DOI: 10.5061/dryad.rv15dv4f4
title: Consumption of pollen contaminated with field-realistic concentrations of fungicide cause sub-lethal effects in common eastern bumble bee (Bombus impatiens [Hymenoptera]: [Apidae]) microcolonies

description: Bumble bees are declining across the globe. Causes of this decline have
 been attributed to a variety of stressors, including pesticides.
 Fungicides are a type of pesticide that have been understudied in the
 context of bumble bee health. As a result, fungicides are often applied to
 flowering plants without consideration of pollinator exposure. Recent work
 demonstrates that fungicides have sublethal effects in bumble bees, but
 little is known about how much fungicide it takes to cause these sublethal
 effects. To address this gap in the literature, we fed microcolonies of
 the common eastern bumble bee (Bombus impatiens) pollen contaminated with
 a range of fungicide concentrations. We chose these concentrations based
 on the range of fungicide concentrations in pollen and nectar that were
 reported in the literature. Results revealed that later-stage pupae and
 newly emerged males are potentially sensitive to fungicide exposure,
 showing smaller size and reduced fat reserves at intermediate levels of
 contamination. Compared to the control, intermediated levels of
 fungicide-contaminated pollen led to increased pupal mortality and delayed
 male emergence. Contrary to expectations, higher fungicide levels did not
 exhibit a linear relationship with negative impacts, suggesting nuanced
 effects. Because body size and emergence timing are important aspects of
 bumble bee reproductive behavior, results have implications for mating
 success, potentially disrupting colony development.
description: To understand how fungicides affect bumble bee microcolony health
 and reproduction, we established five fungicide treatments, which varied
 by the concentration of Pristine® in their pollen diet. We selected
 experimental values of Pristine® of 0 ppb, 150 ppb, 1,500 ppb, 15,000 ppb
 and 150,000 ppb.We exposed bumble bees to fungicide by incorporating it
 into pollen balls, which we made in batches per treatment by mixing 90g of
 honey bee-collected pollen with 35ml of a 50/50 sucrose:water solution
 containing the appropriate concentration of fungicide. We made pollen
 balls for each treatment in separate batches, from a single supply of
 homogenized honey bee-collected pollen. Using callow worker bees from four
 purchased <em>B. impatiens</em> queen-right colonies (Plant
 Products USA, Westland, MI), we established microcolonies consisting of
 five individuals, supplied with a 50/50 sucrose:water solution, a 1 g
 (±0.5 g) fungicide-free pollen ball, and 1-2 g of wax material from their
 natal colony to stimulate oviposition and brood production behavior. We
 replicated each of the five fungicide treatments nine times, for a total
 of 45 microcolonies. We placed microcolonies in a rearing room under red
 light at The Ohio State University’s Rothenbuhler Honey Bee Research
 Laboratory. Rearing room conditions were kept between 30°C-32°C, and
 50%-70% relative humidity. We blocked by location within the rearing room,
 where each block contained microcolonies from each fungicide treatment and
 ensured that all workers within a block originated from the same original
 queenright colony to control for potential confounding genetic
 differences. Our experiment ran from August to October 2022, during which
 time we monitored the pollen consumption, brood production, and mortality
 in the 45 microcolonies. We stored data in Microsoft Excel, and ran all
 analyses in R. 
description: This README file was generated on 2024-05-02 by Emily Runnion. GENERAL
 INFORMATION 1. Title of Dataset: Consumption of pollen contaminated with
 field-realistic concentrations of fungicide cause sub-lethal effects in
 common eastern bumble bee (Bombus impatiens [Hymenoptera]: [Apidae])
 microcolonies 2. Author Information A. Principal Investigator Contact
 Information Name: Emily Runnion Institution: The Ohio State University
 Address: Columbus, OH USA Email:
 [Runnion.4@osu.edu](mailto:Runnion.4@osu.edu) B. Associate or
 Co-investigator Contact Information Name: Jamie Strange Institution: The
 Ohio State university Address: Columbus, OH USA Name: Frances Sivakoff
 Institution: The Ohio State university Address: Columbus, OH USA 3. Date
 of data collection (single date, range, approximate date): 2022 4.
