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OPEN Neonicotinoid pesticide limits

improvement in buzz pollination by
bumblebees
Received: 18 May 2017 P. R. Whitehorn1,2, C. Wallace1 & M. Vallejo-Marin1
Accepted: 13 October 2017
Published: xx xx xxxx Neonicotinoid pesticides have been linked to global declines of beneficial insects such as bumblebees.
Exposure to trace levels of these chemicals causes sub-lethal effects, such as reduced learning and
foraging efficiency. Complex behaviours may be particularly vulnerable to the neurotoxic effects of
neonicotinoids. Such behaviours may include buzz pollination (sonication), in which pollinators, usually
bees, use innate and learned behaviours to generate high-frequency vibrations to release pollen from
flowers with specialised anther morphologies. This study assesses the effect of field-realistic, chronic
exposure to the widely-used neonicotinoid thiamethoxam on the development of sonication buzz
characteristics over time, as well as the collection of pollen from buzz-pollinated flowers. We found that
the pollen collection of exposed bees improved less with increasing experience than that of unexposed
bees, with exposed bees collecting between 47% and 56% less pollen by the end of 10 trials. We also
found evidence of two distinct strategies for maximising pollen collection: (1) extensions to the duration
of individual buzzes and (2) extensions of the overall time spent buzzing. We find new complexities in
buzz pollination, and conclude that the impacts of field-realistic exposure to a neonicotinoid pesticide
may seriously compromise this important ecosystem service.

Understanding the interactions between plants and their insect pollinators is of great practical and politi-
cal relevance, being vital to efforts to ensure food security under rapid global change1. Increased production
is often achieved through the deployment of pesticides but this can compromise sustainability by impacting
beneficial insect pollinators. This conflict is exemplified by the controversy over neonicotinoid insecticides2,3.
Neonicotinoids have become the most widely used insecticides in the world4 but their use is causing widespread
concern, with evidence linking them to the decline of beneficial species such as bumblebees5,6. Beneficial insects
can be exposed to these insecticides when they forage on flowering crops, as well as wild plants growing on agri-
cultural land7–9. This exposure to trace levels of neonicotinoids in nectar and pollen causes a range of sub-lethal
effects in bees, such as reduced foraging efficiency10–12, impaired navigation13, a reduction in learning and
memory14 and reduced reproductive success15,16. These impacts may have substantial impacts at the population
level5,6,17 and therefore adversely affect the plants that rely on insects for pollination18.
A type of pollination that may be particularly sensitive to impairment in learning and memory, due to expo-
sure to pesticides, is buzz pollination19. Buzz pollination is a relatively complex biotic interaction, in which pol-
linators, usually bees, interact with flowers with specialised anther morphologies that require high frequency
vibrations to release pollen20–24. Buzz pollination has evolved independently many times25 and now occurs in
approximately 20,000 plant species, including some of the world’s most important crops, such as tomatoes and
potatoes22. During buzz-pollination, bees grab the anthers with their mandibles, curl their body around the
anther cone and then, decoupling their wings from the indirect flight mechanism, they rapidly contract their
thoracic muscles, which produces a vibration without the wings beating21,26,27. These vibrations, also called buzzes
or sonications due to the audible sound they incidentally produce, are transmitted from the bee’s body to the
anthers, causing the pollen grains to be released on to the bee, where they can be collected24. Previous work has
shown that sonication has both innate and learned components28–30. These studies showed that naïve foragers
are able to effectively sonicate on their first visit to a flower, showing the innateness of the behaviour. However,
after a number of visits, the characteristics of the buzzes change. For instance, Morgan et al.29 found that the peak

1
School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, UK. 2Karlsruhe Institute of Technology (KIT),
Institute of Meteorology and Climate Research–Atmospheric Environmental Research (IMK-IFU), 82467, Garmisch-
Partenkirchen, Germany. Correspondence and requests for materials should be addressed to P.R.W. (email: p.
[email protected])

