[permaculture] Yale Environment 360: Popular Insecticide Reduces Queen Bees’ Ability To Lay Eggs, Study Finds

Lawrence London lfljvenaura at gmail.com
Sat Sep 10 15:28:23 EDT 2016


Sub-lethal effects of dietary neonicotinoid insecticide exposure on honey
bee queen fecundity and colony development

http://www.nature.com/articles/srep32108
Sub-lethal effects of dietary neonicotinoid insecticide exposure on honey
bee queen fecundity and colony development

   - Judy Wu-Smart <http://www.nature.com/articles/srep32108#auth-1>
   -  & Marla Spivak <http://www.nature.com/articles/srep32108#auth-2>


   - *Scientific Reports* *6*, Article number: 32108 (2016)
   - doi:10.1038/srep32108
   - Download Citation <http://www.nature.com/articles/srep32108.ris>
   -
      - Ecology <http://www.nature.com/subjects/ecology>
      - Environmental impact
      <http://www.nature.com/subjects/environmental-impact>

Received:16 March 2016Accepted:25 July 2016Published online:26 August 2016
Abstract

Many factors can negatively affect honey bee (*Apis mellifera* L.) health
including the pervasive use of systemic neonicotinoid insecticides. Through
direct consumption of contaminated nectar and pollen from treated plants,
neonicotinoids can affect foraging, learning, and memory in worker bees.
Less well studied are the potential effects of neonicotinoids on queen
bees, which may be exposed indirectly through trophallaxis, or
food-sharing. To assess effects on queen productivity, small colonies of
different sizes (1500, 3000, and 7000 bees) were fed imidacloprid (0, 10,
20, 50, and 100 ppb) in syrup for three weeks. We found adverse effects of
imidacloprid on queens (egg-laying and locomotor activity), worker bees
(foraging and hygienic activities), and colony development (brood
production and pollen stores) in all treated colonies. Some effects were
less evident as colony size increased, suggesting that larger colony
populations may act as a buffer to pesticide exposure. This study is the
first to show adverse effects of imidacloprid on queen bee fecundity and
behavior and improves our understanding of how neonicotinoids may impair
short-term colony functioning. These data indicate that risk-mitigation
efforts should focus on reducing neonicotinoid exposure in the early spring
when colonies are smallest and queens are most vulnerable to exposure.
Introduction

Honey bees, *Apis mellifera* L., provide pollination services to over 150
different crops worldwide1 <http://www.nature.com/articles/srep32108#ref1>,2
<http://www.nature.com/articles/srep32108#ref2>. In recent years,
beekeepers in the US, Canada, and parts of Europe have experienced
unsustainably high colony losses3
<http://www.nature.com/articles/srep32108#ref3>,4
<http://www.nature.com/articles/srep32108#ref4>,5
<http://www.nature.com/articles/srep32108#ref5>,6
<http://www.nature.com/articles/srep32108#ref6> highlighting a serious
threat to global food security, agricultural productivity, and trade7
<http://www.nature.com/articles/srep32108#ref7>. A number of factors
contribute to managed bee losses, including: *Varroa* mites, bacterial and
viral infections, poor nutrition, migratory stress, queen failure, and
pesticides3 <http://www.nature.com/articles/srep32108#ref3>,8
<http://www.nature.com/articles/srep32108#ref8>,9
<http://www.nature.com/articles/srep32108#ref9>. Neonicotinoid insecticides
are a cause for concern due to their toxicity and pervasive use in
agricultural and urban areas worldwide10
<http://www.nature.com/articles/srep32108#ref10>,11
<http://www.nature.com/articles/srep32108#ref11>,12
<http://www.nature.com/articles/srep32108#ref12>,13
<http://www.nature.com/articles/srep32108#ref13>. Currently, there is heavy
scrutiny of and debate over the field relevance of laboratory-based
results, accuracy of field studies, determination of
environmentally-realistic exposure levels and relevant experimental
dosages, and the interpretation of reported adverse effects of
neonicotinoids on bees, other wildlife, and whole ecosystems11
<http://www.nature.com/articles/srep32108#ref11>,13
<http://www.nature.com/articles/srep32108#ref13>,14
<http://www.nature.com/articles/srep32108#ref14>,15
<http://www.nature.com/articles/srep32108#ref15>,16
<http://www.nature.com/articles/srep32108#ref16>,17
<http://www.nature.com/articles/srep32108#ref17>,18
<http://www.nature.com/articles/srep32108#ref18>.

Neonicotinoids are systemic broad-spectrum insecticides that target sucking
and chewing insect pests. These insecticides may translocate, at varying
concentrations, to all parts of treated plants including the nectar and
pollen. Bees may become unintentionally exposed through dust from seed
coatings created during planting and through foraging on contaminated
pollen, nectar, water, and sap exudates of treated plants19
<http://www.nature.com/articles/srep32108#ref19>,20
<http://www.nature.com/articles/srep32108#ref20>,21
<http://www.nature.com/articles/srep32108#ref21>. Neonicotinoids are
currently registered in over 120 countries and represent 24% (valued at US
$2.6 billion) of the global insecticide market as of 2008. Imidacloprid,
the first registered active ingredient within the neonicotinoid class is
considered “highly toxic” (LD50oral: 13 ng bee−1)8
<http://www.nature.com/articles/srep32108#ref8> to bees. In addition,
imidacloprid (valued at US $1.1 billion) represents 41.5% of the total
neonicotinoid market and is the largest selling insecticide in the world22
<http://www.nature.com/articles/srep32108#ref22>, rendering the potential
for exposure to bees high.

In this experiment, concentrations of imidacloprid fed to honey bee
colonies were based on plant residue studies and selected to simulate
potential exposure on foraging bees collecting contaminated and
uncontaminated nectar over a typical bloom period in nature. The lower
doses, 10 and 20 ppb, approximate residues that are characteristically
found in the nectar and pollen of agricultural crops, such as apples and
cucurbit vegetables, that are treated by soil-drench and foliar spray
applications following label rates23
<http://www.nature.com/articles/srep32108#ref23>,24
<http://www.nature.com/articles/srep32108#ref24>. However, 10 and 20 ppb
may be underestimates, as some crops such as cucurbits can have higher
residue levels (60–80 ppb in pollen) when neonicotinoids are applied via
drip irrigation, foliar spray, or through transplant water24
<http://www.nature.com/articles/srep32108#ref24>. The higher doses, 50 and
100 ppb, represent residues found in urban landscape plants such as
shadbush and rhododendron shrubs (*Amelanchier* spp., *Rhododendron* spp.)
and Cornelian cherry (*Cornus mas*), which are treated by soil-drench or
trunk injections and can express residues in the ppm range25
<http://www.nature.com/articles/srep32108#ref25>,26
<http://www.nature.com/articles/srep32108#ref26>,27
<http://www.nature.com/articles/srep32108#ref27>. Therefore, the
concentrations of imidacloprid treatments represent environmentally
relevant exposure rates for bees foraging in both rural and urban settings.

The body of knowledge on the effects of neonicotinoids is vast and includes
an increasing number of studies on sub-lethal effects, particularly on
neurophysiological and behavioral impairments28
<http://www.nature.com/articles/srep32108#ref28> including metabolic
changes to brain activity, impaired foraging and learning performance, and
motor functions in worker honey bees28
<http://www.nature.com/articles/srep32108#ref28>,29
<http://www.nature.com/articles/srep32108#ref29>,30
<http://www.nature.com/articles/srep32108#ref30>. Notably, little is known
about the effects of neonicotinoids on queen bees. One study has reported
negative effects of neonicotinoids on honey bee queen development and
mating success31 <http://www.nature.com/articles/srep32108#ref31>. Other
laboratory and field studies on bumble bees have shown that sub-lethal
exposure to neonicotinoids (imidacloprid, clothianidin, and thiamethoxam)
can reduce queen production and disrupt colony initiation16
<http://www.nature.com/articles/srep32108#ref16>,32
<http://www.nature.com/articles/srep32108#ref32>,33
<http://www.nature.com/articles/srep32108#ref33>,34
<http://www.nature.com/articles/srep32108#ref34>,35
<http://www.nature.com/articles/srep32108#ref35>,36
<http://www.nature.com/articles/srep32108#ref36>. Field studies examining
colony-level effects on honey bees also have reported higher queen failure
and supercedure rates when colonies were exposed to neonicotinoids35
<http://www.nature.com/articles/srep32108#ref35>,37
<http://www.nature.com/articles/srep32108#ref37>. In those studies the
behaviors of the exposed queen bees were not recorded. The queen bee is the
only individual in the colony that lays fertilized eggs that develop into
worker bees necessary for colony growth and survival. Therefore, it is
important to study the potential effects of neonicotinoids on this key
reproductive individual and the subsequent indirect effects on colony
development.
Results
Measurements during chronic exposure (day 1–22)

