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548 | NATURE | VOL 528 | 24/31 DECEMBER 2015
© 2015 Macmillan Publishers Limited. All rights reserved
LETTER
doi:10.1038/nature16167
Neonicotinoid pesticide exposure impairs crop
pollination services provided by bumblebees
Dara A. Stanley
1
, Michael P. D. Garratt
2
, Jennifer B. Wickens
2
, Victoria J. Wickens
2
, Simon G. Potts
2
& Nigel E. Raine
1,3
Recent concern over global pollinator declines has led to
considerable research on the effects of pesticides on bees
1–5
.
Although pesticides are typically not encountered at lethal levels
in the field, there is growing evidence indicating that exposure to
field-realistic levels can have sublethal effects on bees, affecting
their foraging behaviour
1,6,7
, homing ability
8,9
and reproductive
success
2,5
. Bees are essential for the pollination of a wide variety of
crops and the majority of wild flowering plants
10–12
, but until now
research on pesticide effects has been limited to direct effects on
bees themselves and not on the pollination services they provide.
Here we show the first evidence to our knowledge that pesticide
exposure can reduce the pollination services bumblebees deliver to
apples, a crop of global economic importance. Bumblebee colonies
exposed to a neonicotinoid pesticide provided lower visitation rates
to apple trees and collected pollen less often. Most importantly,
these pesticide-exposed colonies produced apples containing fewer
seeds, demonstrating a reduced delivery of pollination services. Our
results also indicate that reduced pollination service delivery is not
due to pesticide-induced changes in individual bee behaviour, but
most likely due to effects at the colony level. These findings show
that pesticide exposure can impair the ability of bees to provide
pollination services, with important implications for both the
sustained delivery of stable crop yields and the functioning of
natural ecosystems.
Biotic pollination is required by a large proportion of crops world-
wide
10
, disproportionately including those with economically high
values and nutritional content
13
. The contribution of pollination services
to global agriculture has been steadily increasing and was estimated at
US$361 billion in 2009 (ref. 14). In addition, animal-vectored pol-
lination is required by an estimated 87.5% of all angiosperms to
reproduce
11
, making this process fundamental to the functioning of
natural ecosystems. Therefore, any threats to the delivery of pollina-
tion services could have serious consequences for both food security
and wider ecosystem function. Neonicotinoid pesticides, the most
widely used group of insecticides worldwide
15
, are implicated as one
of the contributing factors in the global declines of bee pollinators
3,16
.
Although previous work has shown that bumblebee foraging activity,
colony growth and reproduction can be altered by sublethal exposure
to neonicotinoid pesticides
1,2,5–7
, all research on pesticide effects has
focused on bees as the service providers, but has not assessed the polli-
nation service itself. Therefore it is unknown whether pesticide exposure
actually results in changes to the delivery of pollination services to crops
and wild plants (for a discussion of potential mechanisms see ref. 17).
This information is essential to assess the severity of pesticide effects on
ecosystem services, and to inform actions to mitigate negative effects.
Apples are an important global crop, with 75 million tonnes har-
vested from 95 countries in 2012 and an estimated export value of
US$71 billion (Food and Agriculture Organisation statistics, http://
faostat3.fao.org). Apple crops benefit from insect pollination with
seed number, fruit set, fruit size and shape all improved with increased
pollination services
18
. Bumblebees are major pollinators of apples
19
and many other crops across the world
12
, and are exposed to low levels
of pesticides when foraging in agricultural areas. Here we investigated
how exposure to low, field-realistic levels of a widely used neonicoti-
noid insecticide (thiamethoxam) could affect the ability of bumblebees
to pollinate apple trees. We pre-exposed colonies to 2.4 parts per billion
(ppb) thiamethoxam, 10 ppb thiamethoxam or control solutions (con-
taining no pesticide; rationale for selecting pesticide concentrations
and relevance of results are outlined in Methods and Supplementary
Information) in their nectar source (artificial sugar water) for a period
of 13 days (8 colonies per treatment, that is, 24 colonies in total).
