Behavioral responses of honey bees (Apis mellifera) to natural and synthetic xenobiotics in food

Abstract

While the natural foods of the western honey bee (Apis mellifera) contain diverse phytochemicals, in contemporary agroecosystems honey bees also encounter pesticides as floral tissue contaminants. Whereas some ubiquitous phytochemicals in bee foods up-regulate detoxification and immunity genes, thereby benefiting nestmates, many agrochemical pesticides adversely affect bee health even at sublethal levels. How honey bees assess xenobiotic risk to nestmates as they forage is poorly understood. Accordingly, we tested nine phytochemicals ubiquitous in nectar, pollen, or propolis, as well as five synthetic xenobiotics that frequently contaminate hives—two herbicides (atrazine and glyphosate) and three fungicides (boscalid, chlorothalonil, and prochloraz). In semi-field free-flight experiments, bees were offered a choice between paired sugar water feeders amended with either a xenobiotic or solvent only (control). Among the phytochemicals, foragers consistently preferred quercetin at all five concentrations tested, as evidenced by both visitation frequency and consumption rates. This preference may reflect the long evolutionary association between honey bees and floral tissues. Of pesticides eliciting a response, bees displayed a preference at specific concentrations for glyphosate and chlorothalonil. This paradoxical preference may account for the frequency with which these pesticides occur as hive contaminants and suggests that they present a greater risk factor for honey bee health than previously suspected.

Full text

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Scientific RepoRts | 7: 15924 | DOI:10.1038/s41598-017-15066-5
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Behavioral responses of honey
bees (Apis mellifera) to natural and
synthetic xenobiotics in food
Ling-Hsiu Liao, Wen-Yen Wu & May R. Berenbaum
While the natural foods of the western honey bee (Apis mellifera) contain diverse phytochemicals, in
contemporary agroecosystems honey bees also encounter pesticides as oral tissue contaminants.
Whereas some ubiquitous phytochemicals in bee foods up-regulate detoxication and immunity
genes, thereby beneting nestmates, many agrochemical pesticides adversely aect bee health
even at sublethal levels. How honey bees assess xenobiotic risk to nestmates as they forage is poorly
understood. Accordingly, we tested nine phytochemicals ubiquitous in nectar, pollen, or propolis, as
well as ve synthetic xenobiotics that frequently contaminate hives—two herbicides (atrazine and
glyphosate) and three fungicides (boscalid, chlorothalonil, and prochloraz). In semi-eld free-ight
experiments, bees were oered a choice between paired sugar water feeders amended with either
a xenobiotic or solvent only (control). Among the phytochemicals, foragers consistently preferred
quercetin at all ve concentrations tested, as evidenced by both visitation frequency and consumption
rates. This preference may reect the long evolutionary association between honey bees and oral
tissues. Of pesticides eliciting a response, bees displayed a preference at specic concentrations for
glyphosate and chlorothalonil. This paradoxical preference may account for the frequency with which
these pesticides occur as hive contaminants and suggests that they present a greater risk factor for
honey bee health than previously suspected.
e western honey bee (Apis mellifera) is a eusocial species whose foragers collect food to meet hive requirements
and adjust their food-gathering behavior according to these collective needs. Foragers are the rst members of
the colony to encounter and evaluate potential food resources and to make decisions about whether to bring
them back to the hive. us, the discriminative abilities and behavioral preferences of foragers have tremendous
impacts on the nutrition and health of the entire colony. Relative to other insect genomes, the A. mellifera genome
has a strikingly reduced inventory of gustatory receptors, with the 10 gustatory receptor genes (Grs) representing
only 13–15% of those present in other insect genomes1. Despite this reduced inventory, honey bees are demon-
strably able to dierentiate among select natural and synthetic chemicals2–4.
Phytochemicals in nectar and pollen can both attract pollinators and repel inappropriate oral visitors5,
including honey bees. Quinine, an alkaloid from Cinchona species, is among the best-known phytochemical
repellents for honey bees2. As well, some phenolic compounds in sugar water or nectar can enhance honey bee
visitation6–8, whereas others can, depending on concentration, deter feeding6,8–10. Liu et al.10 speculated that for-
agers can estimate the concentration of phenolics in pollen and change their foraging dynamics accordingly.
ese ndings suggest that bees have the ability to evaluate food quality and use phytochemicals as cues to make
foraging decisions, but whether they rely on phytochemicals that enhance colony health as phagostimulants or
whether social cues from nestmates inuence nectar-gathering behavior has not yet been systematically assessed.
