text boxes: 0
Authorship: AMK, DW, MJA and MA designed the experiment. DW,
MHPP, NW, KM, MF, and JF conducted the field and lab work. DW analysed
the data and wrote the first draft of the manuscript. All authors
commented and edited the draft and approved the final manuscript.
Data accessibility statement : The data supporting the results
will be archived in an appropriate public repository and the data DOI
will be included at the end of the article.
Abstract
Bees
are exposed to various stressors, including pesticides and lack of
flowering resources. Despite potential interactions between these
stressors, the impacts of pesticides on bees are generally assumed to be
consistent across bee-attractive crops, and regulatory risk assessments
of pesticides neglect interactions with flowering resources.
We assessed the interactive impacts of the globally used
azoxystrobin-based fungicide Amistar and three types of flowering
resources (purple tansy, buckwheat, and a floral mix) on Bombus
terrestris colonies in a full-factorial semi-field experiment with 39
large flight cages.
Fungicide exposure through purple
tansy monocultures reduced population (colony) growth, production of
males, and adult worker body mass, while Amistar had no impact on
colonies in buckwheat or floral mix cages. Furthermore, buckwheat
monocultures hampered survival and fecundity irrespective of fungicide
exposure. This shows that flowering resources modulate pesticide impacts
and that B. terrestris requires access to complementary flowers
to gain both fitness and fungicide tolerance.
Introduction
Declines in bee diversity and distribution (Biesmeijer et al.2006; Vanbergen et al. 2013; Potts et al. 2016) are
believed to be driven by a combination of stressors including
pesticides, diseases, and loss of flowering resources (Goulson et
al. 2015; Potts et al. 2016). Research on the effects of
pesticides on bees has mostly focused on insecticides due to the
taxonomic proximity of target pests (insects) to bees (Cullen et
al. 2019). However, fungicides are often the pesticides that bees are
most exposed to (Mullin et al. 2010; Pettis et al. 2013;
McArt et al. 2017a) and despite their image of being relatively
non-toxic to bees, they can have negative effects on bees.
Experiments show that various
fungicides can either magnify the toxicity of insecticides (Goulsonet al. 2015; McArt et al. 2017a; Sgolastra et al.2017) or impair bees independently from other agrochemicals (Ladurneret al. 2005; Zhu et al. 2014; Artz & Pitts-Singer 2015;
Bernauer et al. 2015). Additionally, correlational evidence
indicates that fungicides promoted honeybee colony failure in Belgium
(Simon-Delso et al. 2014) and accelerated the declines of four
bumblebee species in the United States (McArt et al.2017b).
Azoxystrobin is a systemic broad-spectrum fungicidal substance that was
launched in 1996 and became the globally best-selling fungicide within
three years with an 8-fold increase in annual global sales in the
following twelve years (Bartlett et al. 2002; Leadbeater 2014).
It is frequently found in bees, pollen, and honey (Mullin et al.2010; Krupke et al. 2012; Piechowicz et al. 2018), but
only few published studies examined azoxystrobin effects on bees leaving
uncertainties about its risk. In honeybees, azoxystrobin affected gene
expression but not in a dose-response manner (Christen et al.2019) and increased forager mortality but only at concentrations above
field-realistic levels (Fisher et al. 2017). Semi-field studies
with field-realistic exposure found no effects on several honeybee
fecundity and mortality but a reduction in the foraging performance and
pollination services of Bombus terrestris colonies (Tamburiniet al. 2021a, b).
Agricultural
intensification affects bees not only through increased pesticide
exposure but also through altering flowering resource availability.
Reduced flowering plant diversity harms bees through a temporary
reduction in the quantity of available resources, as diversity ensures
continuous flowering, (Papanikolaou et al. 2017; Requier et al. 2017;
Kaluza et al. 2018) and an unbalanced diet (Di Pasquale et al. 2013;
Dance et al. 2017; Filipiak et al. 2017; Sutter et al. 2017; Leza et al.
