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Crop Pollination Exposes Honey Bees to Pesticides Which

Alters Their Susceptibility to the Gut Pathogen Nosema
ceranae Informative, descriptive title in sentence form with a verb.
Please note that not all publications accept sentence titles.
Jeffery S. Pettis1, Elinor M. Lichtenberg2, Michael Andree3, Jennie Stitzinger2, Robyn Rose4,
Dennis vanEngelsdorp2*
1 Bee Research Laboratory, USDA-ARS, Beltsville, Maryland, United States of America, 2 Department of Entomology, University of Maryland, College Park, College Park,
Maryland, United States of America, 3 Cooperative Extension Butte County, University of California, Oroville, California, United States of America, 4 USDA-APHIS, Riverdale,
Maryland, United States of America Overall, abstract is Use of "We"
informative and concise with active
Abstract voice
Recent declines in honey bee populations and increasing demand for insect-pollinated crops raise concerns about
pollinator shortages. Pesticide exposure and pathogens may interact to have strong negative effects on managed honey
bee colonies. Such findings are of great concern given the large numbers and high levels of pesticides found in honey bee
colonies. Thus it is crucial to determine how field-relevant combinations and loads of pesticides affect bee health. We
sentencescollected pollen from bee hives in seven major crops to determine 1) what types of pesticides bees are exposed to when Principal
rented for pollination of various crops and 2) how field-relevant pesticide blends affect bees’ susceptibility to the gut
provide parasite Nosema ceranae. Our samples represent pollen collected by foragers for use by the colony, and do not necessarily objectives
indicate foragers’ roles as pollinators. In blueberry, cranberry, cucumber, pumpkin and watermelon bees collected pollen are clearly
and context.
almost exclusively from weeds and wildflowers during our sampling. Thus more attention must be paid to how honey bees identified
are exposed to pesticides outside of the field in which they are placed. We detected 35 different pesticides in the sampled with
pollen, and found high fungicide loads. The insecticides esfenvalerate and phosmet were at a concentration higher than
their median lethal dose in at least one pollen sample. While fungicides are typically seen as fairly safe for honey bees, we
Results concisely
found an increased probability of Nosema infection in bees that consumed pollen with a higher fungicide load. Our results
highlight a need for research on sub-lethal effects of fungicides and other chemicals that bees placed in an agricultural
setting are exposed to.
Principal conclusion places paper in
Citation: Pettis JS, Lichtenberg EM, Andree M, Stitzinger J, Rose R, et al.appropriate context
(2013) Crop Pollination withBees
Exposes Honey other studies
to Pesticides Whichand
Alters Their Susceptibility
to the Gut Pathogen Nosema ceranae. PLoS ONE 8(7): e70182. doi:10.1371/journal.pone.0070182
highlights areas for future research
Editor: Fabio S. Nascimento, Universidade de Sa˜o Paulo, Faculdade de Filosofia Cieˆncias e Letras de Ribeira˜o Preto, Brazil
Received March 25, 2013; Accepted June 16, 2013; Published July 24, 2013
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: Funding for this study was provided by the National Honey Board ( and the USDA-ARS Areawide Project on Bee Health (http:// = 412796). Neither the Honey Board nor USDA-ARS Program Staff had a role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Dennis vanEngesldorp is a PLOS ONE Editor. All other authors have declared that no competing interests exist. This does not alter the
authors’ adherence to all the PLOS ONE policies on sharing data and materials.
* E-mail:
As appropriate, introduction
has a funnel shape. Foreshadows
Introduction behavior, learning and immune function [9,13,14,16–22]. Re-
Introduction begins broadly duced immune functioning is of particular intereststudy's becauseresearch
Honey bees, Apis mellifera, and provides
are one of the background
most important recent disease-related declines of bees including honey questions
bees [3,23]. and
pollinators of agricultural cropsand
[1]. Recent declines
context: in honey
in this case, bee Pesticide and toxin exposure increases susceptibility to and
populations in many North American and European countries [2–
the importance of mortality from diseases including the gut parasite Nosema spp.
4] and increasing cultivation of crops that require insects for [14,15]. These increases may be linked to insecticide-induced
honeybees to agriculture.
pollination [5] raise concerns about pollinator shortages [5,6]. alterations to immune system pathways, which have been found
Habitat destruction, pesticide use, pathogens and climate change for several insects, including honey bees [22,24–26].
are thought to have contributed to these losses [2,7,8]. Recent Surveys of colony food reserves and building materials (i.e. wax)
research suggests that honey bee diets, parasites, diseases and have found high levels and diversity of chemicals in managed
pesticides interact to have stronger negative effects on managed colonies [18,27,28]. These mixtures have strong potential to affect
honey bee colonies [9,10]. Introduction begins[11,12]
Nutritional limitation to narrow
and inindividual and colony immune functioning. However, almost all
exposure to sub-lethal dosesscope; here,
of pesticides with a
[13–16], in brief review research
particular, of to-date on pesticides’ effects on pathogen susceptibility
may alter susceptibility to orthe literature.