Imidacloprid dose (0, 10, 20, 50 and 100 ppb), colony size (1500, 3000 and
7000 bees), and exposure duration (1, 2, and 3 weeks) affected queen
behavior. Observed responses occurred within the first week of exposure and
effects were sustained throughout the experiment. There was no interaction
effect of time with dose and colony size. Time did not consistently or
significantly affect queen egg-laying behavior (F16,1053 = 0.93; p = 0.54)
or activity as measured by the distance travelled and duration of
immobility (F16,2153 = 1.31; p = 0.18 and F16,1213 = 1.65; p = 0.05,
respectively). In contrast, there was an interaction effect between dose
and colony size for queen egg-laying (F8,1053 = 6.17; p < 0.0001), distance
travelled (F8,2153 = 4.02; p < 0.0001), and duration of immobility (F8,1213
= 3.31; p < 0.001) (Fig. 1
<http://www.nature.com/articles/srep32108#f1>, Figure
S1 <http://www.nature.com/articles/srep32108#s1>). Therefore data were
pooled over the three-week exposure and separated by treatment dose and
colony size in the statistical analysis. Experimental replicates of colony
size and treatment dose were established at different times over the
season, but there were no effects of start date on any of the measures
across the three years (2012–2014).
*Figure 1: Average (SE) number of eggs laid by queens per 15 minute
observation period pooled over three week chronic exposure of imidacloprid
(IMD) (0, 10, 20 50, and 100 ppb) in 1500-, 3000-, and 7000-bee colonies
((dose*size*week) interaction: F16,1053 = 0.93; p = 0.54; (dose*size)
interaction: F8,1053 = 6.17; p < 0.0001).*
[image: Figure 1] <http://www.nature.com/articles/srep32108/figures/1>

Different letters denotes significant statistical differences among
treatment levels within each colony size at α < 0.05. Results indicate that
queens in untreated colonies laid significantly more eggs than queens in
treated colonies at all colony sizes.
Full size image <http://www.nature.com/articles/srep32108/figures/1>
Queen egg-laying

Initial worker populations in each colony were equalized by weight, and
imidacloprid treatments were randomly assigned. While natural colony
variation did occur, pre-treatment measurements showed no significant
differences in queen egg-laying rates among colonies of similar sizes (Table
S1 <http://www.nature.com/articles/srep32108#s1>).

Across colony sizes, there were significant differences in egg-laying
rates; as expected queens in 7000-bee colonies laid more eggs compared to
queens in colonies with fewer bees (F2,1053 = 85.37; p < 0.0001), but there
were no differences in egg-laying rate of queens within colonies of 1500
and 3000 bees (F2,1262 = 80.56; p = 0.6). Queens in untreated 1500-, 3000-,
and 7000-bee control colonies laid on average (SE) 6.5 ± 0.8, 6.0 ± 0.5 and
10.3 ± 0.6 eggs per 15-min observation, respectively, which were
significantly (35–65%) more eggs compared to eggs laid in most treated
colonies of the same colony sizes (F4,1053 = 106.91; p < 0.0001) (Fig. 1
<http://www.nature.com/articles/srep32108#f1>). The exceptions were in
3000-bee colonies treated at 10 ppb (2.1 ± 1.3) and 20 ppb (3.2 ± 0.4),
where egg-laying rates were not significantly different from rates in
control colonies of the same size, due to high variation in queen responses
particularly during the first two weeks of exposure (Table S1
<http://www.nature.com/articles/srep32108#s1>).
Queen activity and immobility

Queens in untreated colonies traversed greater distances per observation
compared to queens in treated colonies of the same size (F4,2153 = 4.53; p
= 0.0012). Queens in untreated colonies were also more active, as
determined by the proportion of time spent immobile per observation
(average ± SE 28% ± 0.04, 34% ± 0.03, and 13% ± 0.02), compared to queens
in 20, 50 and 100 ppb treated colonies (63% ± 0.1, 58% ± 0.06, and 37% ±
0.05) in 1500-, 3000-, and 7000-bee colonies, respectively. Queen
immobility showed greater dose-dependent responses as colony size
decreased: significant differences in queen immobility were observed among
treatments in 1500- and 3000-bee colonies (F4,1213 = 67.92; p < 0.0001) but
not among treated 7000-bee colonies (F4,1213 = 67.92; p > 0.73) (Figure S1
<http://www.nature.com/articles/srep32108#s1>).
Worker foraging behavior

Foraging activity was not recorded for colonies that contained 1500 bees.
There were relatively equal proportions of foragers entering and exiting
colonies within all treatment doses and colony sizes, but the total number
of foragers in the 7000-bee colonies was twice the number of foragers in
3000-bee colonies. There were significantly more workers from control
colonies observed entering (F4,837 = 50.00; p < 0.0001) and exiting the
colony (F4,837 = 44.46; p < 0.0001) compared to colonies that received
imidacloprid. Differences among treatments in foraging activity among
3000-bee colonies were dose-dependent while in the 7000-bee colonies there
were no differences in foraging rate among the treatments (Figure S2
<http://www.nature.com/articles/srep32108#s1>).
Worker hygienic behavior

In-hive activity was assessed by performing a freeze-killed brood test, an
assay for hygienic behavior, which is a behavioral mechanism of disease and
parasite resistance38 <http://www.nature.com/articles/srep32108#ref38>,39
<http://www.nature.com/articles/srep32108#ref39>, pre- and post-
imidacloprid treatment in the larger (7000-bee) colonies only. Before
imidacloprid treatment, the rate of hygienic removal of freeze-killed brood
was not significantly different among all colonies and averaged between
79.7% and 95.8% (F4,18 = 0.32; p = 0.86). After three weeks of imidacloprid
exposure, the colonies treated with 50 and 100 ppb imidacloprid displayed
significantly reduced hygienic removal with 63.3% ± 11.6 and 73.7% ± 9.7 of
the freeze-killed brood removed respectively, compared to 97.4% ± 1.9
(control), 80.8% ± 6.0 (10 ppb), and 97.2% ± 2.1 (20 ppb) (F4,18 = 4.50; p
= 0.011)(Fig. 2 <http://www.nature.com/articles/srep32108#f2>).
*Figure 2: Hygienic behavior assays or percent (SE) of freeze-killed brood
removed in 24 hours by worker honey bees from “7000-bee” colonies before
and after chronic imidacloprid exposure (dose: F4, 18 = 4.5; p = 0.01).*
[image: Figure 2] <http://www.nature.com/articles/srep32108/figures/2>

Different letters denotes significant statistical differences at α < 0.05
among treatment levels for post treatments only (no significant differences
found in pre-treatment assay (F4,18 = 0.3; p = 0.9). Results indicate
significantly lower hygienic behavior in higher treatments (50 & 100 ppb)
only. Hygienic behavior was used as a measure to assess worker activity
inside the hive, and is defined as the ability of worker bees to detect and
remove diseased and mite-infested brood thereby limiting within-colony
transmission of pathogens and parasites.
Full size image <http://www.nature.com/articles/srep32108/figures/2>
Measurements after chronic exposure (day 23)Adult bee population and brood
production

While adult bee populations changed over the course of the 3-week
experiment, the final population sizes were not significantly different
among treatment levels within each colony size (F4,55 = 1.42; p =
0.241) (Figure
S3 <http://www.nature.com/articles/srep32108#s1>). At the end of the
treatment period, the average (SE) number of eggs in small untreated
(1500-bee) colonies was significantly higher (901 ± 126) compared to eggs
in treated colonies (ranging from 222 ± 218 to 489 ± 117) of the same size
(F4,26 = 3.02; p = 0.0398). Imidacloprid-treated colonies that contained
1500 bees showed an inconsistent number of larvae and a decrease in the
number of pupae as dose increased, but differences were not statistically
significant (F4,26 = 2.18; p = 0.104 and F4,26 = 0.54; p ≥ 0.710,
respectively). In 3000-bee colonies, the number of eggs present was not
different between control and 10 ppb treated colonies of same size, but was
significantly higher in control colonies than all other treated colonies (F
4,27 = 4.05; p = 0.0125). The number of larvae in 3000-bee colonies was
only significantly different between control and 100 ppb treatments (F4,27
= 1.26; p = 0.31), and the number of pupae in control colonies was
significantly higher than all treated hives; the only differences in number
of pupae among the treated hives were observed between 20 and 100 ppb
treatments (F4,27 = 11.74; p < 0.0001). As colony size increased, no
significant differences in the number of eggs, larvae, and pupae were
observed among 7000-bee treated and control colonies (F4,23 = 1.57; p =
0.16, F4,23 = 0.71; p = 0.59, F4,23 = 1.62; p = 0.21, respectively) (Table 1
<http://www.nature.com/articles/srep32108#t1>). However, the ratio of adult
worker bees to pupae was dose-dependent and increased from 1.6 ± 0.5 in 10
ppb treated colonies to 4.6 ± 2.2 in 100 ppb treated colonies (untreated =
1.3 ± 0.2).
*Table 1: Least square means (±SE) of brood production at each
developmental stage, food stores (nectar and pollen) in comb cells, and
number of unused cells in “n” number of 1500-, 3000-, and 7000-bee colonies
exposed to imidacloprid (0, 10, 20, 50, 100 ppb) for three weeks.*
Full size table <http://www.nature.com/articles/srep32108/tables/1>
Food stores

There were no statistical differences among any of the colonies in the
number of cells containing stored nectar and honey (F4,115 = 1.70; p =
0.16). However untreated colonies significantly more cells containing
stored pollen (F4,115 = 22.65; p < 0.0001) than treated colonies, except in
1500-bee colonies treated at 10 ppb. The average (SE) number of cells
containing pollen in treated colonies were 61–71% (1500-bee colonies),
94–138% (3000-bee colonies), and 125–161% (7000-bee colonies) lower than
pollen stores found in untreated colonies at the same population size (Table
1 <http://www.nature.com/articles/srep32108#t1>). In addition, significant
differences among treatment levels became more prominent with colony size.
Unused cells

All colonies were started on plastic foundation imprinted with cell bases
on which bees built wax cells for brood and food storage. Unused comb,
defined as unused foundation (cell bases) or empty drawn wax cells, were
quantified to assess space as a limiting factor for colony development and
productivity. There was significantly less unused comb in untreated
3000-bee colonies than in treated colonies of the same size, but there were
fewer differences among the treated and control 1500-bee colonies and no
differences in unused comb among the treated and control 7000-bee colonies
(F4,115 = 11.72; p < 0.0001) (Table 1
<http://www.nature.com/articles/srep32108#t1>).
Brood pattern