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frequency of sonication declined with experience, and Russell et al.30 established that the length and amplitude of
buzzes increased with experience, suggesting a learned component. It is important to note that neither of these
previous studies determined experimentally whether change in sonication characteristics during learning affect
the quantity of pollen that bees can remove from flowers. Therefore, to date, we do not know whether sonication
learning is associated with increased pollen collection. The challenge of manipulating morphologically complex
flowers through the deployment of a multifaceted behaviour potentially makes buzz pollination particularly sen-
sitive to neurotoxic pesticides, such as neonicotinoids.
We know very little about how the ability of bees to buzz-pollinate flowers may be affected by pesticide expo-
sure. In the only study on this to date, Switzer and Combes19 looked at buzz pollination behaviour before and
after an acute dose of the neonicotinoid imidacloprid. The authors found that the lowest dose of 0.0515 ng did not
result in any quantitative changes in the frequency and length of sonication buzzes of Bombus impatiens workers.
Unfortunately, too few bees exposed to the higher doses (0.515 and 5.15 ng) resumed foraging, and it was there-
fore not possible to assess how these concentrations impact buzz pollination. Furthermore, the quantity of pollen
collected by bees in the different treatments was not assessed. Therefore, the effects of field-realistic, chronic
exposure to neonicotinoids on buzz pollination remains to be determined. The present study explores this highly
topical subject and aims to determine the effect of the widely-used neonicotinoid thiamethoxam on (1) the
characteristics of sonication buzzes and changes over time and (2) the collection of pollen from buzz-pollinated
flowers.

Materials and Methods

Two commercial Bombus terrestris audax colonies were obtained from Biobest (Belgium) via Agralan Ltd
(Swindon, UK); the first on 8th June 2016 and the second on 20th July 2016. The experiment was carried out with
bumblebees from the first colony and then repeated with bumblebees from the second colony. On arrival the
majority of workers from the colony were removed and randomly split into three groups, ensuring an approx-
imately equal distribution of worker sizes in each group. These groups of workers were then placed in separate
plastic nest boxes (27 cm × 25 cm × 14 cm) where they established queenless microcolonies. Each microcolony
was supplied with either control nectar or nectar contaminated with the neonicotinoid thiamethoxam at two
concentrations. All groups were provided ad libitum with untreated pollen (Biobest via Agralan Ltd).

Thiamethoxam dilutions.  An initial stock solution (105 µg thiamethoxam L−1) was made by diluting 10 mg
pure thiamethoxam (Sigma-aldrich, UK) in 100 ml acetone. This was further diluted with purified water to 104 µg
thiamethoxam L−1. Aliquots of the diluted stock solution were added to sucrose solution (Biogluc, Biobest via
Agralan ltd) to create concentrations of 2 parts per billion (ppb) and 10 ppb. An equivalent volume of acetone in
purified water was added to the control sucrose solution. The concentrations used were chosen to reflect the range
of values found in the nectar and pollen of crop and wild plants in the field7,9,31,32.

Pre-treatment and training.  The microcolonies were exposed to the treated or control nectar for nine days
before the buzz pollination trials began. During this period, the microcolonies were connected to a flight arena for
training to encourage workers to leave their nest boxes to forage. The flight arena was a 100 cm × 60 cm × 35 cm
wooden box with a Perspex lid. The microcolonies were connected one at a time, each for a total of 14 hours.
During this period the arena contained training flowers (non-poricidal Chrysanthemum sp.).