severity of diverse bee parasites and fed a single chemical to test bees [16]. Because pesticides may have
pathogens. interactive effects on non-target organisms (e.g. [29]), it is crucial
Recent research is uncovering diverse sub-lethal effects of to determine how real world combinations and loads of pesticides
pesticides on bees. Insecticides and fungicides can alter insect and affect bee health. Topic sentence clearly
spider enzyme activity, development, Introduction
oviposition behavior, One pathogen of major concern to beekeepers is Nosema spp.
states the main point of
offspring sex ratios, mobility, navigation and orientation,
continues to feeding The endoparasitic fungal infections of N. apis and N. ceranae
the paragraph.
narrow; here, the
PLOS ONE | relevance of 1 July 2013 | Volume 8 | Issue 7 | e70182

Cucumis sativus) fields was until they could be transferred to a 229uC freezer in the lab. CA. Within each selected field.9 (3. We separate these samples in data analyses. cucumber. Visual pollen traps (Brushy Mountain Bee Farm. we chose the clear nail polish after letting air bubbles escape for 48 h. and watermelon (Table 1).1 (2.b a Apple York Springs. blueberry. We chose Nosema as a model colored pollen pellets were identified (see below). traps after three days. and a This study addresses two important questions. understandable without reference to the text. Research questions studies have characterized the pesticide profile of various materials Past tense used. research model Words like "then" adversely affect honey bee colony health. and covered slides and sealed with based on growers’ needs.5)a. Traps with less than 5methods g remained were on hives until they apple (Malus domestica) orchards that was identified as Malus sp. ME. few have looked at the pollen is appropriate were unable to include it in the pesticide analysis and Nosema in a with numbers. We considered a sample to be dry when its weight did not Pollen was collected from honey bees with permission of the change between two consecutive time points (measured every 4– beekeepers and the land owners. DE.7 (0. a 3g procedures. We then divided each sample into three portions. Sussex County 27.b Watermelon Seaford. NC) to identification of pollen grains through comparison with voucher or these hives.makes active voice Hives were deployed in these fields for pollination services slide with a drop of silicon oil.pone. For slide by grinding 2 pollen pellets in 2 mL water and letting them Use of "we" withwe selected three fields that were separated by at least each crop. apple. however. MD) for the Nosema infection study. NJ. was appropriate contained 5 g of pollen or for 10 days. Berks County 98. doi:10. quantified each color by comparing to Sherwin-WilliamsH We collected pollen carried by foraging honey bees returning to color palettes.7 (0.Funnel shape continues as Justification for Crop Pollens Affect Bee Health introduction becomes more specific. NJ. Kern County 42. Bees use Similarities between closely related pollens.1 (1. table is Table 1.8)a. and than target crop permitted a more inclusive analysis. We used compared the quantity of pollen collected by hives in different Table title is informative. and can result in crops via a Kruskal-Wallis test followed by a help readers post-hoc non- complete colony collapse [30].b 4. Pollen Identification Each 5 g pollen subsample was dehydrated in a drying oven at Ethics Statement 40uC. as Because almond pollen was collected after all other pollens. and species their hind tibiae in flattened regions called corbiculae.c a. A 5 gof following spring [31].6)b.7 (2.1 (11. Quantity and diversity of pollen collected in pollen traps on individual honey bee hives. by weight. We cranberry fields twice: early in the flowering season and late in the also calculated the proportion. To identify pollen types collected by the bees. referring to identified as belonging to the target crop’s genus. Moravian Falls. 2) How do field-relevant pesticides methods infection 2 July 2013 | Volume 8 | Issue 7 | e70182 . and removedExplanation of whyat least them if they contained Because of this limitation. we assumed that all pollen collected in 5 g of pollen.7) 5. Service Laboratory in Gastonia.0 (1.6) 4.2 km. we sorted the pollen in each subsample by Hive Selection and Pollen Collection Addresses method color. pumpkin. we clearly identified inside a honey bee nest [27. Infection with Nosema in the parametric Tukey-type test (using the R package nparcomp the understand autumn leads to poor overwintering and performance the sequence [33]). dissolve to form a slurry. We prepared each cranberry.c Cranberry (late season) Hammonton. Atlantic County 13.2) 7.1) 1.1371/journal. 