Brood pattern (number of empty cells within a standard patch of sealed
pupal cells) is an indicator of queen status and or the quality of brood
care and health. The proportion of empty brood cells was significantly
different among treatments (F4,39 = 10.93; p < 0.0001). However, there was
no effect of colony size or interaction effects between dose and colony
size (F2,39 = 2.1; p = 0.14, F8,39 = 1.3; p = 0.29, respectively). The
overall average (SE) percentage of empty cells in control colonies was
10.2% ± 3.99, which was not significantly different from 10 ppb treated
colonies (22.2% ± 6.26) but was statistically lower than 20, 50 and 100 ppb
treated colonies (23.9% ± 3.86, 30.8% ± 4.6, 48.3% ± 4.7, respectively).
Among the treated hives, 10, 20 and 50 ppb were not different from each
other but were lower than 100 ppb treated hives (Fig. 3
<http://www.nature.com/articles/srep32108#f3>).
*Figure 3*
[image: Figure 3] <http://www.nature.com/articles/srep32108/figures/3>

Examples of brood patterns from colonies chronically exposed to
imidacloprid (0, 10, 20, 50 and 100 ppb) during brood rearing illustrating
a dose-dependent effect where the amount of empty cells in a given brood
area increases with treatment concentration (*A*); parallelogram containing
100 cells used to standardize brood pattern measures (*B*); and the average
percentage (SE) of cells not containing pupae (empty) in a brood area of
100 cells separated by colony size (1500, 3000, and 7000 bees) and
imidacloprid (IMD) dose (0, 10, 20, 50 and 100 ppb) (dose: F4, 39 = 10.9; p
< 0.0001; colony size: F2, 39 = 2.1; p = 0.14; interaction effect: F8, 39 =
1.3; p = 0.3). Greater % of empty cells indicates worse brood patterns and
overall brood health (*C*). Letters denote statistically significant
differences among treatment levels within each colony size at α < 0.05.
Results indicate significantly worse brood pattern (more empty cells),
particularly at higher treatments (50 and 100 ppb), compared to untreated
colonies.
Full size image <http://www.nature.com/articles/srep32108/figures/3>
Chemical residue analysis

Imidacloprid treatment syrup fed to colonies over three weeks was tested to
confirm dosage. No imidacloprid residues were found in untreated syrup. The
average residue level in syrup containing 10, 20, 50 or 100 ppb
imidacloprid was 6.4 ± 4.4, 32.9 ± 2.1, 57.7 ± 6.1 and 94.2 ± 5.2 ppb,
respectively (Table S2 <http://www.nature.com/articles/srep32108#s1>).
Adult worker bees, and stored nectar collected from outside forage sources
also were analyzed to estimate colony exposure. No residues were detected
in bees from untreated colonies of all colony sizes and in 7000-bee
colonies treated at 10 ppb. In contrast, imidacloprid was detected in bees
from all other treated colonies with a positive correlation between the
amount of residues detected and treatment dose within each colony size.
However, residue in bees treated with 100 ppb was significantly higher than
in bees from all other treatments (F4,36 = 9.06; p < 0.0001) (Figure S4
<http://www.nature.com/articles/srep32108#s1>). Imidacloprid residues
detected in stored nectar generally increased with treatment dose within
each colony size, however this was not significant (colony size: F2,34 =
1.52; p = 0.23). Low imidacloprid residues were detected in stored nectar
in one 1500-bee (2.2 ng/g) and one 3000-bee (1.8 ng/g) control colony,
possibly due to bees from these colonies robbing nectar from treated
colonies. Stored nectar from untreated colonies exhibited significantly
lower imidacloprid residues than from 50 and 100 ppb treated colonies and
stored nectar from10 and 20 ppb had significantly lower residues compared
to 100 ppb but not 50 ppb treated colonies (dose: F4, 34 = 12.68; p <
0.0001) (Figure S5 <http://www.nature.com/articles/srep32108#s1>).
Discussion

This study found adverse effects of imidacloprid on honey bee queen
behavior, worker bee activity, brood production, and pollen stores. Not all
responses presented in a dose-dependent or monotonic manner, which is
similar to other toxicological studies on neonicotinoid effects40
<http://www.nature.com/articles/srep32108#ref40>,41
<http://www.nature.com/articles/srep32108#ref41>. In general, effects were
less evident at lower doses in larger colonies likely due to the colony’s
ability to regulate resources fed to the queen and the greater number of
foragers collecting outside (untreated) resources that may dilute
imidacloprid levels, thus lessening potential effects. These findings
elucidate the complexity of quantifying exposure effects on highly social
honey bees and are in line with previous work42
<http://www.nature.com/articles/srep32108#ref42> suggesting that honey bees
are less susceptible or better at detoxifying neonicotinoids compared to
other bee species. Nonetheless our results indicate that small colonies may
not be capable of buffering agrochemical exposure and are therefore at
increased risk.

Environmentally relevant concentrations of imidacloprid, based on plant
residue studies, were fed to colonies. Feeding occurred every *other* day
(pulse exposure) over three weeks and the quantity of syrup fed was
proportional to the colony population, but was insufficient to sustain
colony development. Bees fully consumed or stored each treatment within 24
hours and all colonies were observed foraging for floral resources.
Eighteen of the 216 syrup treatments, mixed over three years, were randomly
tested for residues to provide an estimate of imidacloprid concentration
levels fed to bees. The average residue levels in treated syrup were close
to the intended dosage with the exception of 20 ppb, which was higher than
intended (32.9 ± 2.1 ppb), due either to mixing error during treatment
dilutions or sensitivity of analytical equipment and residue recovery rate
(112.6–119.8%) by testing facilities. The imidacloprid treatments reflect
residues found in the nectar and pollen of some treated agricultural crops
(10–30 ppb) and ornamental plants (50, 100 ppb). Notably, most studies on
seed-treated crops, such as soy, maize, and canola, reported residues at
<10 ppb in nectar and pollen43
<http://www.nature.com/articles/srep32108#ref43>,44
<http://www.nature.com/articles/srep32108#ref44>,45
<http://www.nature.com/articles/srep32108#ref45> thus the results of this
study are more relevant to colonies that are exposed to higher levels in
these or other crops. Regardless, there are examples in which seed-treated
maize, sunflower, and canola have yielded clothianidin residues >10 ppb in
pollen and or nectar45 <http://www.nature.com/articles/srep32108#ref45>,46
<http://www.nature.com/articles/srep32108#ref46>. In addition to collecting
potentially contaminated nectar and pollen sources, bees must collect water
for thermoregulation of the colony47
<http://www.nature.com/articles/srep32108#ref47>. Neonicotinoid
contamination in water puddles near seed-treated maize fields during
planting has been as high as 63.4 μg/L (thiamethoxam)21
<http://www.nature.com/articles/srep32108#ref21>. Other studies have found
guttation, or plant exudates derived from xylem collected by bees as a
water source48 <http://www.nature.com/articles/srep32108#ref48>, of
seed-treated maize can also exhibit high levels of neonicotinoids from >10
mg/L to 346 mg/L19 <http://www.nature.com/articles/srep32108#ref19>,20
<http://www.nature.com/articles/srep32108#ref20>. Another consideration in
estimating relevant exposure to bees is the uptake of neonicotinoids in
non-target wildflowers, such as dandelions (*Taraxacum officinale*) and
clovers (*Trifolium repens*, *Melilotus* spp.) that may exhibit residues
from <10 ppb (dandelions near seed-treated maize) to 89–319 ppb in clover
nectar when turfs were treated with clothianidin spray application32
<http://www.nature.com/articles/srep32108#ref32>,49
<http://www.nature.com/articles/srep32108#ref49>. These examples illustrate
the immense need for more residue data to better assess environmental
exposure and risk of systemic insecticides to bees.

We exposed colonies to imidacloprid and examined potential effects of
*indirect* imidacloprid exposure on queen behavior and colony development.
Neonicotinoids bind to nicotinic acetylcholine receptors (nAChRs) in the
central nervous system activating constant transmission of nerve signals,
an excitatory action that at low doses may cause hyperactivity but with
increasing concentration and exposure time can cause severe tremors or
paralysis in exposed bees as more nAChRs are bound50
<http://www.nature.com/articles/srep32108#ref50>. Queens in treated
colonies exhibited reduced fecundity likely due to imidacloprid acting
directly on sensory and motor functions of the central nervous system that
impacted egg-laying behavior and activity. A recent study, also suggests
that neonicotinoids can compromise the viability and quantity of stored
sperm in mated queens thereby further reducing queen success31
<http://www.nature.com/articles/srep32108#ref31>. In nature, queen bees are
indirectly exposed to environmental toxicants via trophallaxis when fed by
worker nurse bees. Trophallaxis in social insects, such as ants, can
attenuate toxicity of lethal toxicants particularly those that elicit
delayed-action toxicity, such as neonicotinoids, by evenly distributing
toxicants among nestmates and rendering them benign likely through dilution
by other uncontaminated food or bodily fluids already in the gut51
<http://www.nature.com/articles/srep32108#ref51>,52
<http://www.nature.com/articles/srep32108#ref52>. In honey bees, the same
mechanistic explanation may apply for the influence of population size on a
colony’s ability to buffer pesticide exposure and toxicity. Through
trophallaxis, queen bees and brood are fed royal jelly and brood food,
proteinaceous glandular secretions derived from fresh and stored pollen.
Stored pollen, or beebread, is eaten directly by nurse bees to stimulate
production of secretions from the mandibular and hypopharyngeal glands47
<http://www.nature.com/articles/srep32108#ref47>. The pathway by which
contaminated food reaches queens and brood through trophallaxis might
originate from the transfer of toxicants through the mandibular and
hypopharyngeal glands located in the heads of nurse bees. Although little
has been reported about neonicotinoid contamination in glandular
secretions, imidacloprid has been detected in products containing glandular
secretions such as brood food (>170 ppb acetamiprid and thiacloprid)53
<http://www.nature.com/articles/srep32108#ref53> and royal jelly (0.3–1
μg/kg) when bees were fed imidacloprid (100 ug/kg) in supplemental pollen
but not syrup35 <http://www.nature.com/articles/srep32108#ref35>.
Imidacloprid and highly toxic metabolites (olefin and 5-hydroxy
imidacloprid) have been detected in the heads of worker bees where the
glands are located after they were fed 14C-labeled imidacloprid in syrup54
<http://www.nature.com/articles/srep32108#ref54>.