In addition to its nutrient content, honey, the product of processed nectar, provides phytochemicals that can
promote colony health in several ways. Gherman et al.11, e.g., demonstrated that nurse bees infected with Nosema
preferentially consume sunower honey, which has the highest antimicrobial activity among the four types of
honey oered as choices. Additionally, caeine, an alkaloid found in thenectar of species in the Rutaceae and
Rubiaceae, among others, can enhance memory in honey bees3. Moreover, phytochemicals in nectar, honey, pol-
len, or propolis can confer other health benets. e phenolic acid p-coumaric acid, a constituent of many hon-
eys, upregulates both detoxication genes and immunity genes in larval and adult honey bees; bees consuming
p-coumaric acid in sugar diet were capable of 60% higher rates of metabolism of the organophosphate acaricide
Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801-3795, USA.
Correspondence and requests for materials should be addressed to M.R.B. (email: maybe@illinois.edu)
Received: 7 July 2017
Accepted: 16 October 2017
Published: xx xx xxxx
OPEN
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coumaphos than bees consuming sugar diet alone12,13. Quercetin, a avonol found in many honeys, essentially
all pollen, and in propolis in many parts of the world, also upregulates at least 12 genes encoding cytochrome
P450 monooxygenases, including CYP9Q1, CYP9Q2, and CYP9Q3, which detoxify both tau-uvalinate and cou-
maphos14 and enhances longevity of workers exposed to the pyrethroid insecticide β-cyuthrin15. Additionally,
a sucrose diet containing both quercetin and p-coumaric acid enhancedthe longevity of bees exposed to
bifenthrin15.
In contrast with at least some phytochemicals, exposure to pesticides rarely if ever is benecial to bees; rather,
pesticide ingestion is associated with a wide array of negative eects16. Pesticides detected in honey and beebread
in North American hives include insecticides, acaricides, fungicides, and herbicides17,18. Much attention of late
has been focused, understandably, on pesticides that target arthropods, including insecticides and acaricides
that contaminate hives. Neonicotinoids in particular have been shown to have a range of adverse eects on bees
even at sublethal levels; paradoxically, Kessler et al.3 demonstrated that honey bee foragers display a preference
for sucrose solutions laced with neonicotinoid pesticides, absent any electrophysiological evidence that they can
taste these compounds.
For their part, herbicides and fungicides have been comparatively understudied relative to the frequency with
which they are documented as hive contaminants. Chlorothalonil is among the most frequently encountered
contaminant in beehives, especially in wax and in pollen, where it has been found at levels up to 99 ppm18. e
longstanding assumption has been that fungicides and herbicides, with relatively low acute toxicity relative to pes-
ticides formulated to kill arthropods, are considered to be safe for bees. Nonetheless, fungicide and herbicides can
have unexpected undesirable impacts on honey bees. e herbicide atrazine alters acetylcholinesterase activity in
honey bees19 and exposure to glyphosate reduces sensitivity to sucrose and interferes with learning performance20
and navigation ability21. Moreover, bees consuming food contaminated with the fungicide chlorothalonil experi-
ence higher rates of infection by the parasite Nosema22,23, reduced queen body size, fewer workers and lower col-
ony biomass16. Chlorothalonil also synergizes tau-uvalinate, a pyrethroid acaricide used in beehives for varroa
control, and increases its toxicity to honey bees24. Moreover, the phenomenon of “entombed pollen”, whereby bees
seal o cells containing pollen with higher levels of fungicide, suggests that bees may by some means recognize
the presence of fungicides in their hive25. Although foragers bring fungicide-contaminated pollen into the hive,
entombment suggests that nurse bees or other hive workers evaluate the pollen once it is in the hive and make the
decision to cap o contaminated cells.