 Geographic location of data collection: Columbus, Ohio, USA 5. Information
 about funding sources that supported the collection of the data: This work
 was funded by Sustainable Agriculture Research and Education Projects,
 North-Central Region, Grant Agreement GNC21-335 (to ENR) and an OSU CFAES
 Research & Graduate Education Internal Grant (to FSS and JPS). 6.
 Licenses/restrictions placed on the data: CC0 1.0 Universal (CC0 1.0)
 Public Domain 7. Links to publications that cite or use the data: 8. Links
 to other publicly accessible locations of the data: None 9.
 Links/relationships to ancillary data sets: None 10. Was data derived from
 another source? No A. If yes, list source(s): NA DATA & FILE OVERVIEW
 1. File List: A) brood.csv C) drone.health.csv D) pollen.csv E) qro.csv F)
 workers.csv 1. Relationship between files, if important: None 2.
 Additional related data collected that was not included in the current
 data package: None 3. Are there multiple versions of the dataset? No A. If
 yes, name of file(s) that was updated: NA i. Why was the file updated? NA
 ii. When was the file updated? NA
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: brood.csv 1. Number of variables: 24 2.
 Number of cases/rows: 145 3. Variable List: * colony: unique identifier
 for each of the 45 microcolonies * whole.mean: the average amount of
 pollen eaten, per unique microcolony, every 24 hours from beginning to end
 of the experiment * mean.dose - the average amount of Pristine (fungicide)
 consumed per unique microcolony, every 24 hours from beginning to end of
 the experiment * dose: the amount of Pristine (fungicide) added in ppb to
 each 1 gram of pollen fed to each microcolony every 24 hours * treatment:
 Number 1-5, signifying the concentration of Pristine (fungicide) fed to
 each microcolony. Treatment 1 = Control, no fungicide. Treatment 2 =
 150ppb, Treatment 3 = 1500ppb, Treatment 4 = 15000ppb, Treatment 5 =
 150000ppb * replicate: blocking variable * brood_cells = the total count
 of pupae, larvae, egg sacs, and honey pots per microcolony at the
 conclusion of the experiment, following microcolony dissection * honey_pot
 = total count of honey pots per microcolony. Honey pot determined as a
 open pula cell containing nectar. * eggs = total count of eggs per
 microcolony at the end of the experiment following microcolony dissection
 * dead_larvae: total count of dead larvae per microcolony at the end of
 the experiment following microcolony dissection * live_larvae: total count
 of live larvae per microcolony at the end of the experiment following
 microcolony dissection * dead_pupae: total count of dead pupae per
 microcolony at the end of the experiment following microcolony dissection
 * live_pupae: total count of live pupae per microcolony at the end of the
 experiment following microcolony dissection * dead_drones: total count of
 dead drones per microcolony at the end of the experiment following
 microcolony dissection. dead_drones were counted as those males that did
 not successfully emerge, but surpassed the pupal stage, as seen by removal
 of the brood cell capping, but due to deformed wings, unknown illness,
 etc., were not able to exit the brood cell. * live_drones: total count of
 males found outside of brood cells within post-frozen microcolonies and
 subsequent dissection. * drones: total count of successfully emerged new
 male bees per microcolony over the course of the experiment. * qro: unique
 identifier given to the original queenright colony which the workers in
 the matching microcolony were sourced from. * duration: amount of time the
 microcolony had living workers, from the day callow workers were placed
 from the original queenright colony until the day the microcolony was
 frozen. Microcolonies were frozen either 1) when all workers died, 2)
 seven days after the first male emerged, or 3) within two standard
 deviations of the average microcolony duration (this was used for
 microcolonies that did not produce males) * alive: the number of workers
 alive inside the colony when the microcolony was frozen * dead: the number
 of workers out of 5 who were dead when the microcolony was frozen 1.