Buzz pollination trials.  After the training period, the micro-colonies were connected to the flight arena on
a rotational basis, continuously for five weeks (each colony was connected for approximately four hours in either
the morning or the afternoon and all three colonies were sequentially connected in a 1.5 day period, allowing
each colony to alternate between morning and afternoon sessions). When a microcolony was connected, bees
were individually allowed into the flight arena for a ten minute period; a sliding metal door in the connecting
tube was used to control bee entry to the arena. A microphone recorder (Zoom H4n Handy Recorder) was set up
next to a mesh-covered window in the side of the arena and the entire ten minute session was recorded. Before
a bee entered the arena, three Solanum rostratum (Solanaceae) flowers were attached to a vertical stick, which
was placed next to the mesh window, exactly 5 cm from the recorder. After a bee’s first visit to the arena, she was
caught and tagged with a numbered, coloured disc (Opalith, Christian Graze KG, Germany).
Numbered bees were allowed to visit again until they had successfully completed ten arena visits in each
of which they buzz pollinated one or more flowers, after which they were not allowed to return. Typically, in a
successful foraging bout, an individual bee buzz pollinated all three flowers multiple times. If a bee successfully
foraged from the flowers the pollen from her left rear leg was collected at the end of the arena session and stored
in a 1.5 ml Eppendorf tube with 1 ml of 70% ethanol. This pollen was later counted using a haemocytometer – the
number of grains in 0.1 µl was counted and the number of grains in this aliquot was used for statistical analysis.
Three new flowers were then placed in the arena for the next foraging visit.

Sound analysis.  The sound data were analysed using the software Audacity 2.1.2 (www.audacityteam.org).
For each trial, a high pass filter with a roll-off of 12 dB per octave and a cut-off frequency of 100 Hz was used to
reduce background noise. Spectrograms were used to identify the peak frequency (Hz) and corresponding ampli-
tude (dB) for the first five clear sonication buzzes and the first five clear flight buzzes in each trial. A sonication
buzz was defined as a sonication made on the anther of the flower, including all buzz sounds with less than one
second pause between them. Buzz sounds that occurred after more than a one second pause (or were separated by
a flight buzz) were classified as separate sonication buzzes. The number and duration of sonication buzzes during
the first two minutes of sonication were also recorded, from which we calculated the duration of buzzing per

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Total no. Mean & range (min-max) No. bees completing No. bees
Colony Treatment foragers trials completed 10 trials completing > 4 trials.
1 Control 17 4.9 (1–10) 7 7
1 2ppb 13 5.9 (1–10) 7 7
1 10ppb 12 5.5 (1–10) 6 6
2 Control 9 8.2 (1–10 6 8
2 2ppb 10 7.8 (2–10) 6 8
2 10ppb 11 7.6 (1–10) 8 8

Table 1.  Summary of the sample size of workers from each colony.

Colony 1 Colony 2
Parameter Lower Upper Parameter Lower Upper
Pollen collection Estimate 95% C.I. 95% C.I. Estimate 95% C.I. 95% C.I.
Intercept 2.280 1.989 2.571 2.824 2.598 3.050
2ppb 0.724 0.319 1.129 −0.286 −0.623 0.051
Treatment
10ppb 0.563 0.156 0.970 0.633 0.292 0.974
Trial 0.119 0.100 0.139 0.089 0.074 0.104
2ppb −0.101 −0.127 −0.075 −0.019 −0.043 0.005
Treatment x trial
10ppb −0.124 −0.150 −0.098 −0.127 −0.150 −0.104

Table 2.  Parameter estimates and 95% Confidence Intervals (CIs) from the Poisson GLMM for the number of
pollen grains in 0.1 µl aliquot. The parameter estimates shown here are with reference to the control treatment
group and are shown in the log link scale.

minute for each trial. This was then multiplied by the total time the bee spent visiting (the total time in the arena
minus the time to when the bee first sonicated) to arrive at an index of buzzing effort. This gives an estimate of the
time spent sonicating during the trial.

Statistical analysis.  Data were analysed in R, version 3.1.2. (2014 The R Foundation for Statistical
Computing) and were subsetted to only include bees that had completed four or more trials. Preliminary analyses
showed that the two colonies behaved differently and so were analysed separately. A generalized linear mixed
effect model with a Poisson distribution (with log link function) was used to analyse factors affecting the quantity
of pollen collected (i.e. the number of grains). The buzz effort index, buzz duration (Box-Cox transformed to
fulfil normality assumptions) and the peak frequency and amplitude of the pollination and flight buzzes were
analysed with linear mixed effect models. All models were run in the lme4 package33 and individual trial was the
unit of replication, with treatment (a factor with three levels), trial number (a covariate from 1 up to 10) and their
interaction as fixed effects and the individual bee IDs as a random effect. The significance of fixed effects and their
interactions were tested using likelihood ratio tests to compare models with and without the term of interest. 95%
CIs were calculated as +/−1.96*SE of the mean.