Subsequently, colonies were brought to the field and allowed access to
virgin apple trees of a dessert (Scrumptious) variety, along with trees
of a polliniser (Everest) variety, in pollinator exclusion cages in which
we observed both individual- and colony-level behaviour. At the end of
the season, apples from tested trees were collected to assess pollination
service delivery in terms of fruit and seed set.
When whole colonies were given access to apple trees we found
an effect of insecticide treatment on visitation rates to apple flowers
(F
2,86
= 3.1, P = 0.05); colonies exposed to 10 ppb pesticide provided
lower visitation rates to apple flowers than controls (Fig. 1a; Extended
Data Table 1). We also found an effect of treatment on the number of
foraging trips from which bees returned carrying pollen (χ
2
= 9.65,
degrees of freedom (df) = 2, P = 0.008), with fewer bees from colo-
nies exposed to 10 ppb pesticide returning with pollen than work-
ers from control colonies (Fig. 1b). Apple abortion rate was affected
by treatment (χ
2
= 5.94, df = 2, P = 0.05), with trees pollinated by
1
School of Biological Sciences, Royal Holloway University of London, Egham TW20 0EX, UK.
2
Centre for Agri-Environmental Research, School of Agriculture, Policy and Development, University of
Reading, Reading RG6 6AR, UK.
3
School of Environmental Sciences, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Control 2.4 ppb 10 ppb
Treatment
Visitation rate per patch
(no. visits per ower per min)
0.00
0.02
0.04
0.06
0.08
0.10
No. bees carrying pollen
0
2
4
6
8
10
12
Control 2.4 ppb 10 ppb
Treatment
NS
NS
NS
NS
**
ab
Figure 1 | Effects of pesticide treatment on colony-level behaviour.
a, b, Visitation rates provided by colonies to Scrumptious apple flowers
(number of visits per flower per minute) (a) and number of foraging trips
from which bees returned carrying pollen (b), from colonies exposed to
different pesticide treatments. Eight colonies were observed per treatment
group, and means ± s.e.m. are shown, *P < 0.05. NS, not significant.
Results from statistical models are given in Extended Data Table 1.
24/31 DECEMBER 2015 | VOL 528 | NATURE | 549
LETTER
RESEARCH
© 2015 Macmillan Publishers Limited. All rights reserved
2.4 ppb pesticide-exposed colonies aborting more fruit than controls
(Fig. 2a), although overall levels of fruit set did not differ (χ
2
= 4.1,
df = 2, P = 0.13) and there was no difference in the proportion of trees
that produced fruit among treatments (χ
2
= 1.2, df = 2, P = 0.55).
However, we found a significant effect of treatment on the number
of seeds produced per apple, an indicator of fruit quality, (χ
2
= 8.27,
df = 2, P = 0.02); flowers pollinated by colonies exposed to 10 ppb pesti
-
cide produced significantly fewer seeds than those pollinated by 2.4 ppb
colonies (Fig. 2b). These results show that colonies exposed to pesticide
can deliver reduced pollination services to apple crops.
These colony-level effects could be explained by several mecha-
nisms, including individual behavioural changes. Individual bees
exposed to 10 ppb pesticide spent longer foraging (F
2,57
= 3.72, P = 0.03;
Fig. 3a), visited more Scrumptious flowers (χ
2
= 12.79, df = 2,
P = 0.002) and switched more frequently between varieties dur
-
ing each trip (χ
2
= 11.32, df = 2, P = 0.003: Fig. 3b; Extended Data
Table 2), which suggests a modification of their floral preferences
7
.
Neonicotinoids target neurotransmitter receptors in insects and, as
well as causing neuronal inactivation
20
, some have been shown to be
partial neuronal agonists
21
; therefore increases in individual foraging
activity may be explained by acute increases in neuronal activity caus-
ing hormesis (a biphasic response in which low levels of an otherwise
toxic compound can result in stimulation of a biological process
22
).