 Missing data codes: None 2. Specialized formats or other abbreviations
 used: None
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: drone.health.csv 1. Number of variables: 21
 2. Number of cases/rows: 421 3. Variable List: * colony: unique identifier
 for each of the 45 microcolonies * whole.mean: the average amount of
 pollen eaten, per unique microcolony, every 24 hours from beginning to end
 of the experiment * mean.dose - the average amount of Pristine (fungicide)
 consumed per unique microcolony, every 24 hours from beginning to end of
 the experiment * dose: the amount of Pristine (fungicide) added in ppb to
 each 1 gram of pollen fed to each microcolony every 24 hours * treatment:
 Number 1-5, signifying the concentration of Pristine (fungicide) fed to
 each microcolony. Treatment 1 = Control, no fungicide. Treatment 2 =
 150ppb, Treatment 3 = 1500ppb, Treatment 4 = 15000ppb, Treatment 5 =
 150000ppb * replicate: blocking variable * id - unique identifier given to
 each emerged male * emerge_time: time since the initiation of the
 microcolony until this row's male emerged * wet_weight: the weight of
 the newly emerged drone, in grams, while alive * dry_weight: the weight of
 the whole body of the male after freezing, and then being dried in an oven
 at 60 degrees Celsius for 72 hours * radial: the length of the male's
 right radial wing vein, in micrometers * abdomen_dry: the weight, in
 grams, of the dried abdomen of the male. Abdomens removed immediately
 after recording whole-body dry weight. * order_on_sheet: order in which
 the males emerged during the experiment * abdomen_post_ethyl: the weight
 of the male's abdomen, in grams, after being removed following the
 initial 72 hour drying, soaked in ethyl ether for 24 hours, and dried
 again. See Methods for particulars. * fat_content: the fat content of the
 male's abdomen, calculated as the difference between the
 "abdomen_dry" value and the "abdomen_post_ethyl" value
 * relative_fat: the relative fat content of the male's abdomen,
 calculated as the value of "abdomen_post_ethyl" divided by
 "radial" * workers_alive: the number of workers alive inside the
 colony when the male emerged * qro: unique identifier given to the
 original queenright colony which the workers in the matching microcolony
 were sourced from. * duration: amount of time the microcolony had living
 workers, from the day callow workers were placed from the original
 queenright colony until the day the microcolony was frozen. Microcolonies
 were frozen either 1) when all workers died, 2) seven days after the first
 male emerged, or 3) within two standard deviations of the average
 microcolony duration (this was used for microcolonies that did not produce
 males) 1. Missing data codes: NA (data not available) Missing data
 described based on male "id" 1.11R2.1 3.7R2.3 3.9R2.2 3.7R2.2
 4.11R2.1 4.4R2.7 1.4R2.2 4.4R2.8 1.4R2.3 These males had deformed wings
 and we could not measure the radial cell length 4.12R2.2 2.1R2.7 4.3R2.2
 1.5R2.1 3.3R2.3 4.4R2.5 1.7R2.1 1.4R2.1 4.3R2.3 4.4R2.9 4.4R2.3 4.4R2.1
 4.3R2.5 3.3R2.2 4.4R2.4 4.3R2.1 4.3R2.4 5.5R2.1 3.3R2.1 4.4R2.6 1.7R2.2
 4.4R2.2 These males' abdomens were damaged during the ethyl ether
 process 5.12R2.1 4.11R2.2 We forgot to record these male's wet weight
 4.4R2.13 5.7R2.5 1.5R2.6 These males' relative fat values were
 negative and therefore not included 1. Specialized formats or other
 abbreviations used: None
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: pollen.csv These are the data used to
 calculate relative body mass, then analyze change in relative body mass
 for recaptures (i.e., birds captured in the pre and post periods of the
 experiment). 1. Number of variables: 18 2. Number of cases/rows: 933 3.