Complicating the assessment of how honey bees evaluate food quality with respect to its xenobiotic content
is the fact that many of the behavioral studies to date have involved immobilization and/or force-feeding in
no-choice assays. In laboratory tests, restrained bees can be induced to ingest toxic substances (e.g., quinine,
salicin, amygdalin and L-canavanine)2,26 and experience post-ingestion malaise or even death as a result27; bees
presented with no alternative food choices will consume foods that, under choice conditions, were rejected28. In
contrast, free-ying and freely-moving bees generally appear to detect and avoid toxic substances readily2,29–31.
Moreover, forager responses to resources vary according to colony-level demand32. When foragers return from
the eld, they unload the nectar from their crop to receiver (or food storage) bees, which, by taking up the nectar
at dierent rates, signal to foragers that certain food resources are preferred33. us, forager behavioral responses
and decisions reect not only an individual’s assessment of foraging resources but also a forager’s assessment of
colony-level needs. Consequently, to understand forager behavioral responses to xenobiotics in natural situations,
a free-ight assay of foragers that interact with hivemates is most likely to reect natural behavior.
Accordingly, to characterize forager behavioral responses to xenobiotics when alternate food is available, we
assessed their discriminatory behavior in free-ight assays in a semi-eld setting. In these assays, free-ying bees
from a functioning colony with nestmates present were allowed to choose between two identical feeders, one con-
taining a test chemical in sugar water and the other containing sugar water and solvent as the control. is assay
was used to compare honey bee foraging responses to natural phytochemicals and synthetic xenobiotics found as
common contaminants in U.S. beehives.
Results
Of the phytochemicals tested, at least one representative from each chemical class, albeit at varying concentra-
tions, elicited a response indicative of either preference or avoidance (Table1). Colony identity may have con-
tributed to some of the variation in responses (data not shown). Caeine, an alkaloid, was avoided by foragers
according to both visitation frequency ratio at 1 ppm (one-sample t(6) = −2.568, p = 0.042) and consumption
ratio at 0.1 ppm (one-sample t(8) = −4.603, p = 0.002). With respect to phenolic acids, evidence of discrimina-
tive behaviour was found only for sugar water containing caeic acid; foragers showed an avoidance response
according to the visitation frequency ratio at 1 ppm (one-sample t(4) = −2.908, p = 0.044) but showed a preference
according to the consumption ratio at the same concentration (one-sample t(4) = 23.522, p < 0.001).
Among the ve tested avonoids, bees displayed a consistent preference response to quercetin at all ve
concentrations according to both visitation frequency (0.01 mM, one-sample t(7) = 3.162, p = 0.016; 0.05 mM,
one-sample t(7) = 7.146, p < 0.001; 0.1 mM, one-sample t(6) = 2.586, p = 0.041; 0.25 mM, one-sample t(5) = 2.961,
p = 0.032; 0.5 mM, one-sample t(5) = 5.396, p = 0.003) and consumption ratios (0.01 mM, one-sample t(7) = 2.825,
p = 0.026; 0.05 mM, one-sample t(7) = 3.749, p = 0.007; 0.1 mM, one-sample t(6) = 4.424, p = 0.004; 0.25 mM,
one-sample t(5) = 3.969, p = 0.011; 0.5 mM, one-sample t(5) = 4.599, p = 0.006). In 0.1 mM and 0.25 mM querce-
tin trials, foragers collected 35% more sugar water from the quercetin feeder than from the control feeder.
Naringenin at 100 ppm also triggered a similar preference response (one-sample t(5) = 3.955, p = 0.011); foragers
collected 15% more sugar water in the case of naringenin compared with the control feeder, but the visitation
frequency ratio at this concentration did not indicate a preference response (one-sample t(5) = −0.021, p = 0.984).
With respect to chrysin and pinocembin, bees displayed an avoidance response to 0.1 ppm chrysin (one-sample
t(5) = −2.676, p = 0.044) and 1 ppm pinocembrin (one-sample t(7) = −3.539, p = 0.009) according to the visitation
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frequency ratios but neither avoidance nor preference was detected according to consumption ratio (0.1 ppm
chrysin, one-sample t(5) = −0.419, p = 0.693; 1 ppm pinocembrin, one-sample t(7) = 0.833, p = 0.432).