2018). Flowering plants differ in the nutrients they provide and bees,
especially bumblebees, can adapt their foraging behaviour to their
nutritional demands (Ruedenauer et al. 2015, 2020; Somme et
al. 2015; Vaudo et al. 2015, 2016, 2020; Kraus et al.2019). Bumblebees generally prefer protein-rich pollen (Hanley et
al. 2008; Leonhardt & Blüthgen 2012; Ruedenauer et al. 2015;
Vaudo et al. 2015), which can foster their development (Baloglu
& Gurel 2015; Kämper et al. 2016; Roger et al. 2017) and
resilience to pathogens (Roger et al. 2017).
Both the quantity and quality of
food may affect the sensitivity of bees to pesticides. While flower
density and insecticide exposure additively impaired Osmia
lignaria reproduction (Stuligross & Williams 2020), low-sugar diets
and insecticide exposure synergistically decreased food consumption,
haemolymph sugar levels, and survival in honeybees (Tosi et al.2017). The presence of pollen in the diet mitigated pesticide effects on
honeybee survival through upregulation of detoxification-related genes
(Schmehl et al. 2014; de Mattos et al. 2018). It is less
clear what pollen components confer tolerance to pesticide impacts. High
protein content and high pollen diversity have been suggested to
decrease pesticide sensitivity but studies investigating interactions
between pesticides and either pollen differing in protein content or
diversity (polyfloral vs monofloral pollen) found effects ranging from
antagonistic over additive to synergistic (Wahl & Ulm 1983; Archeret al. 2014; Dance et al. 2017; Leza et al. 2018;
Barraud et al. 2020; Crone & Grozinger 2021). Perhaps, flowering
plant diversity reduces pesticide sensitivity particularly when bees can
select flowers themselves. Indeed, a semi-field study mimicking the
effect of flower strips through planting diverse untreated flowering
species close to insecticide-treated oilseed rape found that floral
diversification can offset insecticide impacts on Osmia bicornis(Klaus et al. 2021). However, it remains unclear
whether this buffering effect was caused by a more diverse diet, a
reduction in insecticide exposure, or both. A recent meta-analysis found
overall no interaction between agrochemicals and nutritional stress
(i.e. reduced food quantity or quality) on bee survival, but only three
studies, consisting of 19 data-sets of which 10 showed synergistic
effects, were investigated. For non-lethal endpoints, the sample size
was markedly smaller and especially field-realistic studies are lacking
(Siviter et al. 2021).
To study the interactive effects of the azoxystrobin-based fungicide
Amistar and different flowering plant resources on Bombus
terrestris, we conducted a full-factorial semi-field experiment.
Thereby, we enclosed bumblebee colonies with untreated or
Amistar-treated purple tansy (Phacelia tanacetifolia ),
common buckwheat (Fagopyrum esculentum ), or a floral mix
consisting of several planted and spontaneously flowering species. The
two monocultural species are commonly used in semi-field experiments on
pesticides (Gradish et al. 2016; Scott-Dupree et al. 2017;
Frewin et al. 2019; Knäbe et al. 2020; Franke et
al. 2021) and provide similar amounts of nectar (Knauer et al.unpublished data; Petanidou 2003; Cawoy et al. 2009) but purple
tansy is regarded as a more valuable resource for bees than buckwheat
due to its nearly three times higher crude pollen protein content
(Pernal and Currie, 2000; Somerville, 2001). We hypothesize that (1)
both Amistar and flowering resource type (hereafter simply ‘resource’)
affect bumblebees and (2) colony development and pesticide tolerance are
lowest in colonies feeding exclusively on buckwheat due to the lack of
flowering plant diversity and pollen protein.