Data availability.  The datasets generated during the current study are available from the corresponding
author on reasonable request.

Results
A total of 72 bees were observed carrying out 463 arena visits. 44 of these bees carried out at least four arena visits
and were included in the analyses (Table 1).

Pollen collection.  Bees were observed to collect more pollen with increasing experience, but this ability was
disrupted by exposure to thiamethoxam (trial by treatment interaction in colony 1: χ22 = 93.43, p < 0.001; colony
2: χ22 = 131.12, p < 0.001, Table 2, Fig. 1). By the tenth trial control bees were collecting an average of 95% and
126% more pollen than bees in the 2ppb group and 10ppb groups respectively (Fig. 1).

Buzzing effort.  Buzzing effort increased with experience and in Colony 1 there was no interaction with
treatment, meaning that we found no evidence for different rates of change of buzzing effort among the treatment
groups (χ22 = 1.33, p = 0.515). Furthermore, in this colony, the 10ppb group had a greater overall buzzing effort
than the control group (χ22 = 6.81, p = 0.03, Fig. 2, Table 3). In contrast, the trial by treatment interaction was
significant in Colony 2 (χ22 = 12.03, p = 0.002), with the 10ppb group decreasing their buzzing effort with expe-
rience in contrast to the control and 2ppb groups (Fig. 2). In this colony the control and 2ppb groups increased
their effort at similar rates but the 2ppb group had a lower overall buzzing effort (Table 3).

Buzz duration.  Mean buzz duration increased with experience and, in contrast to the result for buzzing
effort, there was an interaction between treatment and trial for Colony 1 (χ22 = 8.46, p = 0.015) but not for

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Figure 1.  Model predictions from the Poisson GLMM for pollen collected in the three treatments. Lines
represent mean values from model fits and shaded areas correspond to 95% confidence intervals.

Figure 2.  Model predictions from the linear mixed-effects model for buzzing effort across the three treatments.
Shaded areas correspond to 95% confidence intervals.

Colony 1 Colony 2
Parameter Lower Upper 95% Parameter Lower Upper
Buzzing effort Estimate 95% C.I. C.I. Estimate 95% C.I. 95% C.I.
Intercept 1.648 1.389 1.907 2.449 1.841 3.057
2ppb 0.267 −0.002 0.536 −1.342 −2.228 −0.456
Treatment
10ppb 0.340 0.076 0.604 0.731 −0.193 1.655
Trial 0.077 0.043 0.111 0.096 0.012 0.180
2ppb 0.017 −0.067 0.101 0.083 −0.037 0.203
Treatment x trial
10ppb −0.030 −0.114 0.054 −0.144 −0.269 −0.019

Table 3.  Parameter estimates and 95% CIs from the linear mixed effect models for buzzing effort. The
parameter estimates shown here are with reference to the control treatment group. Results in italics denote non-
significant relationships.

Colony 2 (χ22 = 0.79, p = 0.675). In Colony 1 the treated bees did not increase their mean buzz duration to the
same extent as the control bees but in Colony 2 all groups increased their buzz duration at similar rates (Fig. 3,
Table 4).