Synthetic xenobiotics. Results of the free-ight preference tests with atrazine and glyphosate (herbicides)
are shown in Fig.1B,D. Foragers did not show signicantly dierent responses to the atrazine sugar water solu-
tions according to either consumption ratios or visitation frequency ratios. As for glyphosate, foragers displayed a
preference according to consumption ratio for 10 ppb glyphosate-sugar water compared with control sugar water
(one-sample t(5) = 3.289, p = 0.022). At higher glyphosate concentrations, no dierences in consumption ratios
were detected. No dierence in visitation frequency ratios was recorded at any of the tested concentrations.
Results of the free-ight preference tests with boscalid, chlorothalonil, and prochloraz (fungicides) are shown
in Fig.1A,C. Foragers showed strong avoidance responses only to high prochloraz concentrations, i.e., 10 ppm
(visitation frequency ratio, one-sample t(5) = −3.88, p = 0.012; consumption ratio, one-sample t(5) = −5.801,
p = 0.002) and 100 ppm (visitation frequency ratio, one-sample t(5) = −13.616, p < 0.001; consumption ratio,
one-sample t(5) = −108.626, p < 0.001). A preference for chlorothalonil was detected at 0.5 ppb, as indicated
by both consumption ratios (one-sample t(4) = 3.504, p = 0.025) and visitation frequency ratios (one-sample
t(4) = 4.781, p = 0.009). A similar preference for chlorothalonil at 50 ppb was evidenced by the consumption ratios
(one-sample t(4) = 4.316, p = 0.012) but not by the visitation frequency ratios (one-sample t(4) = 1.588, p = 0.188).
Discussion
Among all tested natural xenobiotics, foragers consistently showed a preference for quercetin according to both
visitation frequency ratios and preference ratios at all concentrations. is clear predilection for quercetin under
the conditions of the free-ight assay is indicative of its biological signicance to honey bees. Quercetin is among
Category Chemical name Concentration df
Visitation frequency ratio1Sugar water consumption ratio1
mean± SE mean ± SE
Alkaloid Caeine
0.1 ppm 8 0.99 ± 0.04 0.93 ± 0.02**
1 ppm 6 0.96 ± 0.02*0.97 ± 0.02
10 ppm 8 0.98 ± 0.05 0.98 ± 0.03
Phenolic acid
Caeic acid
0.1 ppm 5 0.97 ± 0.04 0.96 ± 0.02
1 ppm 4 0.91 ± 0.03*1.08 ± 0.00***
10 ppm 5 0.98 ± 0.04 1.04 ± 0.03
Cinnamic acid
5 ppb 4 1.22 ± 0.14 1.11 ± 0.09
50 ppb 1 1.11 ± 0.08 1.21 ± 0.09
[50 ppb]2[2]2[1.11 ± 0.05]2
5000 ppb 2 1.08 ± 0.09 0.85 ± 0.12
p-Coumaric acid
1 ppm 6 0.95 ± 0.02 0.96 ± 0.03
10 ppm 7 0.97 ± 0.02 1.03 ± 0.03
100 ppm 7 0.97 ± 0.03 1.00 ± 0.02
Flavonoid
Chrysin
0.1 ppm 5 0.80 ± 0.08*0.97 ± 0.06
1 ppm 6 1.10 ± 0.09 1.01 ± 0.06
10 ppm 11 1.02 ± 0.04 1.06 ± 0.03
Galangin
0.1 ppm 5 0.95 ± 0.09 1.08 ± 0.05
1 ppm 5 1.08 ± 0.04 1.12 ± 0.05
10 ppm 5 1.00 ± 0.05 1.00 ± 0.02
100 ppm 5 1.11 ± 0.05 0.95 ± 0.02
Naringenin
0.1 ppm 8 1.05 ± 0.15 1.08 ± 0.10
1 ppm 11 0.92 ± 0.05 1.01 ± 0.04
10 ppm 11 1.01 ± 0.07 1.00 ± 0.03
100 ppm 5 1.00 ± 0.10 1.15 ± 0.04*
Pinocembrin
10 ppb 7 1.01 ± 0.13 0.98 ± 0.04
100 ppb 5 0.92 ± 0.09 1.00 ± 0.03
1000 ppb 7 0.82 ± 0.05** 1.04 ± 0.05
Quercetin
0.01 mM 7 1.06 ± 0.02*1.04 ± 0.02*
0.05 mM 7 1.24 ± 0.03*** 1.17 ± 0.05**
0.10 mM 6 1.20 ± 0.08*1.35 ± 0.08**
0.25 mM 5 1.26 ± 0.09*1.37 ± 0.09*
0.50 mM 5 1.18 ± 0.03** 1.17 ± 0.04**
Table 1. Foraging preference of foragers for natural phytochemical xenobiotics. 1A ratio higher than 1 indicates
a preference for the test chemical, and a ratio lower than 1 indicates avoidance of the test chemical. e asterisks
indicate the means are signicantly dierent from 1 (*p < 0.05; **p < 0.01; ***p < 0.001, one-sample t-test).