6 h). we collected pollen from each hive in number of different pollen colors collected from that bee hive. we estimated pollen diversity as the yielded little pollen.1 (1.0070182. Atlantic County 13. sometimes this pollen to make food for larvae inside the nest.6)b. prevent identification to genus or species with this method [39].9 (0.0 (1. Ethics Methods Statement. We placed pollen removed from apple trees. Cumberland County 8.t001 PLOS ONE | www.14] had demonstrated an subsample was sent to the USDA’s Agricultural Marketing interaction with pesticide exposure.0)c Cranberry (early season) Hammonton. NJ. Washington County 4.6 (29. PA. being brought back to the nest. PA.’’ could only be identified to genus. We methods section three honey bee hives with the strongest foraging forces by visually identified each pollen type under 400x magnification by easier to read.0 (9.b Pumpkin Kutztown.5 (1.0) 3. Typically pollen dried in 12–18 h.1 (0. Crop Location Mean grams of pollen collected (se) Mean number of pollen types (se) a. color from each subsample on a separate slide.5) 6. and queens can be superseded soon after experimental subsample was sorted by color and then each group of similarly becoming infected with Nosema [32]. We placed a small amount of slurry on a 3. Many samples them as ‘‘Cranberry early’’ and ‘‘Cranberry late.0 (2. Adams County 26. Pollen traps collect the pollen pellets bees carry on reference specimens is standard in pollination Genusecology [37. pathogen because earlier work [13. of the pollen that was season. In cases where the total amount of pollen section.2)c a. NC for pesticide analysis. so assessing target genus rather We measured the wet weight of each pollen sample. Manyby blends affect bees’ susceptibility to infection journals the Nosema collected from a single colony was less than 6 g all the pollen parasite? require an was used for pesticide analysis. re-weighed after color separationlimitations and fixed each the hive for nine hives in seven crops: almond. We checked names provided.2)a. flight in the bee yard for 5–10 min. Because our first round of pollen trapping in cranberry fields For each subsample.5)c b Blueberry Deblois. and that all pollen in the Cucurbitaceae family from traps in 50 mL centrifuge tubes and stored the samples on ice collected in cucumber (Cucurbitaceae. 1) What types of 10 g subsample was sent to the USDA-ARS Bee Research pesticides might bees be exposed to in major crops? While multiple Laboratory (Beltsville.b 4.b Almond Rosedale.38]. from cucumber flowers.c Letters indicate statistically different groups.c b Cucumber Cedarville.28].0)b.3)b. and attached plastic observing comparing with published reference collections [34–36].

We used these We further tested effects of pesticides in pollen on measured data to determine the total number of pesticides detected in each Nosema prevalence using a generalized linear mixed model with a 3 July 2013 | Volume 8 | Issue 7 | e70182 .6%. oxadiazines and formamidines. category from each sample. consuming pollen with a specific pesticide. We found insecticides and fungicides in all 19. cyclodienes in 52. The number of pesticides detected in trapped pollen cakes were placed in small petri dishes with the laboratory cages. the R popbio package [44] that shows our mixed notmodel directlyoutput. we followed Table is referenced recent recommen- that category.05. fungicides. and applying a sequential Bonferroni correction [40] across H7 = 44. determined whether load varied by pesticide category using a Statistical values p = 0. bee as a random effect. 31. A protein oxadiazines in 10. We (Fig.161. and several insecticide types (carbamates. tested" help orient the reader To look for potential effects of individual pesticides on and Pesticide Analysis Subheadings guide susceptibility to Nosema infection.plosone. varied by the crop in which the bee hives were located (Kruskal- Pollen from either one of the crop fields or one of two control diets Sentences Wallis test: H6 = 12. We thus different crops (Table 1. We only calculated diversity for categories with at dations [42. A relative risk value of one indicates that the cucumber field did not yield sufficient pollen in traps for pesticide probability of infection is equal between exposed and non-exposed analysis.04). presence or absence of N. the dose required to kill fed 1 g of pollen mixed with 0.063. spores. We the target crop.2%. S1).2 ppb). Insufficient degrees of freedom section p = 0. formamidines in 52. 3. and the diversity and bee’s Nosema status as the response variable.0001). Nosema Infection The Nosema infection experiment is similar to published methods Pesticide Analysis [26].0.08)(Fig. We thus selected for different numbers of elements.9. then provided bees than loads of herbicides or any of the insecticide categories with ad libitum access to clean 50% (w/v) sucrose solution. The total number of pesticides present and total load did not meet parametric assumptions. Crop Pollens Affect Bee Health Kruskal-Wallis tests to determine whether either of these measures on infection prevalence. the number of individuals Words like with "further Nosema differed with the crop in which sampled bee hives were placed. GLMM. estimated by quantifying the general linear mixed model with sample as a random effect. we pooled pollen from the three the chance of developing a disease after a particular