Methods
Study design
The experiment was conducted in 2020 and consisted of 39 cages with a
ground cover of 53 m2 (5.9 m × 9 m; height = 2.5 m)
that were erected at a minimum distance of 4 m from each other on a 0.7
ha-large university-owned experimental field in Freiburg, Germany
(48°01’08.5”N 7°49’31.2”E). The three resources – buckwheat, purple
tansy, and the floral mix – were randomly assigned to the cages. For
the floral mix, a custom seed mix from Rieger Hofmann
(Blaufelden-Raboldshausen, Germany, www.rieger-hofmann.de) was sown that
consisted of F. esculentum (40% by weight), P.
tanacetifolia (10%), Centaurea cyanus (20%), Sinapis
arvensis (10%), Malva sylvestris (10%) and Trifolium
resupinatum (10%; Table S1; Appendix A). The latter two, however,
barely germinated. Unlike the monocultures, floral mix cages were not
weeded and contained, therefore, also flowering Achillea
millefolium, Cirsium arvense , Linaria vulgaris, Persicaria
lapathifolia, Plantago lanceolate , Verbascum nigrum, andVicia cracca .
The cages were covered by a nylon
net (mesh size = 0.95 × 1.35 mm). Each cage contained one colony during
a 1-week pre-exposure period (before Amistar application) and a 10-day
exposure period (after Amistar application, Fig. 1). The colonies were
additionally examined after a 13-day post-exposure period, in which they
were allowed to forage freely outside the cages. Stratified random
allocation approaches were used to assign cages to spray treatments
(i.e. Amistar or water/control) and colonies to resource-spray
combinations with strata being based on flower density and number of
adult bees, respectively (Appendix A).
Fungicide application
The fungicide Amistar (active ingredient (a.i.) = azoxystrobin) was
applied at a rate of 250 g a.i. per hectare (=1 L ha-1of formulated product) in the morning of 4 July 2020 in 6 of 13 purple
tansy cages, in 6 of 12 buckwheat cages, and 7 of 14 floral mix cages
(Appendix A, Fig. S1a). Amistar application was done according to label
instructions in EU member states (www.syngenta.com) by a ‘Good
Experimental Practices’-certified spray contractor using a motorized
sprayer equipped with a 3 m-long bar with anti-drift spraying nozzles
during dry weather with low wind speed (<2 m
s-1). During application, the sprayed cage was covered
with plastic sheets to further reduce the probability of spray drift to
adjacent cages. Using different equipment, control cages were sprayed
with water of the same volume as the diluted product. To prevent direct
exposition during bee flight, the exits of all bumblebee nests were
closed early in the morning and opened again directly after the
application.
Experimental bumblebee
colonies
Forty-three B. terrestris terrestris colonies purchased from Katz
Biotech AG were delivered on day - 8 (day 0 = day of Amistar/water
application) and assessed for queen presence, disease signs, and colony
size. No visual signs of pathogens or parasites were detected. The 39
colonies selected for the experiment had on average 36.3 living workers
(standard deviation = 7.4) with no difference between resources or spray
treatments (two-way ANOVA, P >0.93) and contained
only few dead workers (range = 0-3, mean = 0.9, median = 1). On day -7,
the colonies were placed inside the cages on the short side opposite the
entrance, facing South-East (Fig. S1b). A straight path without flowers,
dividing the cage into two halves, allowed easy access to the colonies.
The colonies were housed in the delivered plastic nest boxes placed
within wooden boxes on small wooden stands or bricks. All colonies were
delivered with two syrup containers and a pollen supplement. The larger
syrup container underneath the nest was closed immediately after
delivery, whereas the smaller syrup container and pollen supplement
(both within the nest) were removed during the first assessment in the
field on day -5, two days after the placement of colonies inside the
cages. One colony (buckwheat-control) died on day -3 and was replaced by
a spare colony.