2Missing one sugar water consumption data point.
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the most predictable constituents of nectar, honey, pollen, beebread, and propolis. Along with kaempferol, also
a avonol, quercetin acts as a signaling substance in stimulating pollen germination and pollen tube growth34
and, with its derivatives, is a ubiquitous constituent of propolis in North America35. Beyond its value as a recog-
nition cue indicative of appropriate food, quercetin has demonstrable health benets for bees; among these, it
up-regulates detoxication and immunity genes in honey bees12,36. How quercetin is detected by honey bees is
uncertain; as it is non-volatile37,38, it may be detectable by gustatory receptors. Some nectar phenolics can modulate
gustatory responsiveness in the Asian honey bee A. cerana6,39 and may function similarly in A. mellifera as well.
In terms of the other phenolic acids and avonoids tested, p-coumaric acid elicited neither preference or
avoidance behavior at any concentration, whereas foragers displayed a preference for naringenin at 100 ppm as
indicated by consumption rates and an avoidance response to both 0.1 chrysin and 1 ppm pinocembrin as indi-
cated by visitation frequency. All of the phytochemicals tested here for their behavioral eects were examined by
Mao et al.12 using qRT-PCR for their ability to upregulate CYP9Q3, the honey bee P450 with the broadest known
xenobiotic substrate capacity. In their study, p-coumaric acid was the only one that elicited more than a 1.5-fold
increase in expression relative to control12. Clearly, the ability to upregulate a key xenobiotic-metabolizing P450
gene is not correlated with dierential behavioral responses of foragers to these phytochemicals.
Forager responses to caeine appear to be complex. Honey bee foraging and recruitment to sugar water feed-
ers containing caeine are stimulated at the concentrations at 25 and 100 ppm8,40. Due to the possible pharma-
cological eects of caeine on honey bee neurons41, the neuroactive eects of caeine may be responsible for
increasing foraging and recruitment, possibly for the benet of the plant and to the detriment of the bee40. In
this study, honey bees avoided caeine at low environmental concentrations (0.1 and 1 ppm) consistent with the
report by Singaravelan et al.8 that caeine is repellent to honey bee at high concentrations (150 and 200 ppm).
An individual assay also demonstrated honey bee are more likely to reject sugar water augmented with caeine41.
ese ndings indicate honey bees can detect and avoid caeine in their food, despite its potential benecial
eects in enhancing memory3.
Sugar water contaminated with synthetic xenobiotics may have a discernible taste to bees. Foragers signif-
icantly avoided intake of prochloraz-sugar water at 10 ppm and 100 ppm, as evidenced by both visitation fre-
quency ratios and consumption ratios. Nevertheless, our assays also show a signicant preference for sugar water
Figure 1. Ratios (mean ± SE) as preference indices of forager responses to selected synthetic xenobiotics,
fungicides and herbicides. (A) Consumption ratios for three fungicides-sugar water solutions in dierent
concentrations. (B) Consumption ratios for two herbicide-sugar solutions in dierent concentrations.
(C) Visitation frequencyratios for three fungicide-sugar water solutions in dierent concentrations. (D)
Visitation frequencyratios for two herbicide-sugar water solutions in dierent concentrations. A ratio higher
than 1 indicates a preference for the test chemical, and a ratio lower than 1 indicates avoidance of the test
chemical. e asterisks indicate the means are signicantly dierent from 1 (*p < 0.05; **p < 0.01; ***p < 0.001,
one-sample t-test).