exposure [41]. To permit comparison between categories with affect bees’ susceptibility to Nosema infection. pollen control for the fact that our regression included one data point per traps. we calculated explain whyrisk the relative We determined the identity and load readers through of pesticide the residues and its 95% confidence interval for bees becoming infected methods were after present in pollen samples collected frompaper. Tucson. understood. We fed 5 mL of the Nosema inoculum to Within a sample. ceranae spores by homogenizing After adjusting for multiple comparisons. AZ) and tested as pesticide-free by the be clearly collected by the bees came from seven categories. The pollen control group (‘‘BRL’’) was fed a mixed not (H6 = 11.0014). pesticide diversity and fungal load. 2 in pollen collected by bees in apple orchards (Figs.6%. and herbicides tory. cyclodienes. We obtained 210 disease-free honey bees from each of three All pollen collected in this study contained pesticides (Table 2. was low thus asked whether the pesticide load and diversity varied with (mean6se = 0. in text. df = 8.734. herbicides. Pollen diversity. for pollen analysis described in Mullin et al. cranberry (early and late). with the ten bees in one of loads ranged from 23. Like pollen weights. The Nosema inoculum was freshly prepared by mixing ophosphates in 63. pumpkin or watermelon fields was from the target crop. collected bees 12 days after infection and examined them for the p. from hives in blueberry. 2). and pyrethroids in 100% of pollen Nosema spores isolated from an infected colony (details provided in samples. apple. For each field sampled (n = 19).0001). Each bee was mean 6 se = 9. all crops (except almond). One early-season cranberry field and one here each pesticide.96.43] and a modification of the logi. Relative risk measures appropriate. carbamates in control group was fed an artificial honey bee pollen substitute.6 to 51. This pollen was collected in the desert Southwest (Arizona allowing results to in 23. pollen samples. We thus analyzed how these Results variables differ between crops using non-parametric Kruskal- Pollen Collection Wallis tests.21. the source hive and pesticide variables as fixed effects. Bee colonies collected different amounts of pollen in the pesticide loads did meet parametric assumptions. short and concise. Kruskal-Wallis test: H7 = 29.0 ppb (11. pollen diet prepared by the USDA-ARS Bee Research Labora. hives for analysis.760.86. The proportion of pollen that bees collectedeasier from to prevented us from expanding this model to include crop. model to investigate in detail how pesticides and pollen source description pyrethroids).5%. which they fully consumed in 2–4 days. We calculated PLOS ONE | www. Both neonicotinoids and oxadiazines were present only [26]) with 50% sucrose solution to obtain a concentration of ca. p = 0. each sample’s total pesticide load.plot function in and in parentheses least three chemicals. When separated by category and log-transformed.5 mL of syrup (1:1 sucrose to water half a population within 24 or 48 h) for esfenvalerate and phosmet by weight). pesticide loads did not individual abdomens in 1 mL distilled water. company and the same group and from the same colony housed together in a The maximum pesticide concentration in any single pollen sample location wooden hoarding cage (12612612 cm). this crop for each category using one Kruskal-Wallis test per category proportion dramatically differed between crops (Fig.6. These pollen (Table 2). Table S1).hist. organophosphates.2 different chemicals. To graph logistic regression and load as the total load divided by the number of chemicals in results in a meaningful manner. and the pollen sample fed to the Citationload of ofanother pesticides in each of 10 categories: insecticides. p. Notably.8%. neonicotinoids in 15.3360. 1. range 3–21).6%.5. We found USDA Agricultural Marketing Service prior to use. Insecticides present in pollen Bee Products. to are in in number of differently colored pollen pellets collected parentheses.6% of.0. Collinearity prevented developing a full paper for methodsneonicotinoids. Each group of bees was exceeded the median lethal dose (LD50. Methods follow the LC/MS-MS and GC/MS methods groups. except for almond and read. pollen fungicide loads were significantly higher each cage during the first two days of adult life. we calculated diversity as the analysis two measures that vary with crop and are not nested: total proportion of pesticides from a category found in a given sample. S1). none of the pollen trapped pesticide categories to control for multiple comparisons.0001).310. Pesticide information such placed into as21 groups upon emergence. 4. but the total pesticide are load did were used. varied by crop (Table 1. [27]. Here we focus only vary by crop for any pesticide category (Fig. p = 0. organ- MegaBeeH. million spores per 5 mL. likelihood ratio test: x2 = 121. Includes material healthy colonies at the Bee Research Laboratory. Kruskal-Wallismaking test: H7 results = 23.