In this study, indirect imidacloprid exposure through trophallaxis likely
resulted in a diluted or “filtered” exposure to queens and brood, but we
were unable to quantify the actual exposure levels. Individual queen bees
were tested for residues but no imidacloprid or metabolites were detected
possibly because individual queens (weighing < 1 g) provided insufficient
sample weight to obtain results. Another possibility is that only
metabolites were present in queen bees. Chemical analyses of metabolites
had limits of detection of 10 and 25 ppb for olefin and 5-OH imidacloprid,
respectively. Worker nurse bees attending to queens, or retinue bees, were
observed feeding and grooming queens in all colonies throughout the
experiment, indicating it is unlikely that reduced egg-laying was the
result of poor queen attendance but was rather due to some physiological
effect from exposure to imidacloprid and/or metabolites.

Chemical residue analysis of adult worker bees, and stored nectar or honey
collected from inside comb cells after the chronic exposure period provided
confirmation of imidacloprid exposure and contamination of food stores
within the colony. For each colony size, imidacloprid detection in worker
bees increased with treatment dose as expected. However, residue levels
were lower than the intended dose, particularly in 7000-bee colonies,
possibly due to greater numbers of foragers able to collect outside
(untreated) resources and social nestmate interactions (trophallaxis)
mediating or diluting exposure levels.

The combination of observed responses and chemical analysis indicates that
colony size was a significant factor in reducing the toxicity and degree of
affliction in treated colonies but only between the smaller (1500- and
3000-bees) and larger (7000-bees) colonies. Queens from 7000-bee colonies
exhibited more gradual and graded (dose-dependent) responses, laid twice as
many eggs and travelled greater distances per observation compared to the
smaller colonies of the same treatment further supporting the hypothesis
that imidacloprid concentrations were diluted within the larger colonies (Fig.
1 <http://www.nature.com/articles/srep32108#f1>). Few differences were
observed in egg-laying rates and inactivity of queens between 1500- and
3000-bee colonies indicating that the smaller two sizes were not very
different from each other. In epidemiological terms, social network
interactions that comprise organizational immunity55
<http://www.nature.com/articles/srep32108#ref55>,56
<http://www.nature.com/articles/srep32108#ref56> against pathogen
transmission may be extended to pesticide exposure and attenuation of
toxicity. Organizational immunity has the effect of isolating infected or
intoxicated individuals through reduced social interactions and spatial
segregation of diseased bees55
<http://www.nature.com/articles/srep32108#ref55>,56
<http://www.nature.com/articles/srep32108#ref56>. Though little is
understood about the triggers and mechanisms of organizational immunity,
the induction of detoxification through increased activity of enzymes in
individual honey bees is negatively correlated with population size57
<http://www.nature.com/articles/srep32108#ref57>. Thus small colonies may
rely more on social isolation and metabolic detoxification of older
foraging bees to avoid transmission of toxicants to younger hive bees that
are more sensitive to pesticides and have lower detoxification capacities58
<http://www.nature.com/articles/srep32108#ref58>. In contrast, larger
colonies have more workers that may bring back uncontaminated forage to
directly dilute collected toxicants via trophallaxis with nestmates. It may
thus be more advantageous for larger colonies to increase social
interactions to attenuate toxicants rather than rely on metabolic
detoxification that can be energetically costly, further reinforcing the
“buffering” capacity of population size to environmental toxicants,
although this hypothesis would require validation59
<http://www.nature.com/articles/srep32108#ref59>.

The adverse effects on queen behavior extended to colony level effects.
There was significantly less brood (eggs, larvae and pupae) and more highly
disrupted brood patterns observed in colonies after chronic exposure at all
doses and population sizes compared to untreated colonies. Brood production
is highly correlated with the population of brood-rearing nurse bees and
pollen foragers and thus is a good measure for grading colony health60
<http://www.nature.com/articles/srep32108#ref60>,61
<http://www.nature.com/articles/srep32108#ref61>. Brood pattern is also
used to assess the health of the developing brood and the queen. “Spotty”
or irregular brood patterns often indicate the presence of brood diseases,
a failing queen, poor brood care and or limited pollen60
<http://www.nature.com/articles/srep32108#ref60>,62
<http://www.nature.com/articles/srep32108#ref62>. Brood care (nursing
frequency and duration) of young larvae (<4 days) is strongly correlated
with the amount of pollen in the hive62
<http://www.nature.com/articles/srep32108#ref62>. During times of pollen
deficits, older larvae (>4 days) receive preferential feedings while
younger larvae are more likely to be cannibalized to compensate for the
protein shortage63 <http://www.nature.com/articles/srep32108#ref63>. In
this study, imidacloprid exposure had the strongest effect on the amount of
pollen stored in the hive, particularly in the larger (3000- and 7000-bee)
colonies likely because the amount of brood and the demand for pollen was
greater. Untreated colonies had on average (SE) 4.3% ± 0.34 of all cells
containing stored pollen, 6–17 times the amount compared to all treated
hives, which had <2% of all cells containing pollen (10 ppb: 0.9% ± 0.51;
20 ppb: 1.5% ± 0.32; 50 ppb: 0.6% ± 0.35; and 100 ppb: 0.5% ± 0.36).
Preferential cannibalism of young larvae due to pollen deficits may explain
the high variation observed in larvae compared to eggs and pupae among
treatments. Another explanation for lower amounts of brood in treated
colonies is the potential direct toxicity due to imidacloprid exposure via
contaminated brood food, which can alter the physiology and development of
larvae64 <http://www.nature.com/articles/srep32108#ref64>. The overall
effects on brood production and pattern in this study were likely caused by
a combination of factors, including effects on queen behavior, direct
toxicity from contaminated food, reduced brood care and lack of pollen, but
it is unclear which factors had the greatest impact on brood development.

A number of studies have demonstrated adverse effects of neonicotinoid
exposure on foraging behavior in bees12
<http://www.nature.com/articles/srep32108#ref12>,30
<http://www.nature.com/articles/srep32108#ref30>,65
<http://www.nature.com/articles/srep32108#ref65>,66
<http://www.nature.com/articles/srep32108#ref66>. In our study,
significantly lower foraging activity was observed in 3000- and 7000-bee
colonies exposed to neonicotinoids, regardless of dose. Given there were no
statistical differences in the initial and final worker bee populations it
is likely that low pollen stores in treated colonies (61–161% less than in
untreated colonies) was due to exposed bees being too intoxicated to forage
efficiently or not stimulated to forage at all. Even with similar
population sizes, the treated colonies were set back severely in brood
production and pollen stores compared to untreated colonies. Although the
colonies in our study were smaller than typical field colonies, colonies
containing 4500 and 9000 worker bees can produce more brood per adult bee
than colonies containing 17,000 and 35,000 bees62
<http://www.nature.com/articles/srep32108#ref62>. Therefore population size
was not a limiting factor for brood production and rearing capacity. Our
findings suggest that treated colonies may appear healthy (based on
population size) but may actually be performing poorly in normal colony
functions based on brood and pollen stores, which have long-term
consequences for colony survival and may be better indicators of colony
productivity (pollination services) and health61
<http://www.nature.com/articles/srep32108#ref61>. In addition, in-hive
activity (hygienic behavior) in 7000-bee colonies was disrupted at 50 and
100 ppb treatments. Worker bees with impaired hygienic behavior may have
been unable to detect dead brood or were motor-impaired and possibly
inactive, similar to foragers and queens in treated colonies. Impaired
hygienic performance could affect the colonies ability to prevent
within-colony and apiary transmission of pests and pathogens, potentially
making colonies exposed to neonicotinoids at high levels more susceptible
to robbing by other bees, disease, and parasites66
<http://www.nature.com/articles/srep32108#ref66>,67
<http://www.nature.com/articles/srep32108#ref67>,68
<http://www.nature.com/articles/srep32108#ref68>.

