Name: empirical, metagenomic, and computational techniques illuminate the mechanisms by which fungicides compromise bee health
Uploaded: 2017-10-09T12:30:00
Description: Watch this Scientific Journal Video about Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health at JoVE.com

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...

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 ...

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