p = 0. indicating Nosema prevalence in bees discussed. tables.05). thus providing honey bees with little access to other flowers. two fungicides (chlorothalonil andand not simply pyraclostrobin). df = 1. Chlor.0. insecticides (e. Crops native to the New World.71. source watermelon. p = 0.0.15). and two miticides used by beekeepers to control varroa infestation (amitraz and fluvalinate) had a pronounced repeated. 8 and amounts of pollen from these plants to bring back to the identified colony.pone.). data indicate only flowers from which bees are actively collecting A pollen sample’s fungicide load significantly affected Nosema pollen and not all flowers they visited.9%) found in our pollen Unexpected results samples had limitations relative risk values significantly different from are 1 (Table large 2). However. bees have been introduced.23. and almond orchards are large. Bars show mean 6 se. df = 1. pumpkin and p = 0. Letters the target crop (Fig. Our results show that beekeepers need to consider not only pesticide regimens of the fields in which they are placing their bees. AIC = 612.7. p = 0. df = 2. Discussion begins Crop Pollens withAffect studyBee results.50]. p = 0.2% (mean 6 se) of the per sample total fungicides chlorothalonil and pyraclostrobin suggest that some fungicide load. but pesticide diversity did not in concentrations with sublethal effects on honey and bumble bees (x2 = 1.pesticide repetitive diversity within only those categories containing three or exceptions to this were hives placed in almond and apple orchards.47]. vibrated by visiting bees (buzz pollination) [46. Because pollen traps between collect only associated with higher Nosema prevalence in bees that consumed them. the colony.36). obs. also did not affect Nosema prevalence (x2 = 2. yielded little or no pollen in our fed pollen containing those chemicals (DMPF and fluvalinate) was discussion moreisthan saveddouble the Nosema prevalence in bees that did not samples. where honey relative risk larger than two.Research patterns on are context with other pesticides’ effects on bee health has focused almost exclusively on analyzed. All of the non-target pollen that we were Important. GLMM. the majority were collecting nectar from our focal crops.3%) fed Nosema spores became infected.26] and high pesticide exposure [27. of the crops. A bee’s source colony. lence. representative doi:10.9%) were associated with increased Nosema preva- discussed. Fungicide and neonicotinoid diversities varied by Almond flowers early in the year. The results from this study highlight several patterns that merit further attention. Finally. collect mainly pesticides (22.1371/journal. Honey bees may collect nectar from blueberry and blocking variable. cranberry flowers via legitimate visits or ‘‘robbing’’ through slits df = 2. Bumble bees.52]. The two identified.22. Both control diets had relative risk values not significantly corbicular pollen intended for consumption bysentences. infection rates in bees that consumed greater quantities of the and comprised 50. The combination of high pesticide loads and increased Nosema othalonil was also the most abundant fungicide in our samples. but also spray programs near those fields that may contribute to pesticide drift onto weeds. sometimes ratio test: x2 = 5. case. p = 0. Of the seven pesticides found in pollen It is possible that bees were exposed to pesticides while provide flow from over half. Interest- Results Nosema are prevalence.0. our different from one. Health then broadens in Discussion scope.15.36.3. exposure to obtained the same result (chlorothalonil load: x2 = 5. Honey bees are not capable of buzz pollination and thus are unlikely Addresses study to collect 22 of the 35 pesticides (62.5. likelihood detected pesticides in floral nectar and pollen [49. The bees in our study collected pollen from diverse sources. the two crops that saw high levels of pollen collection by beekeepers to control hive mites (marked with a * in Table 2) had a honey bees are Old World crops that evolved with honey bees as provided but not natural pollinators. First. These findings support other research with honey bees and native bees indicating that in some crops Results are native bees may be more efficient pollinators [45].plosone.0610.0070182. in this Places this paper in effect on bees’ ability to withstand parasite infection. these chemicals. often failing to collect any pollen from Figure 1. df = 1. included as a [51. Replacing fungicide load with chlorothalonil load cut at the base of flower corollas [53].g001 applications S1). Second. 3).19). suggesting the honey bees were collecting significant amounts results are clearly of pollen from weeds surrounding our focal fields.02. despite being rented to pollinate specific crops. 5. Transition words for the next consumesection.14] and thiacloprid [15]). Honey bees rarely collect pollen from blueberry or cranberry Nosema Infection flowers. Several studies have prevalence among bees fed that pollen (Fig. research.g. even when we detected no pollen from those crops. Two of the three detected pesticides applied by ingly. or at least four. pesticides via nectar may be unlikely in cucumber. ours is the first to demonstrate how real world pollen-pesticide blends affect honey bee health.02). fungicides were present at high levels in both crop discussed and non-crop meaningfully pollen collected by bees. chlorothalonil load model AIC = 613. several individual pollen samples contained loads higher than the median lethal dose for a specific pesticide. fipronil [15] and the neonicotinoids imidacloprid [13. Beekeepers often report poor honey production when colony: x2 = 2. data are more shown in chemicals. Third.28].8. which only release large quantities of pollen after being 147 of the 630 bees (23. pesticide diversity: x2 = 1. which can buzz pollinate. but diversities of other pesticide categories did not ( 4 July 2013 | Volume 8 | Issue 7 | e70182 . While multiple studies have shown negative effects of specific pesticides on honey bee individual and colony health [14. df = 1. Pollen collection from the crop where a hive was Clearly identifies located was low for most crops. practical able to identify to genus or species was from wildflowers (Table indicate statistically significant differences (p. 1). crop. fungicides have stronger impacts on bee health than previously PLOS ONE | www. while the remaining 14 were associated with decreased blueberry pollen when placed in blueberry fields [48]. honey bees did not always return to the nest with corbicular pollen from those crops. fungicide load model their hives are placed in these crops (pers.