Interpretation of the environmental relevance of our findings and colony
fate may require additional studies on full-sized field colonies (≥30,000
workers) and longer observation periods to determine whether queens and
colonies can recover from short-term exposures. However, this study
highlights the importance of mitigating neonicotinoid exposure when honey
bee colonies are at low population sizes such as in early spring when
colonies are small due to normal winter losses or when surviving colonies
are divided by splitting the population among daughter colonies to prevent
swarming. In addition, commercially available “packages” containing small
populations from 7000–10000 worker bees are purchased early in spring to
replace dead colonies. Small colonies, as shown in our data, which are
unable to buffer or dilute neonicotinoid exposure are most vulnerable to
queen effects. Risk-mitigation options should focus on reducing exposure
risks when colonies are at their lowest population size due to season or
management practices, for example in the early spring when risk of exposure
to seed-treatment dust is at its highest during planting44
<http://www.nature.com/articles/srep32108#ref44>. This study provides a
mechanistic explanation for how sub-lethal effects of neonicotinoids may
impair short-term colony functioning, and offers insights into potential
effects of imidacloprid exposure on long-term colony survival10
<http://www.nature.com/articles/srep32108#ref10>. The results have
implications for promoting bee health because they offer a potential
explanation for queen failures, which have been identified as a precursor
to colony mortality in commercial beekeeping operations9
<http://www.nature.com/articles/srep32108#ref9>.
Methods
Experimental colonies and treatments

Worker bees and sister queens were removed from healthy field colonies
located in Chaska, MN to set up the experimental observation hives located
at the University of Minnesota campus in Saint Paul, MN (approximately 64
kms away). Field colonies, requeened with queen cells each time, were used
to stock observation hives multiple times each summer between May and
August yielding three or four replicate studies each year over three years
(2012–2014).

One, 2, and 5 frame observation hives were established with wooden
Langstroth-type deep frames and undrawn plastic foundation, and for each
replicate were given a laying queen and roughly 1500, 3000, or 7000 workers
estimated by weight of worker bees (0.5, 1, and 2 lbs., respectively, or
0.23, 0.45, and 0.91 kg). Colonies were provided with 2 to 4 grams of
pollen supplement and sugar syrup (1:1) for 1-2 days before treatment.
Smaller colonies (1500- and 3000-bees) were placed in glass-walled
observation hives (Fig. 4A <http://www.nature.com/articles/srep32108#f4>).
The larger colonies (7000-bees) were placed in Ulster observation hives
(Brushy Mountain Bee Supply, NC) containing a bottom box holding 4 standard
frames and a division board feeder where treatment syrup was provided (Fig.
4B <http://www.nature.com/articles/srep32108#f4>). The early morning of
observation days, the fifth frame, containing the queen, was placed in an
upper section made of clear Plexi-glass and separated from frames below by
a queen excluder. All observation hives were housed in sheds maintained at
constant temperature and relative humidity (23–25 °C and 70%).
Additionally, all hives contained an entrance leading to the outside
allowing bees to freely forage around some agricultural fields and urban
residential neighborhoods.
*Figure 4*
[image: Figure 4] <http://www.nature.com/articles/srep32108/figures/4>

Observation hives used for 1-frame and 2-frame experimental hives
containing 1500 and 3000 worker bees and a laying queen, respectively (*A*).
Ulster (U501) observation hive for 5-frame experimental hives containing
roughly 7000 worker bees and a laying queen. The frame containing the
laying queen was placed in the upper level during observation periods while
the other four frames and a feeder remained in the lower portion of the
box. The entrance of the hive is located in the lower level and is shown
taped closed in this picture (*B*).
Full size image <http://www.nature.com/articles/srep32108/figures/4>

After the pretreatment period and when egg laying was confirmed, each
colony was randomly assigned an imidacloprid treatment (0, 10, 20, 50, and
100 ppb) provided in 50% sucrose syrup. Colonies were given proportional
amounts of sucrose solution containing imidacloprid: 80, 160 and 320 mL for
1500-, 3000-, and 7000-bee colonies, respectively. Syrup was replenished
every other day for 3 weeks. Syrup quantities were designed to supplement,
but not sustain, the colonies so bees were required to freely forage on
other resources. Stock solutions of imidacloprid (100 ppm) were prepared
using 99.5 ± 0.5% technical grade imidacloprid purchased from Chem Service,
Inc (PS-2086) dissolved with agitation in 50% sucrose overnight. Stock
solutions were prepared every two weeks and treatment solutions were
prepared every week. Samples of treatment solutions (3–6 per dose) were
randomly selected and tested for residue concentrations to confirm accuracy
of dosage.

The number of experimental colonies ranged from 8–20 per treatment and
totaled 79 colonies over three years. Queen absconding events, where the
entire colony left the hive, occurred in four smaller colonies (1500- and
3000-bees) treated at 10 ppb and one 7000-bee colony treated at 100 ppb.
All colonies that absconded or had queen events were removed from the
experiment, accounting for smaller sample sizes in some treatment groups.
Measurements during chronic exposure (day 1–22)

Queen behaviors were measured through two 15-minute observations made every
day for 1500- and 3000-bee colonies, and every other day for 7000-bee
colonies to minimize disturbance. Queen observations from the morning (7–11
am) and afternoon (12–4 pm) bouts were averaged to account for any changes
in hive activity due to weather and outside temperature. Queen activity was
monitored by tracing the queen’s travel path with a felt-tipped pen onto
acetate sheets placed over each observation hive, and egg-laying rate was
quantified by recording the position and number of eggs laid. The distance
travelled (cm) by the queen was then quantified using a digital plan
measure tool (Scale Master Pro model 6025). Immobility in queens, or the
time spent “resting” was also measured and defined as when the queens were
not moving or grooming themselves and did not include when queens were
being groomed or fed by nurse bees.

In addition to queen activity, the behaviors of adult worker bees were
observed. Foraging activity was measured by recording the number of workers
entering and exiting the entrance of each 3000- and 7000-bee colony during
one-minute observations twice a day. Observations made on foraging behavior
in 1500-bee colonies were limited and therefore removed from the
experiment. Hygienic behavior was used as a measure to assess worker
activity inside the hive, and is defined as the ability of worker bees to
detect and remove diseased and mite-infested brood thereby limiting
within-colony transmission of pathogens and parasites38
<http://www.nature.com/articles/srep32108#ref38>,39
<http://www.nature.com/articles/srep32108#ref39>. Hygienic behavior was
measured only in 7000-bee colonies pre- and post-imidacloprid treatment.
Hygienic behavior was measured using a freeze-killed brood assay, in which
a 3-inch (7.6 cm) polyvinyl chloride tube was gently pushed into a section
of comb containing a large area of sealed pupal cells (taken from
non-experimental field colonies). The number of empty cells was counted and
recorded before pouring 400 ml of liquid nitrogen to freeze-kill roughly
160 pupae. The frame was then temporarily put into 7000-bee colonies and
the proportion of pupae completely or partially removed from the cells was
quantified after 24 hours to assess hygienic behavior. The removal of
freeze-killed brood is correlated with the removal of diseased and
parasitized brood38 <http://www.nature.com/articles/srep32108#ref38>,39
<http://www.nature.com/articles/srep32108#ref39>.
Measurements *after* chronic exposure (day 23)

After three weeks of imidacloprid exposure each colony was anaesthetized
using carbon dioxide and placed in a −20 °C freezer. Brood production was
assessed by counting all frame cells containing eggs, larvae or pupae.
Brood pattern was assessed by placing a parallelogram (containing 100
cells) over 3-4 areas of sealed pupae within the colony and quantifying the
average proportion of empty cells not containing pupae for each colony (Fig.
3B <http://www.nature.com/articles/srep32108#f3>). Food stores, or the
number of cells completely or partially filled with nectar or honey and
pollen, was also quantified. Final adult worker population was determined
by using the average weight of 10-subsets of ten individual bees to
estimate the total number of adult workers from the total weight of the
worker bee population.
Chemical residue analyses

Treatment syrups (3 ml) were collected after solutions were made up and
immediately stored in a −80 °C freezer. A total of eighteen samples of
imidacloprid treatment syrup (3–6 per treatment level and colony size) were
collected at random across all years. Adult worker bees (3 g) and stored
comb nectar (3 ml) from three colonies per treatment and size were
collected after the experiment was completed. Samples were analyzed for
residues of imidacloprid and metabolites olefin and 5-hydroxy (OH)
imidacloprid by the US Environmental Protection Agency Analytical Chemistry
Branch (ACB) in Washington DC and the USDA Agricultural Marketing Service,
National Science Laboratory (AMS-NSL) in Gastonia NC using the QuEChERS
(Quick, Easy, Cheap, Effective, Rugged, and Safe) (AOAC OMA 2007.01)
pesticide extraction method.

The analytical method performed by EPA-ACB used acetonitrile (15 ml) and
water (10 ml) in the presence of magnesium sulfate MgSO4 (6 g) to extract
samples. Aliquots of the extracts were passed through a C18SPE (1 g) and
the eluents were concentrated to near dryness. For fatty sample matrices,
(whole bees and stored pollen), partitioning against hexane (7 ml) was
employed to remove lipids prior to the C18 SPE cleanup. The sample extracts
were redissolved in deuterated imidacloprid internal standard solution and
passed through a syringe filter. The samples were then analyzed with liquid
chromatography coupled with tandem mass spectrometry detection (LC-MS/MS).
The USDA-AMS-NSL laboratory extracted samples using an acetonitrile and
water solution to test against certified standard reference materials and
analyzed with LC-MS/MS utilizing the parent and confirmatory ions of
(imidacloprid, olefin, and 5-OH imidacloprid) analytes of interest with
limits of detection of 1, 10 and 25 ppb, respectively.
Statistics

Data collected during and after the chronic exposure period were normally
distributed based on quantile-quantile plots and analyzed using a
mixed-effects ANOVA where imidacloprid dose, time, and colony size were
treated as fixed effects and replicate and year were random effects using
[SAS] software, [version University Edition] and R statistical software
[version 3.2.0]69 <http://www.nature.com/articles/srep32108#ref69>. Fixed
effects were analyzed using a mixed effects model rather than as continuous
variables in a regression model for three reasons. First, fitting as
factors avoided imposition of specific curve forms across all treatments.
Second, an ANOVA permits comparison among specific treatments of interest
in pairwise comparisons. Finally, we focus on concentrations administered
as treatments without calculating internal concentrations for each bee that
would be more amenable to a regression approach. When examining the effect
of time (week) on egg-laying, distance travelled and time spent immobile by
queen bees, a first-order autocorrelation structure was used within the
ANOVA to account for temporal dependence. Where significant treatment
effects existed, mean separation procedures were performed using Tukey HSD
at a significance of α = 0.05.
Additional Information

*How to cite this article*: Wu-Smart, J. and Spivak, M. Sub-lethal effects
of dietary neonicotinoid insecticide exposure on honey bee queen fecundity
and colony development. *Sci. Rep.* *6*, 32108; doi: 10.1038/srep32108
(2016).
References

   1. 1.