Azoxystrobin residue
analysis
To quantify azoxystrobin exposure, two foragers from each cage were
collected on either day 1 or 2 using sweep nets and pooled for each of
the six resource-spray combinations and analysed by the Research Centre
for Agriculture and Environment (CREA-AA, Bologna, Italy) using liquid
chromatography/tandem mass spectrometry (LC-MS/MS). The samples were put
on dry ice in the field as well as during transport and were stored at
-20°C. The limit of quantification was 0.01 mg kg-1.
Assessments before and during exposure
The colonies were assessed once in the laboratory (day -8), and eight
times inside the flight cages (three times before and five times after
Amistar application; Fig. 1) for
- colony weight: Colonies (including their nest box) were weighed and
the weight of an empty plastic nest box was subtracted;
- cumulative number of dead adults: Dead adult bees inside the nest were
counted without removing them while visually inspecting the colonies
through the transparent plastic cover;
- number of living adults: Adult bees were counted from a photo of the
nest taken through a transparent acrylic cover. Number of dead adults
was subtracted from this count and estimated numbers of bumblebees
that left during placement of the cover or were foraging, while the
photo was taken, were added.
Flower density was assessed once before and five times after colony
placement. For this, the cages were divided into six equally large areas
(rectangles; Fig. S1b). During each flower density assessment, one of
three rectangles per side of the cage was randomly selected without
replacement until all rectangles were assessed (then random selection
without replacement re-started). Inside these rectangles, a quadrat (1 m
× 1 m) was placed so that it contained a flower cover/composition that
appeared representative either for the whole cage (only in the first
assessment) or for the selected rectangle. Inside the quadrats, the
number of inflorescences per plant species was counted, multiplied by
the mean number of flowers of three representative inflorescences, and
averaged across the two rectangles. In the case of the floral mix, the
process was done for all plant species and summed up.
Final assessment after colony
termination
Colonies were freeze-killed on day 23 when all foundress queens had
died, possibly because flowering resources declined in the study site
and were lacking in the surroundings. The colonies were afterwards
examined for
- numbers of adult males and workers: Adult bees were separated by caste
and counted. These counts included bees that lived until colony
termination or died within the four days before, as dead bees were
removed from the colonies on day 19. As only five colonies (2 purple
tansy – control, 2 purple tansy – Amistar, and 1 floral mix –
control) produced queens, the number of queens was not analysed.
- number of worker and/or male cocoons: Closed cocoons (from which no
bee had emerged yet) with a diameter <12 mm were counted. In
only one terminated colony a queen cocoon (floral mix–control) was
found and not further considered.
- adult worker body mass and intertegular distance: If available, 15
workers that were presumably alive until colony termination were
weighed using a high-precision balance with wind-break and measured
for the distance between the insertion points of the wings using a
digital calliper. Bees that were particularly dry or ridged were
assumed to have died already before colony termination and were
therefore sorted out. Males and queens were not examined due to their
low numbers.
- pupal body mass and developmental stage: Up to 35 cocoons were opened
to obtain 20 pupae that presumably were alive until colony
termination. The cocoons were sexed, weighed and their developmental
stage was rated on a scale from 1-6 based on eye colour, body colour,
and presence/absence of wings (Wintermantel et al., 2018, Table S2).
Pupae last approximately two days in each developmental stage.
Data analysis
The statistical analyses on bumblebee parameters were done separately
for the three assessment phases: pre-exposure period, exposure period,
and the final assessment using (generalized) linear mixed-effects models
((G)LMMs; for parameters with multiple observations per colony) with
colony identity as a random factor or generalized linear models (GLMs;
for colony-level parameters in the final assessment). Square
root-transformed flower density was analysed in a single LMM for both
the pre-exposure and exposure period with colony identity as a random
factor and a three-way interaction (including two-way interactions and
main effects) between resource (categories: buckwheat, purple tansy,
floral mix), spray treatment (categories: control and Amistar) and a
quadratic term (including the linear term) for day as fixed effects.