2.860.23) Coumaphos* Organophosphate 35.08 (1.5 [64] Cr. If multiple values have been published.32.3625.72 [80] Ap. 2. Cu.67) a We divided LD50 values given as mg/bee (g) by 0. 0.31 (0. c Ap = apple.0.9612. Cu 6 2.3) 1.52. 0.46119.1632. Cu 4 0.6. Pu.65){ Indoxacarb Oxadiazine 1.1.05 (0.75){ Fluvalinate* Pyrethroid 1. Cu.5) 0.51 (0.7 (69.21. Cu.13 [81] Ap.4629.31 (0.420) and white lines 0.pone.0070182.36 (0.56 (0.2 (401) 0.31 (0. Pesticides found in pollen trapped off honey bees returning to the nest.69 [16] Ap. Pu 6 0.4 (13.1 (97.16.69 [16] Ap. making information easier to follow.000) 2.43 (1.110) Alternating 0.86 [16] Ap. Table 2.29) Cypermethrin Pyrethroid 0.3) 1.06 [60] Ap 2 1. 1. 0.94){ Cyprodinil . Pu 7 3.41 [84] Ap 2 0.2. but can persist in the soil for extended periods of time. Wa 16 42. Wa 8 1.61){ Pyrethrins Pyrethroid 0.1 (27. Pu. readers to NA indicates information that is not relevant to control diets.060) 2.94 (0.91.8) to obtain ppm when necessary [85].160. Cu. 0.0 (403) 0.82){ Control diets BRL NA NA NA NA NA 0.48){help Pyraclostrobin 573.23.41 (1.13 (1.99){ Insecticides 2.75 (0. Relative risk (95% Pesticide Insecticide family LD50 (ppm)a detectedc Detections mean6se (max) (ppb) CI) Fungicides Azoxystrobin .54. Wa17 4.4 (2.262.9) 1.165.23 [83] Ap 3 2.562.04) metabolite Imidacloprid Neonicotinoid 0.7 (12. in a 1. we include only the smallest.56117.15.75){ Quintozene (PCNB) .660.663.85 (2.21.76772.0 (19.65 [69] Ap.05) Pendimethalin .27. 2.2 (2.74 (0. 1.20. 0.64.04) Methomyl Carbamate .65){ Difenoconazole . 1.6) 1.1) 2. Pu.61) Phosmet Organophosphate 8.11 [74] Pu.05) Deltamethrin Pyrethroid 0.61) THPI Captan metabolite Cr.15. Cu 5 798.35. Cr.78.44 [70] Cr.700) 0.787. Bl. Cr.65){ Bifenthrin Pyrethroid 0.161.05 (0.800) 0. Pu. Cr.54.73){ Methidathion Organophosphate 1.94 [16] Bl.0 (216) 0.56.6 (332) 0.58 (0. Wa 7 14.81){ Chlorothalonil .91){ Cyfluthrin Pyrethroid .7) information 0. Cr.9 (131) 0.56 [82] Bl.97 [72] Cr 1 0. Information reads from left to Crop Pollens Affect Bee Health right. 2. understand doi:10. Cr. Cu 3 832.56.4 (6. 1.31 (1.31 (0.890.3689. 3. Legend allows *Used by beekeepers within the hive for parasitic mite control.11.781.3 (5.4) 1.04. Cu.91){ Endosulfan sulfate Endosulfan metabolite Cr.13 [65] Ap. Cu 7 16.414.16 [16] Cr 1 5.60 (1.85 [85] Cr 1 1.8 (9.gray 0.plosone.69. 3. 2.461. Cu.125 [67] Ap 3 996.5 (85. Cr 3 1.31 [76] Cr.48) MegaBee NA NA NA NA NA 0.6 (31) 1. 2. Cr.8) 0.02) Cyhalothrin Pyrethroid 0.1) 0.30 [77] Ap.560. Wa 10 171.96707.54.3.217. Pu 5 5.6 (259) 1.19) Esfenvalerate Pyrethroid 0.59 (0.660. 0.28 (0.23.667.96734.18–4.6613.5) 0. Cr. Crops in which Quantity detected.91 [86] Wa 1 13.9) 1.t002 abbreviations used in the table.25 [68] Ap 3 171.97){ Endosulfan I Cyclodiene 54.15.7 (570) 2.388.02) Captan .43.47 [60] Ap 3 59. 0. 0. Bl = blueberry.0.5) 1.4 (14.8630. Cu = cucumber.360.282.1371/journal.35. Wa 6 57.5) 0.1. 2.6 (12) 1. 0.1 (15.08. PLOS ONE | www. Wa = watermelon.14){ Endosulfan II Cyclodiene 54.06 [66] Ap.54.62 (0.54.0 (36.5 (12.8 (12.92){ formamide (DMPF)* metabolite Acetamiprid Neonicotinoid 55.161.700) 0.05 (0. 0. Pu 4 separate 2. 0.31 (0.54 (0. 1.05 (0. 2.661.96){ Heptachlor epoxide Heptachlorb (cyclodiene) Cr 1 0. Wa 10 60.000) 2.261. 0. 1.3 (4.05 (0.08 (1.42.65){ Fenbuconazole .89 (0. Cr. Cr.83){ Carbaryl Carbamate 8.0 (17.862. { Relative risk different from 1 at the 95% confidence level. Cu.4 Dimethylphenyl Amitraz (formamidine) Bl. 1.53. Cr. Pu.128 (equivalent to multiplying by 7.28 [73] Ap.560.38 [78] Cr 1 0. Pu = pumpkin. Cu 10 227.59. 1. 2.83 [85] Ap. Wa 2 0. 2. 3.47 (1.15.33 (0.460. Cu. Cr.78 [71] Cr 2 0.4) 0.05) Thiacloprid Neonicotinoid 114.4) 1.79 (0.42 (0. Wa 9 976. Cu.33.42 (0.7 (29.97 (0.54. b Heptachlor has been banned for use on cranberries since 1978 [87].85. 0. Wa 3 6.5 (9) 0.470) long table.0 (2.360.491.160.49.66){ Chlorpyrifos Organophosphate 5 July 2013 | Volume 8 | Issue 7 | e70182 .39 [79] Cr 1 4.04) Diazinon Organophosphate 1.4 (5. 2.163.35 (0.16531.130. 0.2 (1.564.82){ Herbicides Carfentrazone ethyl .59 [75] Ap. Cr = cranberry.05 (0.8 (53.

