The author(s) thank the University of Wisconsin Biotechnology Center DNA Sequencing Facility for providing amplification and sequencing facilities and services, Caitlin Carlson, Jennifer Knack, Jake Otto, and Max Haase for providing technical assistance with molecular analysis. This work was supported by USDA-Agricultural Research Service appropriated funds (Current Research Information System #3655-21220-001). Further support was provided by the National Science Foundation (under Grant No. DEB-1442148), the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494), and the USDA National Institute of Food and Agriculture (Hatch project 1003258). C.T.H. is a Pew Scholar in the Biomedical Sciences and an Alfred Toepfer Faculty Fellow, supported by the Pew Charitable Trusts and the Alexander von Humboldt Foundation, respectively. The authors thank Hannah Gaines-Day and Olivia Bernauer for their contributions to our earlier, published field work.

1. Examine the Effect of Fungicide Exposure on Bumble Bee Colony Success Using Field Cage Experiments
<ol>
	<li>Set up ten mesh cages in a field planted with oats. Dig a trench around each cage, and dig all four edges of the mesh cage into the ground to ensure that bees cannot escape. Stock the cages with potted, flowering plants that are known to be attractive to bees (e.g. buckwheat, borage, alyssum, cosmos, and sunflowers) (Figure 2).</li>
	<li>Supplement the cages with a singl...

The authors have nothing to disclose.

Investigations into the effects of fungicides on bee health have remained an understudied aspect of pest management strategies. Our study aims to bridge this knowledge gap by using a suite of complementary techniques that explicitly isolate the potential factors driving bee declines. The planning, rationale, and rendering of these experiments are detailed below.
It is important to ensure that no bees are allowed to escape the mesh of the cage experiments, since this would compromise demography...

Managed and wild bee species are experiencing widespread declines, with major implications for both natural and agricultural systems1. Despite concerted efforts to understand the causes of this problem, the factors driving honey bee declines are still not well understood2,3,4. For certain species of wild, native bees, the situation has become dire5,6. If bee populations cannot be sustained when they intersect with industrial agriculture, their populations will continue to fall, and the crops requiring pollinators (35% of worldwide production7) will endure reduced harvests.
While many potential factors such as pesticide exposure, disease, and habitat loss1,4,8,9,10 have been implicated in honey bee decline, relatively little is known about the interactive effect of these stressors on native bees health, within or near agricultural systems. Many current research efforts continue to focus on insecticides, (e.g., neonicotinoids11,12), although past research indicates that fungicides may also play a role in bee decline by impairing memory formation, olfactory reception13, nest recognition14, enzyme activity and metabolic functions15,16,17. Globally, fungicides continue to be applied to flowering crops during bloom. Recent studies have documented that bees commonly bring fungicide residues back to the hive18, indeed, studies have shown a large proportion of tested hives contained fungicide residues19,20. Further work has revealed that fungicide residue is associated with high rates of honey bee larval mortality21,22,23 and the presence of &#34;entombed pollen&#34; within colonies, which although non-toxic, is devoid of microbial activity and is nutritionally compromised24. Despite the fact that fungicides have long been considered &#34;bee-safe,&#34; there is now evidence that exposure to fungicide alone can cause severe colony losses in a native bumble bee species, Bombus impatiens25.
To establish causality between fungicide exposure and colony mortality, the modus operandi of these chemicals need to be determined. As evidenced in soils26, sediments27, and aquatic environments28, by targeting fungi, fungicides most likely alter fungal abundance and diversity within pollen-provisions, thereby invoking a major community shift that may strongly favor bacteria. Without fungal competitors or antagonists, certain pathogenic bacteria can proliferate relatively unchecked, facilitating the spoilage of pollen-provisions. Past research has demonstrated that microorganisms, particularly yeasts and filamentous fungi, serve as nutritional symbionts for bees29,30,31, protect against parasites and pathogens32,33, and provide long-term preservation of pollen stores. Fungicides, therefore, may indirectly harm immature bees by disrupting the microbial community that is needed to provide these services and/or by increasing susceptibility to opportunistic pathogens and parasites12. With increasing demands on food production, crops worldwide are being sprayed each year with fungicides during bloom, underscoring the need to understand the magnitude of such fungicide-induced effects.
To-date, the primary knowledge gaps relating to native bee microbial ecology can be represented by the following questions: To what extent does fungicide change the microbial community within bee pollen-provisions? What are the downstream impacts of consuming pollen with a profoundly altered microbial community? In keeping with these ecologically germane questions, experiments were developed with the primary goals of revealing 1) that fungicide residue alone can cause severe colony decline in a native bee species; 2) the degree to which microbial communities in pollen-provisions are altered by fungicides, and 3) how bee health is affected by a severely altered microbial community. The experimental objectives were defined to address the above questions using a combination of laboratory- and field-based experiments. Using state-of-the-art metagenomic and molecular techniques alongside traditional methods of field observation, this research aims to piece together the potential effects of fungicides on bee health.
The first objective of this study is to demonstrate that fungicide exposure alone can cause significant colony losses among native bee species. A study involving large field cages was used to investigate the effects of fungicide exposure on the colony growth of Bombus impatiens, a ubiquitous, abundant native bee in the US (Figure 1, Figure 2, Figure 3). It was hypothesized that fungicide-treated hives would present lower fitness, and atypical demography compared to non-exposed hives. Data obtained from this experiment supported this hypothesis, demonstrating that fungicide residues within pollen can be the sole cause of profound colony losses in a native bumble bee species25. The second objective of this study is to investigate the response of the pollen microbiome to fungicide exposure. It is hypothesized that the community composition of microbes within pollen-provisions exposed to fungicides will be different from that of untreated pollen. While fungal abundance and diversity are expected to decline significantly, bacteria and/or a single dominant fungal species will likely grow unchecked in the absence of other competing fungi. Through a series of in-vivo trials, these shifts in microbial community composition will be analyzed using metagenomics.