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contaminated with certain fungicides and herbicides at least at some concentrations. e preference detected,
however, although statistically signicant, is not overwhelming, representing a dierence of 1–5% between a
treated feeder and a control feeder. It may be that only a subset of foragers can detect and respond behaviorally
to these compounds; how they are detected, however, remains to be determined. De Brito Sanchez et al.42 have
shown that taste perception of honey bees is more complex than assumed from the relatively low number of
gustatory receptors. ey suggest that there exist post-ingestive mechanisms in honey bees that might be as
important as simple reexive responses to chemicals; such mechanisms may have been operative in our assays.
The eusocial nature of the honey bee, however, raises a question as to which workers may experience
post-ingestive malaise; whether discrimination is exercised at the ower or at the point of trophallactic contact
between a returning forager and receiver bees, who then store the nectar in cells, is an open question. Honey bee
colonies are known to use a complex system to signal and provide feedback to regulate foragers43. During trophal-
lactic interactions between a forager and receiver bee, receiver bees learn about the nectar quality, e.g., the sugar
concentration, and the odor of a food source44–46. A forager may collect contaminated sugar water and return
to the hive, delivering it to receiver bees, which may then ingest the compounds and experience post-ingestive
malaise or well-being. ese receiver bees, as well as the forager itself, have some capacity to signal to foragers
that certain food resources should be avoided or collected by the rate at which food is unloaded33. Our experi-
ments were not designed to detect social feedback, but other studies suggest that this mechanism may function
in guiding forager behavior; foragers, for example, can remedy colony nutritional deciencies by searching for
complementary protein sources47.
If honey bees can perceive the presence of xenobiotics by gustation or any other means, another explanation
of xenobiotic preference may be novelty-seeking behavior, which has been well-documented in both food scouts
and nest scouts48. Such novelty-seeking behavior allows discovery of new resources that can enhance colony t-
ness. A reward system in the brain of food scout foragers could act to insure a steady supply of adequate nutrition
as oral community composition changes.
Irrespective of whether food chemicals are natural or synthetic, honey bees show concentration-dependent
choice patterns. Bees may well avoid a chemical in high concentrations that is preferred or ignored when present
in low concentrations, such as prochloraz and naringenin, respectively. Singaravelan et al.8 found that relatively
low concentrations of nicotine (2.5 ppm in 2.5–20 ppm assay and 0.5, 1 ppm in 0.5–5 ppm assay) elicited a signif-
icant feeding preference in honey bees. Köhler et al.49 observed similar preferences for nicotine at low concentra-
tions and repellency at high concentrations. ey also demonstrated behavioral response thresholds to nicotine
may vary with sugar water concentrations.
Preferences for synthetic xenobiotics that are potentially detrimental can become problematical for honey
bees when they are used as managed pollinators, particularly in orchard systems, where fungicides are oen
applied during the blooming season to prevent fungal diseases. In order to protect pollinators, fungicides are
typically applied at night, with the assumption that the overnight interval is sucient for avoiding adverse out-
comes. However, in addition to the risk of direct exposure, this study suggests that the concentration of residues
that persist through the next day would in fact potentially make contaminated oral resources more attractive
to foragers, thereby increasing the quantity of pesticide brought back to hives. e preference for chlorothalonil
on the part of the foragers demonstrated in this study, e.g., may well explain its high frequency and abundance
as a contaminant in beehives18. Moreover, some fungicides and herbicides interact not only with other agro-
chemicals50 but also with phytochemicals; although there is abundant evidence that toxicity can be enhanced by
combinations of xenobiotics51, how these combinations aect foraging decisions has yet to be assessed, despite
the implications for colony health.
Methods and Materials
Experimental animals. Experiments were performed with A. mellifera, the western honey bee. Colonies
used in assays were from several satellite apiaries maintained by the University of Illinois Bee Research Facility
located northeast of the UIUC campus in Urbana, IL. Colonies were relocated to the free-ight cage before use
in the assay.
Bees were subjected to an acute toxicity pretest in order to determine optimal concentrations for free-ight
preference assays. For these pretests, bees were collected from two hives in the same apiary. Individuals were
collected at the colony entrance as they returned from foraging; ve to seven foragers were placed in a small cage
(12.7 cm × 5.1 cm) aer collection and kept in the same cage for the assay to reduce handling stress. As a means of
further reducing stress, cages were kept in the dark.