Bars show the total number of pesticides in each category found in each crop.pone. White bars show pesticide diversity. pyrethroids.1371/journal.01. H6 = 17. neonicotinoids. e. Exposure to fungicides can also consumed pollen containing pyraclostrobin were almost three times as likely (relative risk = 2.g004 only via apple pollen.01. doi:10. The pesticide with the same degree as imidacloprid [56]. Indeed. 0.0070182. p = 0.g003 PLOS ONE | www. d. 0.03. [21.9. cucumber e.1371/journal.pone. and watermelon d. A * indicates that the total number of pesticides varied between crops within that pesticide category. we found fungicides at high loads in our sampled crops. where letters indicate statistically significant differ- ences: apple a. Post-hoc testing found the following groups.plosone.05. Sequential Bonferroni adjusted critical values are: 0. d. p = 0. Figure 3. blueberry c. H6 = 16.3.g.025. H6 = 14. Load varied by pesticide category.85. an insecticide with numerous highest relative risk was the fungicide pyraclostrobin.0167. especially weighted by the number of chemicals in that category. In our study." as likely (relative "While.75. This result pollen samples) and myclobutanil increases gut cell mortality to the is likely driven by chlorothalonil loads.g002 Transition words such thought. cyclodienes. Crop Pollens Affect Bee Health Figure 2.007.33. the doses [58]. reducing medial lethal still have potential for lethal [55] and sub-lethal effects. However. We only compared pesticide diversities for categories containing at least three chemicals. cranberry_late b. doi:10. f. While fungicides are typically less lethal to bees than insecticides (see LD50 values in Table 2). organophosphates.0070182.57]). p = 0.8. The total load for each category is bees has focused almost entirely on insecticides.0125.2) in bees that consumed these fungicides and than in bees that increase "Indeed" Figure 4. p = 0. pumpkin c." risk . Research on the sub-lethal effects of pesticides on honey sentences. Kruskal-Wallis test statistics comparing pesticide diversity between crops are: fungicides.26. consuming pollen with higher fungicide fungicides chlorothalonil (found at high concentrations in our loads increased bees’ susceptibility to Nosema infection.0070182. b. cranberry_early d. Table 2) Study conclusions are discussed in relation to previous research. p = 0. Bees that sub-lethal effects (e.9.16–3. H6 = 6. gray bars show pesticide load (mean 6 se). doi:10. Pesticide diversity found in pollen samples. f. varied by crop. 0. In our study. flow between statistically significant differences. Nosema infection was more than twice as "However. H6 = 7.1371/journal. 95% CI 2.1.pone. Letters indicate did not. to facilitate neonicotinoids [54]. these chemicals make bees more sensitive to acaricides. Fungicide and neonicotinoid diversities varied by 6 July 2013 | Volume 8 | Issue 7 | e70182 . but not pesticide load. 0. neonicotinoids entered the nest comparison across categories.