   Morse, R. A. & Calderone, N. W. The value of honey bees as pollinators
   of U.S. crops in 2000. *Bee Culture Magazine.* *128*, 1–16 (2000).
   -

- 2.

Thapa, R. B. Honeybees and other insect pollinators of cultivated plants: a
review. *J. Inst. Agric. Anim. Sci.* *27*, 1–23 (2006).

   -

3.

vanEngelsdorp, D. *et al.* Colony collapse disorder: a descriptive study.
*PLosONE* *4*, e6481 (2009).

   -

-

   -
      - Article <http://dx.doi.org/10.1371/journal.pone.0006481>

- 4.

Lee, K. V. *et al.* A national survey of managed honey bee 2013–2014 annual
colony losses in the USA. *Apidologie* *46*, 1–14 (2015).

   -

-

   -
      - Article <http://dx.doi.org/10.1007/s13592-015-0356-z>

- 5.

Laurent, M., Hendrikx, P., Ribiere-Chabert, M. & Chauzat, M.-P. A
pan-European epidemiological study of honeybee colony losses 2012–2014.
*EPILOBEE* *2*, 1–44 (2016).

   -

- 6.

Kozak, P. *et al.* Canadian Association of Professional Apiculturists
(CAPA) National Survey Committee (2014) *Statement on Honey Bee Wintering
Losses in Canada.* 1–5 (2014).

   -

- 7.

Kevan, P. G. & Phillips, T. P. The economic impacts of pollinator declines:
an approach to assessing the consequences. *Conserv. Ecol.* *5*, 8–25
(2001).

   -

- 8.

Sánchez-Bayo, F. *et al.* Are bee diseases linked to pesticides?-A brief
review. *Environ. Int.* *89–90*, 7–11 (2016).

   -

9.

vanEngelsdorp, D., Tarpy, D. R., Lengerich, E. J. & Pettis, J. S. Idiopathic
brood disease syndrome and queen events as precursors of colony mortality
in migratory beekeeping operations in the eastern United States. *Prev.
Vet. Med.* *108*, 225–233 (2013).

   -

-

   -
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=22939774&dopt=Abstract>
      - Article <http://dx.doi.org/10.1016/j.prevetmed.2012.08.004>

- 10.

Bryden, J., Gill, R. J., Mitton, R. A. A., Raine, N. E. & Jansen, V.
A. A. Chronic
sublethal stress causes bee colony failure. *Ecology Letters* *16*,
1463–1469 (2013).

   -

-

   -
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=24112478&dopt=Abstract>
      - Article <http://dx.doi.org/10.1111/ele.12188>

- 11.

Cresswell, J. E. A meta-analysis of experiments testing the effects of a
neonicotinoid insecticide (imidacloprid) on honey bees. *Ecotoxicology* *20*,
149–157 (2010).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BC3MXjtFChtA%3D%3D&md5=94b0b1e50e8acc2e89b19e35b21afff7>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=21080222&dopt=Abstract>
      - Article <http://dx.doi.org/10.1007/s10646-010-0566-0>

- 12.

Gill, R. J., Ramos‐Rodriguez, O. & Raine, N. E. Combined pesticide exposure
severely affects individual- and colony-level traits in bees. *Nature* *491*,
105–108 (2012).

   -

-

   -
      - ISI
      <http://links.isiglobalnet2.com/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=000310434500039&DestApp=WOS_CPL>
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BC38XhsFCnsLzP&md5=263bddd51204f03f630c71a450a90209>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=23086150&dopt=Abstract>
      - Article <http://dx.doi.org/10.1038/nature11585>

- 13.

Goulson, D. REVIEW: An overview of the environmental risks posed by
neonicotinoid insecticides. *J. Appl. Ecol.* *50*, 977–987 (2013).

   -

-

   -
      - Article <http://dx.doi.org/10.1111/1365-2664.12111>

- 14.

Carreck, N. L. & Ratnieks, F. L. W. The dose makes the poison: have “field
realistic” rates of exposure of bees to neonicotinoid insecticides been
overestimated in laboratory studies? *J. Apicult. Res.* *53*, 607–614
(2014).

   -

-

   -
      - Article <http://dx.doi.org/10.3896/IBRA.1.53.5.08>

- 15.

Chagnon, M. *et al.* Risks of large-scale use of systemic insecticides to
ecosystem functioning and services. *Environ. Sci. Pollut. Res.* *22*,
119–134 (2015).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BC2cXht1Wmu7bJ&md5=8c8eb99c43f94e144467f3837e0b7e19>
      - Article <http://dx.doi.org/10.1007/s11356-014-3277-x>

- 16.

Goulson, D. Neonicotinoids impact bumblebee colony fitness in the field; a
reanalysis of the UK’s food & environment research agency 2012 experiment.
*PeerJ* *3*, e854 (2015).

   -

-

   -
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=25825679&dopt=Abstract>
      - Article <http://dx.doi.org/10.7717/peerj.854>

- 17.

Mason, R., Tennekes, H., Sanchez-Bayo, F. & Jepsen, P. U. Immune
suppression by neonicotinoid insecticides at the root of global wildlife
declines. *J. Environ. Immunol. Toxicol.* *1*, 3–12 (2013).

   -

-

   -
      - Article <http://dx.doi.org/10.7178/jeit.1>

- 18.

van der Sluijs, J. P. *et al.* Conclusions of the worldwide integrated
assessment on the risks of neonicotinoids and fipronil to biodiversity and
ecosystem functioning. *Environ. Sci. Pollut. Res.* *22*, 148–154 (2014).

   -

-

   -
      - Article <http://dx.doi.org/10.1007/s11356-014-3229-5>

- 19.

Girolami, V. *et al.* Translocation of neonicotinoid insecticides from
coated seeds to seedling guttation drops: a novel way of intoxication for
bees. *J. Econ. Entomol.* *102*, 1808–1815 (2009).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BD1MXhtlCns7zJ&md5=7be12d9cdb25e692a45af0564b32e826>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=19886445&dopt=Abstract>
      - Article <http://dx.doi.org/10.1603/029.102.0511>

- 20.

Tapparo, A. *et al.* Rapid analysis of neonicotinoid insecticides in
guttation drops of corn seedlings obtained from coated seeds. *J. Environ.
Monit.* *13*, 1564–1568 (2011).

   -

-

   -
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=21509402&dopt=Abstract>
      - Article <http://dx.doi.org/10.1039/c1em10085h>

- 21.

Samson-Robert, O., Labrie, G., Chagnon, M. & Fournier, V.
Neonicotinoid-contaminated
puddles of water represent a risk of intoxication for honey bees. *PLoS ONE*
*9*, e108443 (2014).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BC2MXhtleksrs%3D&md5=53a6185439bda4a04a1d8bf52a34a235>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=25438051&dopt=Abstract>
      - Article <http://dx.doi.org/10.1371/journal.pone.0108443>

- 22.

Jeshke, P., Nauen, R., Schindler, M. & Elbert, A. Overview of the Status
and Global Strategy for Neonicotinoids. *J. Agr. Food Chem.* *59*,
2897–2908 (2011).

   -

-

   -
      - Article <http://dx.doi.org/10.1021/jf101303g>

- 23.

Doering, J., Maus, C. & Schoening, R. Residues of Imidacloprid WG 5 in
Blossom and Leaf Samples of Apple Trees After Soil Treatment in the Field.
Application: 2003, Sampling: 2004. *Bayer CropScience AG Report. No.
G201819* (2004).

   -

24.

Dively, G. & 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).

   -

-

   -
      - ISI
      <http://links.isiglobalnet2.com/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=000303696000005&DestApp=WOS_CPL>
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BC38XksFChtbs%3D&md5=46ac557bd06cbbb33c5e90575ad4963d>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=22452667&dopt=Abstract>
      - Article <http://dx.doi.org/10.1021/jf205393x>

- 25.

Doering, J., Maus, C. & Schoening, R. Residues of Imidacloprid WG 5 in
Blossom Samples of Rhododendron sp. after Soil Treatment in the Field.
Application: Autumn 2003, Sampling: 2004. *Bayer CropScience AG Report. No.
G201820* (2004).

   -

- 26.

Doering, J., Maus, C. & Schoening, R. Residues of Imidacloprid WG 5 in
Blossom and Leaf Samples of Amelanchier sp. after Soil Treatment in the
Field. Application: 2003, Sampling: 2004 and 2005. *Bayer CropScience AG
Report. No. G201799* (2005).

   -

- 27.

Doering, J., Maus, C. & Schoening, R. Residues of Imidacloprid WG 5 in
Blossoms and Samples of Cornus mas after soil treatment in the field.
Application 2003, Sampling: 2005. *Bayer CropScience AG. Report. No.
G201801* (2005).

   -

28.