 Variable List: * colony: unique identifier for each of the 45
 microcolonies * treatment: Number 1-5, signifying the concentration of
 Pristine (fungicide) fed to each microcolony. Treatment 1 = Control, no
 fungicide. Treatment 2 = 150ppb, Treatment 3 = 1500ppb, Treatment 4 =
 15000ppb, Treatment 5 = 150000ppb * dose: the amount of Pristine
 (fungicide) added in ppb to each 1 gram of pollen fed to each microcolony
 every 24 hours \ * pristine/g: the amount of Pristine (Fungicide) per gram
 in each pollen ball * replicate: blocking variable * count: the pollen
 ball count per microcolony. For example, "4" would mean this was
 the 4th pollen ball supplied to the microcolony. * start_date: the day the
 pollen ball was placed in the microcolony * start_time: the time the
 pollen ball was placed in the microcolony * start_weight: the weight, in
 grams, of the pollen ball when placed in the microcolony * end_date: the
 day the pollen ball was removed from the microcolony * end_time: the time
 the pollen ball was removed from the microcolony * end_weight: the weight
 of the pollen ball, in grams, after being removed from the microcolony
 following a period of 48 hours of allowing the workers free access to eat
 the pollen * whole_dif: the raw difference between
 "start_weight" and "end_weight" in grams * difference:
 the weight difference between the "start_weight" and
 "end_weight" values after adding back in the weight calculated
 lost from moisture using the amount of moisture lost from the evaporative
 control pollen ball assigned to that microcolony that week *
 percent_consumed: the percent of the pollen ball consumed by the
 microcolony ("end_weight" divided by "start_weight") *
 dose_consumed: the calculated amount of Pristine (fungicide) consumed by
 the microcolony from that pollen ball, in Pristine/g * pid: unique
 identifier for each individual pollen ball * bees_alive: the amount of
 workers alive in the colony at the time the "end_weight" was
 recorded 1. Missing data codes: NA (data not applicable) Missing data
 described based on "pid" R2.3.7-21 This pollen ball was not
 removed from the box because there were eggs in it R2.1.9-17 R2.3.11-5
 R2.4.12-4 R2.5.12-12 These pollen balls were not included in analysis
 because they contained too much frass to be adequately measured 1.
 Specialized formats or other abbreviations used: None
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: qro.csv 1. Number of variables: 2 2. Number
 of cases/rows: 45 3. Variable List: * colony: unique identifier for each
 of the 45 microcolonies * qro: unique identifier given to the original
 queenright colony which the workers in the matching microcolony were
 sourced from. 1. Missing data codes: None 2. Specialized formats or other
 abbreviations used: None
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: workers.csv 1. Number of variables: 17 2.
 Number of cases/rows: 224 3. Variable List: * colony: unique identifier
 for each of the 45 microcolonies * whole.mean: the average amount of
 pollen eaten, per unique microcolony, every 24 hours from beginning to end
 of the experiment * mean.dose - the average amount of Pristine (fungicide)
 consumed per unique microcolony, every 24 hours from beginning to end of
 the experiment * treatment: Number 1-5, signifying the concentration of
 Pristine (fungicide) fed to each microcolony. Treatment 1 = Control, no
 fungicide. Treatment 2 = 150ppb, Treatment 3 = 1500ppb, Treatment 4 =
 15000ppb, Treatment 5 = 150000ppb * replicate: blocking variable *
 start_wet_weight: the weight of the live worker in grams on the day they
 were first placed in the microcolony * alive_at_end: a logical variable,
 coded "1" for yes/true and "0" for no/false, to
 signify if the worker bee was alive when the microcolony was shut down *
 shut_down_dt: the day the microcolony was placed in the freezer *
 dry_weight: the whole body dry weight of the worker bee in grams after
 being frozen and dried for 72 hours at 60 degrees Celsius * days_alive:
 the number of the days the worker bee was alive during the duration of the
 experiment * colony_duration: amount of time the microcolony had living
 workers, from the day callow workers were placed from the original
 queenright colony until the day the microcolony was frozen. Microcolonies
 were frozen either 1) when all workers died, 2) seven days after the first
 male emerged, or 3) within two standard deviations of the average
 microcolony duration (this was used for microcolonies that did not produce
 males) * qro: unique identifier given to the original queenright colony
 which the workers in the matching microcolony were sourced from. * alive:
 the number of workers alive inside the colony when the microcolony was
 frozen * dead: the number of workers out of 5 who were dead when the
 microcolony was frozen 1. Missing data codes: none 2. Specialized formats
 or other abbreviations used: None
 \#########################################################################