Our results show the necessity of can also affect bee activity levels. and may in the same direction. organophosphates (couma- by feeding pollen with real-world pesticide blends and levels that phos. there is a pressing need for PLOS ONE | www.pone. In our is expected to reduce miticide levels in Citations study and those listed above. research suggests that simultaneous exposure to multiple pesticides decreases lethal doses [58.0169 ppm for esfenvalerate. doi:10. While the mean loads for these pesticides are well below their respective median lethal doses (0. would slow control hive pests were present in a large within the sentence proportion of the questions. pesticides can have sub-lethal effects on development. was from the target crop. bees exposed three the pyrethroids (bifenthrin and fluvalinate. reproduction.0098 ppm [21.58]. however. samples. [27] resulted in almost triple the pollen on foragers returning to the hive is 7 July 2013 | Volume 8 | Issue 7 | e70182 . of resistance to these chemicals. weather conditions undoubtedly differed between than bees consuming pollen without this chemical to become crops. Esfenvalerate (LD50 = 0." "Second.0365 ppm. An increasingly Significance number of pesticides we found.13 ppm) was measured at 0. however. to pesticides per sample (7. Our study improves on previous methodologies prid. thus outcomes the fungicide load in the pollen they were fed. The path from in-hive application of these miticides to paper discussed.18. and foraging behavior. when appropriate. First. diverse sampling of Mullin et al. miticides clearly stated and analyses of pollen collected by honey bees or honey bee nest applied by beekeepers to help control the highly-destructive Varroa material [16.83 ppm) at 14. directional separation by insecticide family.1371/journal. and will hopefully decrease spread of these chemicals to the environment. Given the diverse routes ofto effects on honey bee immune functioning. Crop flowering timing and landscape-level floral availability infected after Nosema exposure.60] or increases supersedure (queen replacement) rate [61]. additional sampling is necessary to determine how robust this pattern is.0070182. learning and memory. Second. Our pollen samples contained an average of nine different pesticides. but not to imidacloprid and Nosema can have lower spore counts than bees esfenvalerate) were associated with an increased risk of Nosema only infected with the pathogen but also exhibit hindered immune infection. Research looking at additive and synergistic effects between multiple pesticides is clearly needed. imidacloprid and thiacloprid). but the average number of popular of practice. The formamidine (DMPF) and two of the depend on the pesticide or dose used. diazinon and phosmet) and the oxadiazine (indoxacarb) truly represents the types of exposure expected with pollination of were associated with reduced risk of Nosema infection.7987 ppm for phosmet). Third.27]. For example. our data indicate some bee colonies are being exposed to incredibly high levels of these chemicals. pesticides’ effects on Nosema infection dynamics [13–15] indicate that a detrimental interaction occurs when honey bees are exposed relative risk values significantly different than one were almost all to both pesticides and Nosema. Histograms show the number It is not surprising that total pollen collection varied by crop.plosone. pesticides applied are placed by beekeepers to broader research hives. 0. however. such as the diversity of testing more broadly than the insecticides that are the targets of pollen types found in samples and the proportion of a sample that most current research. and phosmet (LD50 = 8. Conclusions are A similarly large increased risk of Nosema infection was Our results are consistent with previously published pesticide associated with consumption of DMPF and fluvalinate. The moresignificance ofgeographically more intensive and mite [3]. rotating combs out of hives to remove resultsaccumulated in relationpesticides. ranging as high as 21 pesticides in one cranberry field. Esfenvale- agricultural crops. Because of the relatively small number of pesticides we found in each insecticide family.g005 cloud cover or humidity during data collection. two pollen samples contained one pesticide each at a concentration higher than the median lethal dose.216 ppm in pollen collected by bees in a cucumber field. Thus published LD50 values may not accurately indicate pesticide toxicity inside a hive containing large numbers of pesticides. These Significance indicates a pressing need for further research on lethal and ofsub- patterns suggest that insecticides’ modes of action have differential work in relation lethal effects of fungicides on bees. The large numbers of pesticides found per sample and the high concentrations of some pesticides are concerning." Crop Pollens Affect Bee Health and "Third" guide readers through the paper. and advocate for variables less affected by these factors. respectively) are higher than some published imidacloprid concentrations with sub-lethal effects on honey and bumble bees Figure 5.001–0. The curve shows the of short-term measurements may be sensitive to temperature.62]). and increasing other research evidence that pesticide blends harm bees [16. Probability of Nosema infection increased with (0. We focused our analyses on testing for sub-lethal effects of pesticides on bees. fungicide load in consumed pollen. Because we collected pollen samples from different parts of the country and on different days.18. Use of "First. Within a family. exposure to pesticides we show. The significant increase in Nosema infection rate and coumaphos have previously been found to be associated following exposure to the fungicides in pollen we found therefore with apiaries without Colony Collapse Disorder [59]. Specific results vary. avoids overstating Insecticide relative risk values showed an interesting pattern: Our results combined with several recent studies of specific the implications. all neonicotinoids (acetami- functioning [13]. The carbamate (carbaryl).0028 and 0.1.7 ppm in one apple orchard. predicted probability of Nosema infection. Potential extra-nest sources. of bees with (top) and without (bottom) Nosema spores as a function of Bee foraging activity levels vary with weather [63]. often in quantities higher than most(and of thenot pesticides that efforts to reduce miticide accumulation and slow the development at the end) the word "suggests" are applied to crops. The mean and maximum imidacloprid loads in our samples (0.1) is slightly lower than our 9.54.

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