Acoustics of pollination and flight buzzes.  The peak frequency of the pollination buzzes declined with
increasing experience, but this occurred to a lesser extent in the 10ppb group of Colony 1 and in the 2ppb group
of Colony 2 (trial by treatment interaction in Colony 1: χ22 = 11.56, p = 0.003; Colony 2: χ22 = 9.88, p = 0.007,
Table 5, Fig. 4). The peak frequency of flight buzzes also declined over the course of the experiment, but to a
lesser extent (Fig. 4). In Colony 2, this decline in flight peak frequencies was dependent on treatment because the
10ppb group did not show a reduction over time (χ22 = 7.86, p = 0.02). Colony and treatment had no effect on

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Figure 3.  Model predictions from the linear mixed-effects model for mean buzz duration (Box-Cox
transformed) across the three treatments. Shaded areas correspond to 95% confidence intervals.

Colony 1 Colony 2
Parameter Lower Upper Parameter Lower Upper 95%
Buzz duration Estimate 95% C.I. 95% C.I. Estimate 95% C.I. C.I.
Intercept 1.046 1.025 1.067 1.126 1.107 1.145
2ppb 0.031 0.001 0.061 −2.04E-04 −0.037 0.037
Treatment
10ppb 0.038 0.009 0.067 −0.001 −0.039 0.037
Trial 0.005 0.002 0.008 0.004 0.002 0.006
2ppb −0.004 −0.008 0.000 −9.42E-05 −0.005 0.006
Treatment x trial
10ppb −0.005 −0.009 −0.001 0.002 −0.003 0.007

Table 4.  Parameter estimates and 95% CIs from the linear mixed effect models for mean buzz duration. The
parameter estimates shown here are with reference to the control treatment group. Results in italics denote non-
significant relationships.

Colony 1 Colony 2
Parameter Lower Upper Parameter Lower Upper
Peak frequency Estimate 95% C.I. 95% C.I. Estimate 95% C.I. 95% C.I.
Intercept 346.18 337.57 354.79 344.11 335.64 352.58
2ppb 5.69 −6.50 17.88 3.81 −9.19 16.81
Treatment
10ppb −1.94 −14.12 10.24 12.46 −1.18 26.10
Trial −1.67 −2.13 −1.21 −2.04 −2.55 −1.53
2ppb 0.58 −0.08 1.24 1.10 0.33 1.88
Treatment x trial
10ppb 1.10 0.47 1.73 −0.07 −0.87 0.73

Table 5.  Parameter estimates and 95% CIs from the linear mixed effect models for pollination peak frequency.
The parameter estimates shown here are with reference to the control treatment group.

the amplitude (dB) of pollination buzzes (χ21 = 1.65, p = 0.200 & χ22 = 0.58, p = 0.748 respectively). The ampli-
tude of pollination buzzes did, however, significantly decline with increasing experience (χ21 = 13.81, p < 0.001).
The amplitude of the flight buzzes was also significantly affected by trial number, but in this case the amplitude
increased over time (χ21 = 10.39, p = 0.001). Again, colony and treatment had no effect on the amplitude of these
buzzes (χ21 = 0.81, p = 0.369 & χ22 = 2.22, p = 0.330 respectively).

Discussion
We found that exposure to field realistic doses of the neonicotinoid thiamethoxam negatively impacted buzz
pollination. Exposed bees showed less improvement in pollen collection with increasing experience than unex-
posed bees, and therefore collected 47% and 56% less pollen in the 2ppb and 10ppb groups respectively by the
end of 10 trials (Fig. 1). This finding supports previous studies that have observed a reduced foraging efficiency in
neonicotinoid exposed bees either by bringing back smaller pollen loads and/or foraging for pollen less often10–12.

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Figure 4.  Model predictions from the linear mixed-effects model for peak frequency of the pollination
and flight buzzes for both colonies across the three treatments. Shaded areas correspond to 95% confidence
intervals.