The colony that was replaced on day -3 was excluded from the data
analyses (but its replacement was included). During the exposure period,
the foundress queens of eight colonies died; these queen losses were
quite balanced between resources and spray treatments (1-2 queen losses
per resource-treatment combination; Table S3). Data collected after the
death of the queen were excluded from analyses of the exposure period
and only data of colonies whose queens lived throughout the exposure
period were considered in the final assessment. For analyses of
individual-level measures, bees that showed signs of disease were
excluded (parasitism, necrosis, or deformed wings); these were however
only few (Table S3). In addition, three adult workers were excluded from
analyses of body mass as body parts had fallen off. Sample sizes of all
endpoints and analyses are listed in Table S4.
All analyses were conducted in R version 3.6.3. Colony weight, body
mass, intertegular distance, and flower density were analysed using LMMs
fitted with the function lmer of the lme4 package (Bates et al.2015). All GLMMs were fitted with the glmmTMB function/package (Brookset al. 2017). Number of dead adults was analysed using GLMMs with
a Poisson distribution. For number of living adults, GLMMs with a
quasi-Poisson distribution (specified as nbinom1) were used for the
pre-exposure period, to account for overdispersion, and GLMMs with
Poisson distribution were used for the exposure period. The final number
of cocoons and adult males and workers were analysed using GLMs with a
negative binomial distribution to account for overdispersion using the
glm.nb function of the MASS package (Venables & Ripley 2002). Models
with a quasi-Poisson or negative binomial distribution had an ln-link
function. (G)LMMs were fit with maximum likelihood during model
selection and with restricted maximum likelihood when selected models
were evaluated.
Models for the pre-exposure period contained an interaction (including
main effects) between resource and day (continuous variable) as fixed
effects. For the exposure period, models contained a three-way
interaction (including all two-way interactions and main effects)
between resource, spray treatment, and day as fixed effects. Pupal body
mass was fitted using an LMM containing a three-way interaction
(including all two-way interactions and main effects) between resource,
spray treatment, and developmental stage (continuous variable). All
other models on the final assessment contained an interaction (including
main effects) between resource and spray treatment as fixed effects.
In all of these models, an interaction between flower density and
resource (including main effects) was included if a likelihood ratio
test showed P <0.05 and the root-mean-square error
decreased. For the pre-exposure and exposure period, flower density was
an interpolated variable across assessment days, whereas in the final
assessment the mean of all flower density assessments during the period
where colonies were encaged was used. Both of these variables were
centered to mean = 0 and standardised to standard deviation = 1.
Flower density was interpolated using the approx function of the stats
package and data from all flower density assessments. However, in models
on number of living adults or number of dead adults, on day ‑8, flower
density was assumed to be the mean interpolated flower density of all
cages on day -7, as on day ‑8 colonies were assessed in the laboratory
(and therefore not exposed to flowers). For colony weight, the
assessment period started with the first field assessment on day -5
(after pollen and nectar supplies were removed).
Models were evaluated by calculating estimated marginal means (EMMs)
using the emmeans (for simple/main effects) and emtrends (for slopes)
functions of the emmeans package. A Tukey post-hoc correction was
applied when analysing differences between any pair of resources. In the
pre-exposure period, straightforward comparisons between resources were
made (pairwise~ resource). In the exposure period and
final assessment, differences between resources and spray treatments
were determined relative to the other of these two factors
(pairwise~resource|spray treatment or
pairwise~spray treatment|resource). To avoid
confounding effects with spray treatment, resource effects in the
exposure period and final assessment are only reported for the control
group. To compare differences between spray treatments (over time),
Amistar and control cages of the same resource were compared
(pairwise~spray treatment|resource) using
emmeans (main effects) or emtrends (interaction with day).