Standard ve-frame colonies (containing ca. 4,000 worker bees with a naturally mated queen) were used for
the free-ight preference assay in September-October, 2013 and June-August, 2014 at the University of Illinois
Pollinatarium, located on the UIUC campus. Tested colonies were provided with a dish of ground bee pollen
(Betterbee, Greenwich, NY) and a water feeder in front of their hives for the duration of the experiment. A hive
inspection was carried out every two weeks to insure that the colony remained healthy and functioning normally.
e colonies were replaced approximately every four weeks, when foraging activity began to decline.
Chemicals. Two herbicides, atrazine (45330, Sigma-Aldrich) and glyphosate (45521, Sigma-Aldrich); and
three fungicides, boscalid (33875, Sigma-Aldrich), chlorothalonil (36791, Sigma-Aldrich), and prochloraz
(45631, Sigma-Aldrich), were obtained from Sigma-Aldrich (Milwaukee, WI). Caeine (C0750, Sigma-Aldrich)
and three phenolic acids, caeic acid (C0625, Sigma-Aldrich), cinnamic acid (C6004, Sigma-Aldrich), and
p-coumaric acid (C9008, Sigma-Aldrich), as well as four avonoids, chrysin (C80105, Sigma-Aldrich), narin-
genin (N5893, Sigma-Aldrich), pinocembrin (P5239, Sigma-Aldrich) and quercetin (Q4951, Sigma-Aldrich),
were also purchased from Sigma-Aldrich (Milwaukee, WI). One avonoid, galangin (50-908- 908, Indone
Chemical Company, Inc.), was obtained from Indone Chemical Company (Hillsborough, NJ).
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ese ve synthetic xenobiotics and nine natural xenobiotics were selected for testing because they are com-
mon contaminants or constituents, respectively, of honey, pollen and propolis in U.S. hives18,52. e specic phy-
tochemicals were selected because they are known to up-regulate detoxication genes12.
Free-ight preference assay. e acute toxicity of each chemical-containing sugar water diet at each con-
centration was tested in small indoor cages (12.7 cm × 5.1 cm, modied from 2820D,BioQuip Products Inc.)
before carrying out free-ight preference assays in the outdoor ight cage. is pre-test was conducted to ensure
that the concentrations of the chemicals in our test did not cause acute toxicity. Foragers from a colony with a
sister queen of the tested colonies were collected at the hive entrance when they returned from their foraging trip;
ve to seven foragers were collected and placed into a small cage, which was also used for running the tests for
48 hours. Tests of each concentration of each chemical were replicated ve times. Only concentrations causing
no signicant dierence in mortality compared with the control group and promoting at least 80% survival aer
48 hours (e.g., Xavier et al.53) were considered as having no actual toxicity on bees and were used in the free-ight
preference assay.
In the free-ight preference assay, a large outdoor ight cage measuring 6 m × 20 m × 3 m was divided in
half to yield two ight cages measuring 3 m × 20 m × 3 m. A standard ve-frame colony was placed at the center
of each ight cage. Articial feeders with unscented 25% sugar water (w/v) were set up in two end corners
of the ight cage equidistant from the hive (10 m). e articial feeders had a feeder dish (14.75 cm with 24
one-mm-deep grooves that radiated from the center which allowed the bees to collect sugar water from the edge
of the feeder), a 5 . oz. (147.87 ml) feeder cup (FC5-00090, 5.8 cm height, 7.1 cm width, Solo Cup Operating
Corporation), and a feeder cup cover. e feeder cup cover was the same size as the feeder cup and had an inner
foil and an opaque gray outer layer made of tape. e foil was used to prevent chemical breakdown due to expo-
sure to sunlight; the outer tape layer insured that the feeders appeared identical to the bees so as to prevent color
cues from the dierent sugar water from inuencing the bees’ behavior.