However, we found no effect of treatment on whether flowers visited
by these individual bees produced apples (χ
2
= 0.88, df = 2, P = 0.64),
showed higher rates of fruit abortion (χ
2
= 0.42, df = 2, P = 0.81) or
different levels of seed set (χ
2
= 0.11, df = 2, P = 0.95). This suggests
that bees exposed to pesticide must somehow be behaving differently
on flowers, in a way that was not readily observable in our experiment
(for example, changes in stigmatic contact
23
), such that increased visit
frequency did not result in better pollination service delivery at the
individual level.
Our results suggest that effects on pollination service delivery are
not due to individual behavioural modification, but instead are most
likely due to changes in colony activity levels as evidenced by reduced
floral visitation rates and pollen collection. Bees collecting pollen may
be more effective pollinators as they can deposit more pollen on plant
stigmas
24
; therefore if pesticide-exposed colonies are collecting less
pollen they are also likely to be depositing less on stigmas than bees
from control colonies. While individual bees exposed to pesticides
visited more flowers, overall pesticide-exposed colonies provided lower
visitation rates and collected less pollen, thus explaining why reduced
pollination services were delivered. Gill & Raine
7
found that control
(untreated) bees improved their pollen foraging performance over time,
whereas imidacloprid-treated bees became less successful foragers;
foragers in our colony-level experiment may have carried out multiple
trips and become more experienced foragers, potentially explaining
why we find effects on pollen collection here but not in the individual-
level experiment. Interestingly, for almost all parameters measured in
this study we found significant effects on both individual behaviour
and colony-level function following 10 ppb thiamethoxam exposure,
but not at the 2.4 ppb level. This suggests that there are dose-dependent
effects that lie between these two exposure levels. Both these exposure
levels are highly relevant as they are within the range measured in the
field, but further work is necessary to elucidate the lowest level at which
these effects become significant (for further discussion of rationale for
exposure and relevance of results, see Methods and Supplementary
Information).
A 36% reduction in the number of seeds produced in apples polli-
nated by colonies exposed to 10 ppb pesticide in comparison to control
colonies has important agronomic implications for crop production.
The number of seeds in apples is closely linked to fruit crop quality in
most, but not all, varieties
18,25
and the enhancement of fruit quality,
particularly the proportion of Class 1 fruit, underpins the economic
value of UK orchards
26
: growers must typically thin out their apple
crops making the quality of each fruit very important. Therefore
impacts on seed set and fruit quality have direct implications for apple
production value, and as seed set and fruit set are positively linked
in many varieties, reduced seed set can have direct negative implica-
tions for fruit set and total crop yield
26,27
. As certain apple varieties in
the UK currently experience pollination deficits
19,26
, mitigating the
effects of pesticides on bumblebee pollinators could improve polli-
nation service delivery. Apple crops are visited by a wide variety of
pollinator groups, and neonicotinoid pesticides differentially affect
insect taxa
4,28
. Apart from bumblebees, one of the other main polli-
nator groups that visit apple flowers are solitary bees
19
, and it has been
suggested that pesticide sensitivity of solitary bees is likely to be higher
than for larger, social species like bumblebees
4,5,17,29
. Therefore, apple
pollination in a field setting could be more vulnerable to pesticide
exposure than measured here.
Bumblebees are essential pollinators of many important crops other
than apples, including field beans, berries, tomatoes and oilseed rape
12,26
.
If exposure to pesticides alters pollination services to apple crops, it
is likely that these other bee-pollinated crops would also be affected.
Most importantly, the majority of wild plant species benefit from insect
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Proportion of fruit set
May
Control
2.4 ppb
10 ppb
Control 2.4 ppb 10 ppb
Treatment
No. seeds produced per apple
0
1
2
3
4
5
6
September
†
ab
*
NS
Figure 2 | Effects of pesticide treatment on fruit and seed set.
a, b, The change in proportion of fruit set for trees (48 trees in total, 16 per
treatment) pollinated by colonies exposed to different pesticide treatments
measured early (May) and late (September), which represents fruit
abortion level (a), and number of seeds produced per apple (134 apples in
total; 53 in control, 46 in 2.4 ppb and 35 in 10 ppb pesticide treatments)
pollinated by colonies exposed to different pesticide treatments (b). Eight
colonies were observed per treatment group, and means ± s.e.m. are
shown, *P < 0.05, † indicates a difference of P = 0.06 between control and
10 ppb. NS, not significant. Results from statistical models are given in
Extended Data Table 1.