To determine effects on colony weight change over the pre-exposure
period (days -5 to -1) or the exposure period (days 0 to 10), regression
slopes were compared. For the estimation of effect sizes (and their
confidence intervals), models for colony weight were refit with a
different time variable (instead of day), where a unit equals the length
of the regarded assessment period. As number of living adults and number
of dead adults were modelled on the ln-scale, slope coefficients were
less meaningful, and therefore back-transformed model estimates on day
-1 (for the pre-exposure period) and day 10 (for the exposure period)
were compared for the estimation of effect sizes and their confidence
intervals.
Results
Impact of flowering
resources
To avoid confounding effects with spray treatment, resource effects were
assessed by comparing colonies from untreated cages (i.e. any in the
pre-exposure period and control cages in later assessments). While there
were no differences between purple tansy and floral mix colonies,
monofloral buckwheat adversely impacted several parameters in comparison
to monofloral purple tansy and/or the floral mix. In the pre-exposure
period, buckwheat colonies showed higher mortality than purple tansy or
floral mix colonies with 3.7 (i.e. >200%) more dead adults
(Fig. 2, Fig. S2). Buckwheat colonies ended this period with 9.8 (i.e.
23.6%) fewer living adults than purple tansy colonies and decreased
21.4 g (i.e. 22.5%) in weight (P <0.001), while floral
mix colonies maintained a stable weight (P =0.57, Fig. S2;
difference in weight change: 23.3 g; Fig. S2)). Colony weight declined
also in purple tansy (-9.2 g, P =0.039), but no difference to the
other resources was determined (P >0.1). Buckwheat
colonies ended the exposure period with over 25 (i.e. 30%) fewer living
adults than colonies of the other two resources, despite no difference
in number of dead adults (Fig. 2). In addition, buckwheat colonies
continued to lose weight (15.3 g i.e. 19%) in the exposure period
(P =0.04), while purple tansy and floral mix colonies gained 58.8
g (i.e. 50%) and 32.4 g (i.e. 28%), respectively
(P <0.001). At the end of the experiment, buckwheat
colonies had over 150 (i.e. 86%) fewer cocoons than purple tansy or
floral mix colonies (Fig. 2) and 53.0 (i.e. 57%) fewer adult workers
than purple tansy colonies (Fig. 2).
Impact of Amistar
exposure
Amistar negatively affected bumblebee colonies in purple tansy cages,
but no effects were found in buckwheat or floral mix colonies (Fig. 3).
Colonies exposed to Amistar through purple tansy gained 22.5 g less
weight compared to control colonies (Fig. 3, Fig. S2). At the end of the
experiment, colonies exposed to
Amistar through treated purple
tansy had 51.5 (i.e. 55%) fewer adult workers, 7.0 (i.e. 88%) fewer
adult males and a by 21 mg (i.e. 14%)
reduced body mass of adult workers
(Fig. 3, Fig. S3). Amistar had no apparent effect on the shape of the
distribution of worker body mass but shifted the mean so that
Amistar-exposed colonies in purple tansy tended to have more light and
fewer heavy workers than control colonies (Fig. S4).
Residue analysis confirmed that foragers of the Amistar group were
exposed to azoxystrobin during the exposure period (Table 1).
Quantifiable levels of azoxystrobin were also found in foragers of the
buckwheat control group but these were 76% lower than the azoxystrobin
concentration detected in foragers of Amistar-treated buckwheat cages.
Flower density and its
impact
Flower density in all three resources exhibited a non-linear growth
pattern (quadratic term: P <0.001) with an increase at
the beginning of the experiment and a decline starting within the
exposure period (Fig. 4). Already at the start of the experiment (day =
-7), the floral mix had a higher flower density than the other two
resources (P <0.001), and all three resources developed
differently over time (differences in linear terms:P >0.07, differences in quadratic terms:P <0.009).
In contrast, flower density did not differ between cages assigned to
different spray treatments at the start of the experiment (day -7;P >0.35 in all resources). In addition, spray
treatments did not differ in the change of flower density over time
(differences in linear and quadratic terms between spray treatments in
any resource: P >0.23).