<table>
	<tbody>
		<tr>
			<td>Natupol Beehive</td>
			<td>Koppert</td>
			<td>USRESM1</td>
			<td>16 hives</td>
		</tr>
		<tr>
			<td>Propiconazole 14.3</td>
			<td>Quali-Ppro</td>
			<td>60207-90-1</td>
			<td>Propiconazole 14.3%</td>
		</tr>
		<tr>
			<td>Abound</td>
			<td>Syngenta</td>
			<td>4033540</td>
			<td>Azoxystrobin 22.9%</td>
		</tr>
		<tr>
			<td>Chlorothalonil</td>
			<td>Syngenta</td>
			<td>3452</td>
			<td>Fungicide used for trials</td>
		</tr>
		<tr>
			<td>Pollen granules</td>
			<td>Bee rescued</td>
			<td>B004D5650C</td>
			<td>3X 16oz bottles, pollen for trials</td>
		</tr>
		<tr>
			<td>Bacterial strains for inoculation</td>
			<td>Currie Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast strains for inoculation</td>
			<td>Hittinger lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer pairs</td>
			<td>UW Biotech Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Isolation Kit</td>
			<td>Mo Bio</td>
			<td>12830-50</td>
			<td>Commercial DNA isolation kit</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Thermo Fisher</td>
			<td>Q32851</td>
			<td>DNA quantification tool</td>
		</tr>
		<tr>
			<td>Select Master Mix for CFX</td>
			<td>Thermo Fisher</td>
			<td>4472952</td>
			<td>Used to perform real-time PCR using SYBR GreenER dye.</td>
		</tr>
		<tr>
			<td>Real-Time PCR Detection System</td>
			<td>Bio Rad</td>
			<td>1855196</td>
			<td>Instrument used for PCR amplification</td>
		</tr>
		<tr>
			<td>PCR Clean-Up Kit,</td>
			<td>Axygen</td>
			<td>10159-696</td>
			<td>Used for efficient removal of unincorporated dNTPs, salts and enzymes</td>
		</tr>
		<tr>
			<td>DNA 1000 Kit</td>
			<td>Agilent</td>
			<td>5067-1504</td>
			<td>Used for sizing and analysis of DNA fragments</td>
		</tr>
		<tr>
			<td>MiSeq Sequencer</td>
			<td>Illumina</td>
			<td></td>
			<td>Used for next-generation sequencing</td>
		</tr>
		<tr>
			<td>Assorted glassware (beaker, flasks, pipettes, test tubes, repietters)</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

empirical, metagenomic, and computational techniques illuminate the mechanisms by which fungicides compromise bee health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Field cage study:
Data obtained from the cage experiments showed that the bumble bee colonies had a significant response to fungicide exposure. The fungicide-treated hives produced significantly fewer workers (12.2 &#177; 3.8, mean &#177; SE) than the control hives (43.2 &#177; 11.2, F1,9= 6.8, p = 0.03) (Figure 4). Additionally, the bee biomass of the fungicide-tr...

Watch this Scientific Journal Video about Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health at JoVE.com