Control 2.4 ppb 10 ppb
Treatment
Length of time spent foraging (s)
0
500
1,000
1,500
2,000
2,500
No. switches between varieties
0
1
2
3
4
5
6
†
Control 2.4 ppb 10 ppb
Treatment
NS
NS
NS
NS
*
*
ab
Figure 3 | Effects of pesticide treatment on individual bee behaviour.
a, b, Time spent foraging per foraging trip (seconds; n = 68 bees) (a) and
number of switches between Scrumptious and Everest apple varieties
(n = 93 bees) (b) for individual bees exposed to different pesticide
treatments. Means ± s.e.m. are shown, *P < 0.05, † indicates a difference
of P = 0.06 between control and 2.4 ppb. NS, not significant. Results from
statistical models are given in Extended Data Table 2.
550 | NATURE | VOL 528 | 24/31 DECEMBER 2015
LETTER
RESEARCH
© 2015 Macmillan Publishers Limited. All rights reserved
pollination services
11
. Therefore reduced pollination by pesticide-
affected colonies, as evidenced by reduced seed set, also has significant
implications for pollination in wild systems. Many wild plant species
are both self-incompatible and pollen limited
30
, so any reduction in the
delivery of pollination services could have substantial effects on wild
plant communities and therefore wider ecosystem function.
Concerns over global bee declines are strongly driven by the need for
the essential pollination services they provide to both crops and wild
plants. The use of neonicotinoid pesticides presents a potential threat to
bee health and, although the evidence base reporting sublethal (behav-
ioural) effects of pesticides on bees is mounting
3
, we have shown for the
first time that there is also an important effect of pesticide exposure on
the pollination services bees provide. This information provides a new
perspective when trying to fully understand the trade-offs involved
when using insecticides, showing that both the potential benefits and
the true costs of pest control options need to be considered.
Online Content Methods, along with any additional Extended Data display items and
Source Data, are available in the online version of the paper; references unique to
these sections appear only in the online paper.
Received 8 July; accepted 23 October 2015.
Published online 18 November 2015.
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Supplementary Information is available in the online version of the paper.
Acknowledgements We thank technicians at the University of Reading for
assistance in apple collection and seed counting, and E. van Leeuwen and
colleagues at Royal Holloway University of London for useful discussions. This
study was supported by UK Insect Pollinators Initiative grants BB/I000178/1
awarded to N.E.R. and BB/1000348/1 awarded to S.G.P. (funded jointly by the
Living with Environmental Change programme, Biotechnology and Biological
Sciences Research Council (BBSRC), Wellcome Trust, Scottish Government,
Department for Environment, Food and Rural Affairs (Defra) and Natural
Environment Research Council (NERC)). N.E.R. is supported as the Rebanks
Family Chair in Pollinator Conservation by The W. Garfield Weston Foundation.
Author Contributions D.A.S. and N.E.R. conceived the project, D.A.S., N.E.R. and
M.P.D.G. designed the research, D.A.S., J.B.W and V.J.W. carried out the research,
D.A.S., N.E.R., M.P.D.G. and S.G.P. contributed equipment for the research, D.A.S.
analysed the data, all authors were involved in writing the manuscript.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial
interests. Readers are welcome to comment on the online version of the
paper. Correspondence and requests for materials should be addressed to
D.A.S. (darastanley@gmail.com) or N.E.R. (nraine@uoguelph.ca).