Desneux, N., Decourtye, A. & Delpuech, J.-M. The sublethal effects of
pesticides on beneficial arthropods. *Annu. Rev. Entomol.* *52*, 81–106
(2007).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BD2sXhtFWnurw%3D&md5=446f4a34ffac6d00c27bb93722521f8f>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=16842032&dopt=Abstract>
      - Article <http://dx.doi.org/10.1146/annurev.ento.52.110405.091440>

- 29.

Decourtye, A. *et al.* Imidacloprid impairs memory and brain metabolism in
the honeybee (Apis mellifera L.). *Pestic. Biochem. Phys.* *78*, 83–92
(2004).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BD2cXkvFyjtg%3D%3D&md5=2f3158451bc73edb235708a7ec0652b1>
      - Article <http://dx.doi.org/10.1016/j.pestbp.2003.10.001>

- 30.

Yang, E. C., Chuang, Y. C., Chen, Y. L. & Chang, L. H. Abnormal foraging
behavior induced by sublethal dosage of imidacloprid in the honey bee
(Hymenoptera: Apidae). *J. con. Entomol.* *101*, 1743–1748 (2008).

   -

31.

Williams, G. R. *et al.* Neonicotinoid pesticides severely affect honey bee
queens. *Sci. Rep.* *5*, 14921 (2015).

   -

-

   -
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=26442598&dopt=Abstract>
      - Article <http://dx.doi.org/10.1038/srep14921>

- 32.

Larson, J. L., Redmond, C. T. & Potter, D. A. Assessing insecticide hazard
to bumble bees foraging on flowering weeds in treated lawns. *PLoS ONE* *8*,
e66375 (2013).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BC3sXhs12qu7bM&md5=375f5a2c668af6340b1fee1055e5f1e6>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=23776667&dopt=Abstract>
      - Article <http://dx.doi.org/10.1371/journal.pone.0066375>

- 33.

Scholer, J. & Krischik, V. Chronic exposure of imidacloprid and clothiandin
reduce queen survival, foraging, and nectar storing in colonies of Bombus
impatiens. *PLoS ONE* *9*, e91573 (2014).

   -

-

   -
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=24643057&dopt=Abstract>
      - Article <http://dx.doi.org/10.1371/journal.pone.0091573>

- 34.

Whitehorn, P. R., O’Connor, S., Wackers, F. L. & Goulson, D. Neonicotinoid
pesticide reduces bumble bee colony growth and queen production. *Science*
*336*, 351–352 (2012).

   -

-

   -
      - ISI
      <http://links.isiglobalnet2.com/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=000302995400047&DestApp=WOS_CPL>
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BC38XlslOqtbo%3D&md5=b4a6b88b718a37323a06af1f3f4c175b>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=22461500&dopt=Abstract>
      - Article <http://dx.doi.org/10.1126/science.1215025>

- 35.

Dively, G. P., Embrey, M. S., Kamel, A., Hawthorne, D. J. & Pettis, J.
S. Assessment
of chronic sublethal effects of imidacloprid on honey bee colony health. *PLoS
ONE* *10*, e0118748 (2015).

   -

-

   -
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=25786127&dopt=Abstract>
      - Article <http://dx.doi.org/10.1371/journal.pone.0118748>

- 36.

Laycock, I., Lenthall, K. M., Barratt, A. T. & Cresswell, J. E. Effects of
imidacloprid, a neonicotinoid pesticide, on reproduction in worker bumble
bees (Bombus terrestris). *Ecotoxicology* *21*, 1937–1945 (2012).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BC38Xht1OmtLnI&md5=1c225bda90ac348b99ff72cd61a294d3>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=22614036&dopt=Abstract>
      - Article <http://dx.doi.org/10.1007/s10646-012-0927-y>

- 37.

Sandrock, C. *et al.* Impact of chronic neonicotinoid exposure on honeybee
colony performance and queen supersedure. *PLoS ONE* *9*, e103592 (2014).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BC2cXhs1aksrbK&md5=b23122be246f4853fefbf5d4b5c4b44a>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=25084279&dopt=Abstract>
      - Article <http://dx.doi.org/10.1371/journal.pone.0103592>

- 38.

Rothenbuhler, W. Behavior genetics of nest cleaning behavior in honeybees.
I. Response of four inbred lines to disease killed brood. *Anim. Behav.*
*12*, 578–583 (1964).

   -

-

   -
      - Article <http://dx.doi.org/10.1016/0003-3472%2864%2990082-X>

- 39.

Spivak, M. Honey bee hygienic behavior and defense against Varroa jacobsoni.
*Apidologie* *27*, 245–260 (1996).

   -

-

   -
      - Article <http://dx.doi.org/10.1051/apido:19960407>

- 40.

Caron-Beaudoin, É. & Sanderson, J. T. Effects of neonicotinoids on
promoter-specific expression and activity of aromatase: implications for
the development of hormone-dependent breast cancer. *Cancer Microenviron*
*3*, e1216 (2016).

   -

41.

Azevedo-Pereira, H. M. V. S., Lemos, M. F. L. & Soares, A. M. V. M. Effects
of imidacloprid exposure on *Chironomus riparius* Meigen larvae: Linking
acetylcholinesterase activity to behavior. *Ecotox Environ Safe* *74*,
1210–1215 (2011).

   -

-

   -
      - Article <http://dx.doi.org/10.1016/j.ecoenv.2011.03.018>

- 42.

Rundlöf, M. *et al.* Seed coating with a neonicotinoid insecticide
negatively affects wild bees. *Nature* *521*, 77–80 (2015).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BC2MXnsVWrtbc%3D&md5=8bd539fbeb6c808a8239fa32dfd15de2>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=25901681&dopt=Abstract>
      - Article <http://dx.doi.org/10.1038/nature14420>

- 43.

Cutler, G. C. & Scott-Dupree, C. D. Exposure to clothianidin seed-treated
canola has no long-term impact on honey bees. *J. Econ. Entomol.* *100*,
765–772 (2007).

   -

-

   -
      - ISI
      <http://links.isiglobalnet2.com/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=000246950300018&DestApp=WOS_CPL>
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BD2sXpsFKgsro%3D&md5=c10623129512f69edf95143cff64b0a4>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=17598537&dopt=Abstract>
      - Article
      <http://dx.doi.org/10.1603/0022-0493%282007%29100[765:ETCSCH]2.0.CO;2>

- 44.

Krupke, C. H., Hunt, G. J., Eitzer, B. D. Andino, G. & Given, K. Multiple
routes of pesticide exposure for honey bees living near agricultural fields.
*PLoS ONE* *7*, e66375 (2012).

   -

-

   -
      - Article <http://dx.doi.org/10.1371/journal.pone.0029268>

- 45.

Schmuck, R., Schöning, R. Stork, A. & Schramel, O. Risk posed to honeybees
(Apis mellifera L, Hymenoptera) by an imidacloprid seed dressing of
sunflowers. *Pest. Manage. Sci.* *57*, 225–238 (2001).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BD3MXhs1Krtb8%3D&md5=4d999b4bf6ab8f7856271f01c3282124>
      - Article <http://dx.doi.org/10.1002/ps.270>

- 46.

Laurent, F. M. & Rathahao, E. Distribution of C14 imidacloprid in
sunflowers (Helianthus annuus L.) following seed treatment. *J. Agr. Food
Chem.* *51*, 8005–8010 (2003).

   -

-

   -
      - Article <http://dx.doi.org/10.1021/jf034310n>

- 47.

Seeley, T. D. The wisdom of the hive: the social physiology of honey bee
colonies. *Cambridge, MA* : Harvard University Press (1995).

   -

- 48.

Joachimsmeier, I., Pistorius, J., Heimbach, U., Schenke, D. &
Kirchner, W. Water
collection by honey bees- how far will foragers fly to use water sources
like guttation drops? A first distance trial using cerals and oilseed rape.
11th Symposium of the ICP-BR Bee Protection Group, Wageningen (The
Netherlands) *Hazards of Pesticides to Bees*, *Nov 2011. Julius*-*Kühn*-
*Archiv*, *437* (2012).

   -

49.

Botías, C. *et al.* Neonicotinoid residues in wildflowers, a potential
route of chronic exposure for bees. *Environ. Sci. Technol.* *49*,
12731–12740 (2015).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BC2MXhs1SrtLjF&md5=ab02fad579a56f0f5ddb8c305af88516>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=26439915&dopt=Abstract>
      - Article <http://dx.doi.org/10.1021/acs.est.5b03459>

- 50.

Suchail, S. Guez, D. & Belzunces, L. P. Discrepancy between acute and
chronic toxicity induced by imidacloprid and its metabolites in Apis
mellifera. *Environ Toxicol Chem* *20*, 2482–2486 (2001).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BD38XjtVersA%3D%3D&md5=a2857e598f16c1f33e179863dfd25a20>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=11699773&dopt=Abstract>
      - Article
      <http://dx.doi.org/10.1897/1551-5028%282001%29020%3C2482:DBAACT%3E2.0.CO;2>

- 51.

Klotz, J. H. & Reid, B. Oral toxicity of chlordane, hydramethylnon, and
imidacloprid to free-foraging workers of Camponotus pennsylvanicus
(Hymenoptera: Formicidae). *J. Econ. Entomol.* *86*, 1730–1737 (1993).

   -

-

   -
      - Article <http://dx.doi.org/10.1093/jee/86.6.1730>

- 52.

Rondeau, G. *et al.* Delayed and time-cumulative toxicity of imidacloprid
in bees, ants and termites. *Sci. Rep.* *4*, 1-8-(2014).

   -

53.