Initially, the foragers were trained to the feeders for one or two days, aer which the assays began. A trial was
conducted as follows: rst, 30 to 60 minutes with 25% sugar water feeders followed by 60 minutes with a 25%
sugar water feeder with solvent (0.25% DMSO) vs. a treatment feeder containing 25% sugar water containing a
test chemical in solvent. In order to minimize microenvironment and location eects, the locations of the control
and treatment feeders were switched in the second 60 minutes. e same chemical with the same concentration
was tested in both halves of the ight cages, and the treatment feeders were always placed in opposite corners of
the cage (southwest vs. northeast or northwest vs. southeast) to reduce microenvironment (lights or wind) eects.
Every feeder containing the sugar water to be tested was weighed at the beginning and end of every experimental
step to measure the consumption of sugar water. Visitation frequency at each feeder dish was recorded by a digital
time-lapse camera with snapshots at one-minute intervals. Because our pretest showed that foragers generally
take ve to seven minutes to return to the feeder between two successive visiting, only the pictures recorded at
6-minute intervals were used to calculate the number of bees on the feeder dish.
Two herbicides (atrazine and glyphosate) and three fungicides (boscalid, chlorothalonil, and prochloraz) as
well as one alkaloid (caeine), three phenolic acids (caeic acid, cinnamic acid, and p-coumaric acid), and ve
avonoids (chrysin, galangin, naringenin, pinocembrin and quercetin) were tested. To make stock solutions, phe-
nolic acids and avonoids were dissolved in DMSO and caeine was dissolved in water. Every tested sugar water
diet was made fresh at the tested concentration from the chemical stock solution before a test. At least three con-
centrations were tested for each chemical. A naturally occurring concentration of a chemical was generally tested
rst. Next, a ten-fold higher concentration was tested, followed by a 100-fold higher concentration. Each chemical
was tested three to 12 times at each concentration with two to four colonies (usually three replicates for each con-
centration in each colony and at two to three concentrations per colony). e nal trial numbers varied because
foraging was aected by varying weather and hive conditions. Low foraging frequency can occur during severe
weather or when a hive is weak, which can bias results; accordingly, those low foraging trial data were discarded.
We chose to test eects of phytochemicals on feeding preferences in a 25% sugar water solution because this
concentration represents an average value in at least some plant communities. Chalco et al.54, e.g., reported the
mean nectar concentration in 26 species in a South American temperate forest species as 29.9%, ranging from
12% to 52%).
e amount of sugar water consumed from each chemical treatment feeder in two hours (one trial period) was
divided by the amount of sugar water consumed from its paired control feeder to calculate the ratio as an index of
preference. e sum of the number visiting each chemical’s treatment feeder in two hours was also divided by the
sum of the number visiting its paired control feeder to calculate the ratio of visitation frequency. If the chemical
treatment feeder and its paired control feeder were equally attractive to foragers, the ratio of sugar water con-
sumption and the visitation frequency ratio should be equal to 1. A ratio higher than 1 indicates a preference for
the test chemical, and a ratio lower than 1 indicates avoidance of the test chemical. Both the ratio of sugar water
consumption and the ratio of visitation frequency were tested for normality and the mean values were tested by
the one-sample t-test using OriginPro soware (ver. 9.0, OriginLab Corporation) to test if the mean of the ratio
was equal to 1.
Data availability. e datasets generated during this study are available from the corresponding author on
reasonable request.
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Acknowledgements
We thank Catherine Dana, Sarah Vaughan, Freddie Stavins, Keelan Lang, and Jacob Herman for assistance with
the assay, Maminirina Randrianandrasana, Allen Lawrance, Joe Wong, Alan Yanahan and Jared omas for bee
dome preparation, Jodi Flaws and Susan Schantz for suggestions, Charley Nye for help with hive-work, and Gene
Robinson for advice and access to UIUC apiaries. We also thank Lesley Deem and the UI Pollinatarium for access
to the bee dome and indoor space. is project was funded in part by USDA Agriculture and Food Research
Initiative 2010-03760 and 2017-67013 to May Berenbaum, the Interdisciplinary Environmental Toxicology
Program at UIUC, and the Almond Board of California to May Berenbaum.
Author Contributions
L.L. and M.B. designed the experiment and wrote the manuscript text. L.L. and W.W. conducted the experiments,
carried out the data analysis, and prepared the gure and table.
Additional Information
Competing Interests: e authors declare that they have no competing interests.
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