LETTER
RESEARCH
© 2015 Macmillan Publishers Limited. All rights reserved
METHODS
Pesticide preparation. A stock pesticide solution was made by dissolving 100 mg
thiamethoxam (PESTANAL, Analytical Standard, Sigma Aldrich) in 100 ml
acetone (1 mg ml
−1
). Aliquots of stock solution were added to 40% sucrose to
create treatment solutions of 10 μg l
−1
(10 ppb) and 2.4 μg l
−1
(2.4 ppb) thiameth-
oxam. These concentrations were chosen as field-realistic; the lower concentration
(2.4 ppb) was based on thiamethoxam concentrations found in nectar pots of
bumblebee colonies foraging in agricultural areas in the UK
31
and in pollen
collected by honeybees
32
, and the higher concentration (10 ppb) is within the range
measured in pollen and nectar and of a variety of treated crops
33–35
and contam-
inated wild flowers
35–37
, and has been used in previous studies examining effects
of another neonicotinoid (imidacloprid) on bumblebee behaviour
1,7
. A control
solution was also made by repeating the process outlined above but using an aliquot
of 10 ppb acetone only (that is, no pesticide).
Experimental setup. Twenty-four commercially reared Bombus terrestris audax
colonies were obtained from Biobest (Westerlo, Belgium) at the start of April 2014,
each containing a queen and an average of 99 workers (range 57–133).
Colonies were weighed on arrival to estimate the overall colony size, and each
assigned sequentially to one of three treatment groups (2.4 ppb thiamethoxam,
10 ppb thiamethoxam and control) based on decreasing mass (but randomly
assigned within block). Each day, three colonies (one from each treatment)
were assigned to treatment groups, until after 7 days all colonies were receiv-
ing treated sucrose (16 colonies exposed to thiamethoxam and 8 to control
solution). We chose this sequential exposure regime to mimic subsequent
field testing and ensure all colonies had comparable durations of exposure
to their treatment. Colonies were fed treated sucrose solution from a gravity
feeder inserted at the base of the nest box. Feeders were initially refilled every
2–3 days, and then every 1–2 days when the colonies had grown significantly.
Untreated, defrosted honeybee-collected pollen was provided to colonies every
2–3 days. Colonies were exposed to treatments for an average of 13 days (range
12–15) before field testing. Before being moved to the field, colonies had access
to a feeder containing sucrose (40%) in a laboratory flight arena for 48 h to
become accustomed to leaving the nest to forage. There was no difference in
colony weights at the start (ANOVA: F
2,21
= 0.091, P = 0.91) or end (ANOVA:
F
2,21
= 0.88, P = 0.43) of the experimental period, indicating no treatment effect
on colony size.
Field testing. Cage experiments were carried out at Sonning Farm, University
of Reading, UK. 100 apple trees of a commercial dessert apple (Scrumptious
variety) were moved into holding pollinator exclusion cages in mid-March 2014
before flowering to prevent insect visitation. Field experiments began when
trees were entering full flower in mid-April. Each day, one colony from each
treatment was taken from the laboratory, placed individually in one of the
three test cages and observed simultaneously (with one observer per cage) in
a randomized block design (see below for details of observations). Each day a
different treatment was assigned to each observer. Cages were 4.8 × 2.1 × 2.1 m
frames covered in polyethylene mesh (gauge size = 1.33 mm, Extended Data
Fig. 1). Observations were carried out on 8 dry, bright days from 16–26
April 2014 spanning the peak flowering of apples (daily means: maximum
temperature 16 °C, rainfall 2.5 mm). This flowering period limited the number of
days on which testing could be carried out, and therefore the number of colonies
that could be tested; as a result no statistical methods were used to predetermine
sample size. The investigators were not blinded to allocation during experiments
and outcome assessment.
Individual-level measurements. Each morning, three cages were pop-
ulated with two virgin Scrumptious trees each from the holding cages
(mean ± s.e.m. = 130 ± 8.5 flowers per tree) as well as two polliniser trees (Everest
variety, mean ± s.e.m. = 305 ± 15 flowers per tree, Extended Data Fig. 1). The
number of flowers of each variety was standardized across cages to ensure equal
floral density each day, and 40 open and receptive flowers were marked with
cable ties on each Scrumptious tree for subsequent estimation of pollination ser-
vices (fewer flowers were marked on the last day of observations as there were
no longer 40 full-bloom flowers—flower numbers on these days were noted).
The nest boxes in each cage were then opened to allow a single worker to exit.