Flower density in purple tansy cages positively affected colony weight
in both the pre-exposure and the exposure period (Table S5). Flower
density in the floral mix positively affected colony weight in the
exposure period and negatively affected final number of cocoons (Table
S5).
Discussion
Our semi-field experiment reveals that flowering resource type can
strongly impact B. terrestris colonies directly and by modulating
the effect of the azoxystrobin-based fungicide Amistar.
We find that overall fitness and
fungicide tolerance are promoted by different plant resources and thatB. terrestris require therefore access to diverse resources.
As hypothesized, colonies confined with untreated buckwheat developed
less well than colonies confined with a floral mix or monocultural
purple tansy. Although buckwheat
is an excellent nectar source, similar to purple tansy (Knauer et
al. unpublished data; Petanidou 2003; Cawoy et al. 2009), its
pollen is considered of relatively low quality due to its low protein
content (11%) compared to purple tansy (30%) and the other abundant
species of the floral mix (field mustard: 22%, cornflower: 23%)
(Pernal & Currie 2000; Somerville 2001; Baloglu & Gurel 2015; Radev
2018). High protein diets foster bumblebee development (Baloglu & Gurel
2015; Kämper et al. 2016; Roger et al. 2017) and
immunocompetence (Roger et al. 2017). The availability of diverse
flowers should benefit bees through a more balanced diet (Di Pasqualeet al. 2013; Dance et al. 2017; Filipiak et al.2017; Leza et al. 2018) and allow bumblebees, which are
particularly selective and exhibit a preference for protein-rich pollen
(Hanley et al. 2008; Leonhardt & Blüthgen 2012; Ruedenaueret al. 2015; Vaudo et al. 2015), to select resources of
high nutritional value. Although buckwheat flowers provide about 95%
less pollen than purple tansy flowers (Knauer et al. unpublished
data) and floral mix cages had a higher flower density, the adverse
impacts of buckwheat seem not to be driven by a lack of flowers. Flower
density in buckwheat did not affect any bumblebee parameter while flower
density in purple tansy and the floral mix affected colony weight gain
and number of cocoons.
Amistar applied on purple tansy
monocultures negatively affected B. terrestris colonies as
manifested by reduced population size, production of males, and body
mass of adult workers. Amistar may cause these effects by impairing
foraging behaviour and metabolism. Azoxystrobin acts on fungi by
inhibiting mitochondrial respiration and consequently energy supply.
This effect is, however, not limited to fungi, as it was also found in
fish (Olsvik et al. 2010). In honeybees, azoxystrobin altered the
expression of genes involved in energy generation and hormonal
regulation, which may disrupt the development of bees and impair their
foraging efficiency (Christen et al. 2019). Indeed, Amistar can
reduce the foraging rate of B. terrestris (Tamburini et al.
2021), damage their guts and cause a decline in sucrose consumption,
weight gain, and consequently survival rate (Straw & Brown 2021).
Our study shows for the first time that the effects of a fungicide on
bees are modified by flowering resources, as only colonies foraging
exclusively on purple tansy were impacted. The absence of Amistar
effects on colonies in floral mix cages aligns with the hypothesis that
floral diversity can mitigate pesticide effects (Wahl & Ulm 1983; Klauset al. 2021). However, contrary to our expectations, we found no
Amistar effects in colonies feeding exclusively on buckwheat. We
detected azoxystrobin residues in foragers from untreated buckwheat
cages, but do not think that these explain the absence of Amistar
effects in buckwheat colonies. The contamination likely occurred during
the handling of bee samples on the same table. Furthermore, azoxystrobin
levels were substantially higher in treated than in untreated cages in
all resources with the absolute difference being largest in buckwheat.