Improvement in pollen foraging performance over time has also been shown to be negatively impacted by expo-
sure to imidacloprid34. This reduction in efficiency is thought to be due to altered interactions between bees
and wildflowers35. Our study takes this research further and looks at both the interactions between bumblebees
and flowers and also at how these interactions change over time. This offers further explanation for the negative
impacts of neonicotinoids on foraging behaviour and pollination services.
Our findings suggest that the changes in bees’ abilities to buzz pollinate can arise in different ways, illumi-
nating the complex nature of this form of pollination. It is already known that the peak amplitude and duration
of sonication buzzes influences the amount of pollen released by S. rostratum flowers27, and that peak frequency
declines with increasing experience, perhaps as part of a strategy to conserve energy29. We also found that the peak
frequency of the pollination buzzes declined with experience but this was impacted by neonicotinoid exposure,
with exposed bees in the 10ppb group of Colony 1 and in the 2ppb group of Colony 2 not reducing the frequency
to the same extent. Further effects were, interestingly, colony-specific. Although both of the colonies used here
responded to thiamethoxan exposure similarly in terms of the pollen they collected (Fig. 1), they responded quite
differently in terms of buzz duration and effort (Figs 2 and 3). Mean buzz duration was impacted by treatment
in Colony 1, with the control bees increasing the duration of their buzzes with increasing experience more than
the treated bees (although this effect was marginal with the 2ppb group). This effect was not found in Colony 2,
where treatment instead impacted the overall buzzing effort, with the control bees showing either an overall
greater buzzing effort (than 2ppb-treated bees) or an increase in buzzing effort with experience (compared to a
decrease, as in the case of 10ppb-treated bees).
Our findings therefore suggest that pesticide exposure impairs bees’ ability to improve pollen collecting ability
as they gain experience. They may also indicate the existence of two distinct strategies for maximising pollen col-
lection, as exemplified by these colonies: (1) extensions to the duration of buzzes (Colony 1); and (2) extensions
of the overall time spent buzzing (Colony 2). Nevertheless, we found no indication that bees could overcome the
apparent impact of exposure on their peak buzzing frequency. It is known that large inter-colony differences exist
in bumblebees in life history traits such as colour preferences, learning and foraging performance36,37 but such
marked differences in pollination strategies have not been previously noted, and represent a clear priority for
further research.
Interestingly, in the early stages of our experiments the bees exposed to thiamethoxam appeared to make
greater foraging efforts, particularly in Colony 1 where both treated groups had stronger buzzing efforts and
longer buzz duration, ultimately collecting more pollen, in contrast to their performance at later stages. This is
similar to findings in other experiments where bees exposed to thiamethoxam showed increased flower visitation
rates14,35. This is thought to be a result of hormesis, where low doses of pesticide actually stimulate biological
processes38 and has previously shown that other neonicotinoids can slightly improve learning and memory in
honeybees39 and orientation behaviour in moths40. Our experiment has shown that although these effects might
be initially observed, they show no benefit over a period of time in this instance.
It is unclear whether the reduction in pollen-collecting abilities found here would directly impact the fitness
of the plant species that depend upon buzz pollination. However, reductions of this scale in the resources brought

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back to the colony by worker bees will stunt colony growth and impair the rearing of new queens (an effect of
neonicotinoids previously reported15). Such a reduction in fitness is likely to have a negative impact on popula-
tions over larger temporal and spatial scales. Indeed, Woodcock et al.5 found evidence of increased population
extinction rates in wild bees in response to neonicotinoid usage. This loss of pollinators is very likely to have
detrimental effects for the crops and wild flowers that so depend on them. As a result, better understanding of the
process of buzz pollination and its sensitivities to the impacts of neonicotinoid pesticides is an urgent require-
ment for sustainable agriculture.

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Acknowledgements
We would like to thank Rachael Kinnaird, Jane Devlin and Andrew Laing for experimental assistance and James
Weir for constructing the flight arena. PRW was funded by an Impact Research Fellowship held at the University
of Stirling.

Author Contributions
P.R.W. & M.V.-M. designed the experiment and wrote the manuscript, C.W. & P.R.W. carried out the experiment,
P.R.W. conducted the analysis and all authors contributed to drafts of the manuscript.

Additional Information
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ScIenTIfIc REPOrtS | 7: 15562 | DOI:10.1038/s41598-017-14660-x 8