This bee was observed for the duration of its foraging trip (until it attempted
to return to the nest), or until 60 min had elapsed (Extended Data Fig. 2). The
duration of the foraging trip, the number of flowers of each apple variety visited,
and the handling time for each flower visit was recorded using Etholog software
(EthoLog: Behavioural observation transcription tool, University of Sao Paulo,
Brazil, 2011). If the individual bee did not visit any flowers within the first 20 min,
it was assumed not to be a forager and was captured, returned to the colony and
another bee released. All bees that foraged were paint-marked before they were
returned to the colony to ensure the same individuals were not observed twice.
This process was repeated until all cages had the same number of active foragers
recorded (3–5 bees per colony each day). Individual level observations took place
between 10:00 and 16:30.
Colony-level measurements. After individual-level observations, the two focal
Scrumptious trees in each cage were removed and replaced with two new virgin
trees. Again we standardized the number of flowers of each variety across cages
with 40 open and receptive flowers on each tree marked with cable ties. Colony
boxes were opened to allow free entry and exit to all active bees for a period of
60 min. This time period was chosen to avoid over-pollination of test flowers based
on pilot observations. Colony activity was monitored at the nest entrance using
video cameras. After an initial 10-min period to allow the bees to become accus-
tomed to the setup, four 10-min focal observations were carried out on separate
patches of Scrumptious flowers in each cage to estimate visitation rates. At the
end of the 60-min period, the Scrumptious trees were immediately removed to
prevent further visitation. Colony level observations were carried out between
14:30 and 18:30.
Estimation of pollination services. At the end of both the individual and colony
observation periods, all test trees were returned to holding cages in which they
were not visited by any other insects until apples were harvested at the end of the
season. An initial assessment of fruit set from marked flowers (indicating flowers
open during cage tests) was made at the end of May for all test Scrumptious trees to
assess how many flowers were proceeding to fruit set stage (and how many aborted,
Fig. 2a). Marked apples were collected on 27 August, and a final assessment made of
the proportion of marked flowers that had produced mature fruit (Extended Data
Fig. 2). In the lab, seed number was counted per apple for all collected fruit (274
apples from 96 trees across both experiments). Details of all data analyses carried
out are given in the supplementary information.
Data analysis. Individual level. Measures of the number of flowers visited,
numbers of switches between apple varieties, duration of total time in cage
(from when the bee left the colony box until it returned/end of 60 min period)
and time taken to visit the first flower (latency) were recorded for all indi-
vidual bees. For 68 of 93 bees observed (evenly distributed across cages and
treatments) a number of additional response variables were also recorded
including mean duration of the first 5 flower visits, number of inter flower
intervals longer than 60 s, mean duration of flower visits, mean period of time
between flower visits, length of time spent foraging (time between first and last
flower visit) and total time spent on flowers (sum of durations for all individual
flower visits). We tested for differences in these measures among treatments by
constructing mixed-effects models with pesticide treatment as a fixed effect.
As several variables differed among days, including weather, floral abundance
and the identity of colonies used, day of testing was included as a random
blocking factor in all models. Data were analysed in R version 3.1.0 (ref. 38),
using either linear mixed effects (LME) models with the lmer function in the
nlme package for continuous data
39
, generalized mixed effects (GLMM) models
with Poisson distribution used for response variables that were counts using the
glmer function in the lme4 package
40
, or the glmmPQL function in the MASS
package
41
when data were overdispersed. Models were validated by plotting
standardized residuals versus fitted values, normal qq-plots and histograms of
residuals, and continuous response variables were logarithmically transformed
(log (X + 1)) if necessary to improve residual fit. If treatment was significant,
Tukey’s post hoc tests were performed using the glht function in the multcomp
package
42
.