Differences in flower morphology
may explain the diverging results. Purple tansy corollae are narrower
and deeper compared to buckwheat (Vattala et al. 2006), which
impedes access and resulted in noticeably longer durations that
bumblebees spent on single flowers. Hence, foraging on buckwheat was
perhaps not chllenging enough for potential effects on foraging ability
to translate into reduced body size and population growth.
Amistar may also have affected bees by attacking specific microorganisms
present on purple tansy. Plant species differ in the microbial
(including yeast and other fungal) communities they harbour, which bees
can acquire through foraging and feeding (Manirajan et al. 2016;
McFrederick & Rehan 2019). Beneficial microorganisms can alter the
durability of nectar and pollen, increase plant attractiveness to
pollinators, protect bees from pathogens and promote detoxification
(Koch & Schmid-Hempel 2011; Herrera et al. 2013; Pozo et
al. 2015; Zheng et al. 2016; Vollet-Neto et al. 2017;
Raymann & Moran 2018; Leonard et al. 2020). Pesticides can
impact microorganisms found on floral resources or within bee guts. For
instance, azoxystrobin reduced yeast growth in nectar with potential
implications for nectar chemistry and attractiveness (Bartlewiczet al. 2016).
Lastly, pollen protein (or its ratio to other nutrients) may affect
fitness and development differently than fungicide tolerance.
Although earlier studies suggested
that a high pollen protein content can decrease pesticide sensitivity
(Wahl & Ulm 1983; Archer et al. 2014), recent studies did not
find such an effect in bumblebees (Barraud et al. 2020) or even
an increase in pesticide sensitivity in honeybees feeding on pollen with
a high protein-to-lipid ratio (Crone & Grozinger 2021).
Our semi-field experiment indicates that the risk of Amistar, which is
characterized as ‘non-hazardous to bees’, should be re-evaluated.
Interestingly, we found now in two semi-field experiments that Amistar
applied on purple tansy can affect B. terrestris , while we found
no effects in a similar study on honeybees (Tamburini et al.2021a, b). This indicates that B. terrestris may be more
sensitive to Amistar than honeybees, which was previously also found for
other pesticides (Cresswell et al. 2014; Rundlöf et al.2015; Wintermantel et al. 2018; Osterman et al. 2019). The
active ingredient azoxystrobin was first approved at a time when risk
assessments on pollinators were exclusively done with honeybees and we
are unaware of any regulatory testing of azoxystrobin on bumblebees that
was done since then. Besides, the guidance document on the risk
assessment of plant protection products in the EU that includes testing
on bumblebees was never fully implemented due to the opposition of some
member states (EFSA 2013; More et al. 2021). In addition, a
recent study indicates that surfactants (alcohol ethoxylates) found in
Amistar (and other products) rather than the active ingredient
(azoxystrobin) damage bumblebee guts and increase their mortality (Straw
& Brown 2021). The risk of such co-formulants is, however, not assessed
in regulatory pesticide risk assessments of the EU (EFSA 2013).
Our study has shown that the effects of a pesticide on bees can depend
on the forage plants it is applied on. This has potential implications
for the regulatory assessment of pesticides. The European food safety
authority recommends that exposure to an active ingredient is evaluated
in multiple crops, but requests impacts to be tested only in a single
plant species (and only if lower-tier testing indicated a potential
risk) although the active ingredient is to be registered for a range of
crops (EFSA 2013). However, in our experiment, differences in effects
between plant species/mixture did not seem to be driven by differences
in exposure levels but rather by nutrition or plant morphology.
Therefore, resource differences should arguably be considered more
strongly in the risk assessment of pesticides on bees.
Our findings call for further research to evaluate the role that
different resources play in mitigating pesticide effects and to identify
how plant morphology as well as pollen macro- and micro-nutrient
contents, influence the fitness and pesticide resilience of bumblebees
and other pollinators. Agricultural landscapes may then be modified
accordingly, for instance by cultivating flower strips containing
beneficial species, to limit pesticide effects on pollinating insects.
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