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

The overall goal of this multidisciplinary project is to isolate the specific mechanisms by which fungicide residue on pollen may cause colony declines in bumblebees. This method can help answer key questions relating to the high mortality among larval bumblebees as affected by the fungicide mediated disruption of bee microbes symbioses. The main advantage of this technique is that it investigates the indirect effect of fungicides on bees by tracking bee demographics alongside shifts in the microbial communities within bee pollen.Although these methods provide insight into native bee health, it can also be applied to other pollinators such as honeybees, which play a critical role in crop pollenization globally, thereby emphasizing the wise application of fungicides to flowering crops. Demonstrating the procedures in this collaborative study will be Prarthana Dharampal, Luis Diaz-Garcia and Caitlin Carlson. To begin, set up mesh cages in a field planted with oats.Prepare one per replicate. Plan to make at least 10 for a single treatment experiment. First dig a trench around each cage and dig all four edges of the mesh cage into the ground to ensure that the bees can not escape.Then stock the cages with potted flowering plants that are known to be attractive to bees, such as buckwheat, borage, alyssum, cosmos and sunflowers. Also supplement each cage with a single tray of in-bloom clover. Cluster all the floral resources within one corner of the cage, occupying about 2.5 square meters.Vegetate the remaining cage surface area with oats. Before loading the colonies into the field cages, make certain that each contains workers and a single queen. Then load one colony per cage for a 29 day period.In the cage, orient the colony boxes such that the colony's openings point to the south to provide the bees with optimal navigational conditions. Next apply the experimental treatment to half the cages. In this case use a hand-held pesticide sprayer to apply chlorothalonil based fungicide at a field relevant level to the flowering plants.This treatment is applied at day zero and again on day 13. Coat the flowers uniformly such that no additional liquid adheres to floral surfaces. At the end of the study period, remove the colonies from the cages and anesthetize the bees by placing the colonies in a minus 20 degrees Celsius freezer for 20 minutes.Once anesthetized, remove the bees using sterile forceps and record the number of larvae, pupae, adult females and adult males, and the Mother Queen. Then use an analytical balance and record the dry weight of the Mother Queen and other classes of bees. To begin, pulverize commercially purchased pollen into a fine powder using a standard laboratory ball mill.Then to sterilize the powdered pollen, soak it in 70 per cent ethanol. Allow the ethanol to evaporate overnight under UV light in a bio-safety cabinet. The next day, verify the sterility of pollen by plating a small amount on general purpose agar media using sterile toothpick.Incubate the plate at 28 degrees Celsius for 48 hours and only use it if there is no growth. Next, using sterile pipettes, add a field-relevant dose of fungicide to the sterilized pollen and thoroughly mix the two together using sterile wooden sticks. Use sterile water for a control.Now prepare the hives for the experiment in sample sizes of at least three. On a daily basis, asepetically prepare the pollen aliquots for each hive. Then via the trap door in the box enclosing each hive, introduce the pollen.In addition to the pollen, on a weekly basis provide each hive with sterilized sugar solution. Perform this feeding regime for four weeks. As before, anesthetize the hives at the end of the test period.Then very gently pry open and exhume the den contents without letting the pollen or larval bees fall away deeper into the nest. Perform the same counting and weighing metrics for the hive as before but also scrape out the pollen provisions contained within the brood chambers using sterilized forceps. Collect the pollen in sterile tubes, then store it at minus 80 degrees Celsius for later analysis.Data obtained from the cage experiments showed that the bumblebee colonies had a significant response to fungicide exposure. The fungicide treated hives produced significantly fewer workers than the control hives. The overall biomass was also reduced in fungicide treated hives including the Mother Queen's mass.Then number and mass of larvae, pupae and adult males was unaffected. The laboratory based experiments mirrored the field-based results. Taken together, these results are consistent with previously published results and indicate fungicide exposure affects colony fitness.Our data show that microbial diversity and evenness were reduced in the fungicide treated hives. Metagenomic analysis of pollen provisions was used to analyze changes in the microbial communities of fungicide treated hives. There was an enormous decrease in the abundance of commonly isolated Streptomycetales and Enterobacteriales in the fungicide treated hives whereas bacterial members of the order Rickettsiales, which includes common pathogens of arthropods were in much greater abundance in the fungicide treated hives.Fungicide treated hives also had a lower abundance of fungi belonging to the orders Eurotiales, Sordariales and Saccharomycetales, while actually increasing the abundance of fungi in the orders Capnodiales and Ascosphaerales. These results indicate that fungicides can alter the microbiome of pollen provisions, effectively disrupting bee microbe symbiosis and compromising bee health. After watching this video, you should have a good understanding of how to culture bumblebees in the field, dissect bee nests and identify the microbial communities of bee pollen provisions, thereby assessing the role of fungicides on bee colony health.Once mastered, these techniques can be used to demonstrate the fungicide-mediated disruption of the pollen microbiome and its indirect effect on the overall health of bumblebee colonies. While attempting this procedure, it's important to remember to maintain aseptic conditions during the preparation of the pollen meals to avoid infections and infestations. Following this procedure, the description of the microbic community structure can be further improved by relaxing the fraction of the total differences between sequences and reducing the number of taxa by merging those with low relative abundance.

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9.6K visualizações.  USDA-ARS. The overall goal of this multidisciplinary project is to isolate the specific mechanisms by which fungicide residue on pollen may cause colony declines in bumblebees. This method can help answer key questions relating to the high mortality among larval bumblebees as affected by the fungicide mediated disruption of bee microbes symbioses. The main advantage of this technique is that it investigates the indirect effect of fungicides on bees by tracking bee demographics alongside shifts in the m...