To assess differences in apple production on trees visited by pesticide exposed
and control bees, we examined a number of variables including the number
of fruits produced at the start of the season (May) and at the end (September;
Fig. 2a), the change in proportion of apples forming from marked flowers per tree
between the start and end measures (fruit abortion levels) and number of seeds
per apple (measured in early September; Fig. 2b). Models were run as described
previously with treatment as a fixed effect, although the tree on which fruits
were produced, the number of bees released and date of testing were included
as random effects. As a number of trees produced no fruit, seed set data were
analysed in two steps. First, we tested whether there was a treatment difference
in the number of trees that produced any fruit. Second, we tested for treatment
differences in seeds per apple (a measure that only included trees that had
produced some fruit).
Colony level. We tested for differences in colony activity levels (the combined
number of entries and exits by workers to the colony box) and the number
of bees carrying pollen among treatments using GLMM models in the MASS
package
41
, with Poisson distribution for count data. Treatment differences in
flower visitation rate to Scrumptious trees were tested using LME models
39
. Date
of testing was used as a random effect in all models (and patch included as a
random effect in the flower visitation rate model), and models were validated as
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described above. Fruit abortion and seed set variables were analysed as described
for the individual level experiment, using tree and date of testing as random
effects.
31. Thompson, H. et al. Eects of neonicotinoid seed treatments on bumble bee
colonies under eld conditions. (Food and Environment Research Agency
(FERA), 2013).
32. Pilling, E., Campbell, P., Coulson, M., Ruddle, N. & Tornier, I. A four-year eld
program investigating long-term eects of repeated exposure of honey bee
colonies to owering crops treated with thiamethoxam. PLoS One 8, e77193
(2013).
33. Castle, S. J., Byrne, F. J., Bi, J. L. & Toscano, N. C. Spatial and temporal
distribution of imidacloprid and thiamethoxam in citrus and impact on
Homalodisca coagulata populations. Pest Manag. Sci. 61, 75–84 (2005).
34. Dively, G. P. & Kamel, A. Insecticide residues in pollen and nectar of a cucurbit
crop and their potential exposure to pollinators. J. Agric. Food Chem. 60,
4449–4456 (2012).
35. Botías, C. et al. Neonicotinoid residues in wildowers, a potential route of
chronic exposure for bees. Environ. Sci. Technol. 9, 12731–12740 (2015).
36. Krupke, C. H., Hunt, G. J., Eitzer, B. D., Andino, G. & Given, K. Multiple routes of
pesticide exposure for honey bees living near agricultural elds. PLoS One 7,
e29268 (2012).
37. Stewart, S. D. et al. Potential exposure of pollinators to neonicotinoid
insecticides from the use of insecticide seed treatments in the
mid-southern United States. Environ. Sci. Technol. 48, 9762–9769
(2014).
38. R Development Core Team. R: A language and environment for statistical
computing. R Foundation for Statistical Computing, Vienna, Austria.
http://www.R-project.org (2011).
39. Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D. & R Development Core Team.
Package “nlme”: Linear and nonlinear mixed eects models. R package
version 3.1-104 (2012).
40. Bates, D., Maechler, M., Bolker, B. & Walker, S. lme4: Linear mixed-eects
models using Eigen and S4. R package version 1.1-7 http://CRAN.R-project.
org/package= lme4 (2014).
41. Venables, W. N. & Ripley, B. D. Modern Applied Statistics with S. 4th edn
(Springer, 2002).
42. Hothorn, T., Bretz, F. & Westfall, P. Simultaneous inference in general
parametric models. Biom. J. 50, 346–363 (2008).
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Extended Data Figure 1 | An example of the experimental setup at the Sonning Farm field site. Experimental pollinator exclusion cages containing a
bumblebee colony (located in the corner of the cage) and potted experimental apple trees are shown. Photos: D.A.S.
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Extended Data Figure 2 | An experimental bumblebee (Bombus terrestris) worker visiting an apple flower (left), and an example of an apple
produced from a marked (yellow cable tie) apple flower (right; Scrumptious variety). Photos: D.A.S. and C. L. Truslove.
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Extended Data Table 1 | Results from the colony-level experiment
Signicant dierences (P ≤ 0.05) are highlighted in bold.
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Extended Data Table 2 | Results from the individual-level experiment
Signicance dierences (P ≤ 0.05) are highlighted in bold.