Giroud, B. A. V., Vulliet, E., Wiest, L. & Bulete, A. Trace level
determination of pyrethroid and neonicotinoid insecticides in beebread
using acetonitrile-based extraction followed by analysis with
ultra-high-performance liquid chromatography-tandem mass spectrometry. *J.
Chromatogr. A* *1316*, 53–61 (2013).

   -

-

   -
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=24120025&dopt=Abstract>
      - Article <http://dx.doi.org/10.1016/j.chroma.2013.09.088>

- 54.

Suchail, S., De Sousa, G., Rahmani, R. & Belzunces, L. *In vivo*
distribution and metabolisation of 14C-imdacloprid in different
compartments of Apis mellifera L. *Pest. Manage. Sci.* *60*, 1056–1062
(2004).

   -

-

   -
      - Article <http://dx.doi.org/10.1002/ps.895>

- 55.

Cremer, S., Armitage, S. A. O. & Schmid-Hempel, P. Social Immunity. *Curr.
Biol.* *17*, R693–R702 (2007).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BD2sXpt1Knt7w%3D&md5=141a6a7661a5bb353be870a5d20f1891>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=17714663&dopt=Abstract>
      - Article <http://dx.doi.org/10.1016/j.cub.2007.06.008>

- 56.

Stroeymeyt, N., Pérez, B. C. & Cremer, S. Organisational immunity is social
insects. *Curr. Opin. Insect Sci.* *3*, 1–15 (2014).

   -

-

   -
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=25401083&dopt=Abstract>
      - Article <http://dx.doi.org/10.1016/j.cois.2014.09.001>

- 57.

Smirle, M. J. The influence of colony population and brood rearing
intensity on the activity of detoxifying enzymes in worker honey bees.
*Physiol.
Entomol.* *18*, 420–424 (1993).

   -

-

   -
      - Article <http://dx.doi.org/10.1111/j.1365-3032.1993.tb00616.x>

- 58.

Smirle, M. J. & Winston, M. L. Detoxifying enzyme activity in worker honey
bees: an adaptation for foraging in contaminated ecosystems. *Can. J. Zool.*
*66*, 1938–1942 (1988).

   -

-

   -
      - Article <http://dx.doi.org/10.1139/z88-283>

- 59.

Straub, L., Williams, G. R., Pettis, J., Fries, I. & Neumann P. Superorganism
resilience: eusociality and susceptibility of ecosystem service providing
insects to stressor. *Curr. Opin. Insect Sci.* *12*, 109–112 (2015).

   -

-

   -
      - Article <http://dx.doi.org/10.1016/j.cois.2015.10.010>

- 60.

Harbo, J. R. Effect of population size on brood production, worker survival
and honey gain in colonies of honeybees. *J. Apicult. Res.* *25*, 22–29
(1986).

   -

-

   -
      - Article <http://dx.doi.org/10.1080/00218839.1986.11100687>

- 61.

Eckert, C. D., Winston, M. L. & Ydenberg, R. C. The relationship between
population size, amount of brood, and individual foraging behaviour in the
honey bee Apis mellifera L. *Oecologia* *97*, 248–255 (1994).

   -

-

   -
      - Article <http://dx.doi.org/10.1007/BF00323157>

- 62.

Schmickl, T. & Crailsheim, K. How honeybees (Apis mellifera L.) change
their broodcare behaviour in response to non-foraging conditions and poor
pollen conditions. *Behav. Ecol. Sociobiol.* *51*, 415–425 (2002).

   -

-

   -
      - Article <http://dx.doi.org/10.1007/s00265-002-0457-3>

- 63.

Schmickl, T. & Crailsheim, K. Cannibalism and early capping: strategy of
honeybee colonies in times of experimental pollen shortages. *J. Comp.
Physiol. A* *187*, 541–547.53 (2001).

   -

-

   -
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=11730301&dopt=Abstract>
      - Article <http://dx.doi.org/10.1007/s003590100226>

- 64.

Derecka, K. *et al.* (2013). Transient exposure to low levels of
insecticide affects metabolic networks of honeybee larvae. *PLoS ONE* *8*,
e68191.60 (2001).

   -

65.

Henry, M. *et al.* A common pesticide decreases foraging success and
survival in honey bees. *Science* *336*, 348–350 (2012).

   -

-

   -
      - ISI
      <http://links.isiglobalnet2.com/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=Nature&SrcApp=Nature&DestLinkType=FullRecord&KeyUT=000302995400046&DestApp=WOS_CPL>
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BC38XlslOqtb8%3D&md5=d6b8319cc00ee6f45f0373cdb70d5607>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=22461498&dopt=Abstract>
      - Article <http://dx.doi.org/10.1126/science.1215039>

- 66.

Alburaki, M. *et al.* Neonicotinoid-Coated Zea mays seeds indirectly affect
honeybee performance and pathogen susceptibility in field trials. *PLoS ONE*
*10*, e0125790 (2015).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BC2MXhvVertrvK&md5=2ab47fd90bcfb6e020f7052d392eb57b>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=25993642&dopt=Abstract>
      - Article <http://dx.doi.org/10.1371/journal.pone.0125790>

- 67.

Di Prisco, G. *et al.* Neonicotinoid clothianidin adversely affects insect
immunity and promotes replication of a viral pathogen in honey bees. *Proc.
Natl. Acad. Sci. Early Ed.* *110*, 18466–18471 (2013).

   -

-

   -
      - Article <http://dx.doi.org/10.1073/pnas.1314923110>

- 68.

Vidau, C. *et al.* Exposure to sublethal doses of fipronil and thiacloprid
highly increases mortality of honeybees previously infected by Nosema
ceranae. *PLoS ONE* *6*, e21550 (2011).

   -

-

   -
      - CAS
      <http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=npg&version=1.0&coi=1:CAS:528:DC%2BC3MXosVaitLs%3D&md5=3fc40c639df5336c40b361f31756da3a>
      - PubMed
      <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=21738706&dopt=Abstract>
      - Article <http://dx.doi.org/10.1371/journal.pone.0021550>

- 69.

Chambers, J. M., Freeny, A. & Heiberger, R. M. Analysis of variance;
designed experiments. *Chapter 5 of Statistical Models in S* eds Chambers,
J. M. & Hastie, T. J.Wadsworth & Brooks/Cole (1992).

   -

Download references
<http://www.nature.com/articles/srep32108-references.ris>
Acknowledgements

J.W.-S. acknowledges and thanks Brian Aukema for statistical guidance,
Michael Goblirsch and Morgan Carr-Markell for manuscript review, and Karine
Pouliquen for data collection and technical support. J.W.-S. acknowledges
support from the US EPA Science to Achieve Results (STAR) Graduate
Fellowship and CA Beekeepers Association Research Grants and Brushy
Mountain Bee Farm for discounting research supplies.
Author information
Affiliations

   1. University of Nebraska-Lincoln, Entomology, Lincoln, 68583, USA
      - Judy Wu-Smart
   2. University of Minnesota, Entomology, Saint Paul, 55108, USA
      - Marla Spivak

Contributions

J.W.-S. wrote the main manuscript text and prepared figures. M.S.
contributed to manuscript preparation. J.W.-S. and M.S. reviewed the
manuscript.
Competing interests

The authors declare no competing financial interests.
Corresponding author

Correspondence to Judy Wu-Smart
<http://www.nature.com/articles/srep32108/email/correspondent/c1/new>.
Supplementary informationPDF files1.Supplementary Information
<http://www.nature.com/article-assets/npg/srep/2016/160826/srep32108/extref/srep32108-s1.pdf>

http://www.nature.com/article-assets/npg/srep/2016/160826/srep32108/extref/srep32108-s1.pdf

On Sat, Sep 10, 2016 at 3:24 PM, Lawrence London <lfljvenaura at gmail.com>
wrote:

> Neonicotinoid Insecticide Reduces Queen Bees’ Ability To Lay Eggs by
> Two-Thirds - "...jeopardizing the health and stability of entire bee
> colonies"
> <http://e360.yale.edu/digest/insecticide_neonicotinoids_queen_bee_eggs/4801/>
> (e360.yale.edu <https://www.reddit.com/domain/e360.yale.edu/>)
> Yale Environment 360: Popular Insecticide Reduces Queen Bees’ Ability To
> Lay Eggs, Study Finds
> http://e360.yale.edu/digest/insecticide_neonicotinoids_
> queen_bee_eggs/4801/
> e360 digest
> 09 Sep 2016: Popular Insecticide Reduces
> Queen Bees’ Ability To Lay Eggs, Study Finds
> A queen bee surrounded by members of her colony.
> A new study has found neonicotinoids, the world’s most commonly used
> insecticide, cause queen honeybees to lay as much as two-thirds fewer eggs,
> jeopardizing the health and stability of entire bee colonies. The research,
> conducted by scientists at the University of Nebraska-Lincoln and the
> University of Minnesota, was recently published in the journal *Scientific
> Reports* <http://www.nature.com/articles/srep32108>. "The queens are… the
> only reproductive individual laying eggs in the colony," said lead author
> Judy Wu-Smart
> <http://news.unl.edu/newsrooms/today/article/that-stings-study-finds-insecticide-hurts-queen-bees-egg-laying-abilities/>.
> "If her ability to lay eggs is reduced, that is a subtle effect that isn't
> (immediately) noticeable, but translates to really dramatic consequences
> for the colony." The scientists also found colonies exposed to
> imidacloprid, a type of neonicotinoids, collected and stored less pollen
> than insecticide-free colonies, and removed just 74 percent of
> mite-infested or diseased pupae that can infect the entire hive, compared
> to 95 percent removal by unexposed bees.
>
>


-- 
Lawrence F. London, Jr.
lfljvenaura at gmail.com
https://sites.google.com/site/avantgeared


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