Name: assessing agrochemical risk to mated honey bee queens
Uploaded: 2021-03-03T14:30:00
Description: Watch this Scientific Journal Video about Assessing Agrochemical Risk to Mated Honey Bee Queens at JoVE.com

Thank you to Dr. Amy Cash-Ahmed, Nathanael J. Beach, and Alison L. Sankey for their assistance in carrying out this work. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. This research was supported by a grant from the Defense Advanced Research Projects Agency # HR0011-16-2-0019 to Gene E. Robinson and Huimin Zhao, USDA project 2030-21000-001-00-D, and the Phenotypic Plasticity Research Experience for Community College Students at the University of Illinois at Urbana Champaign.

1. QMC assembly
<ol>
	<li>Assemble QMCs from parts (Figure 1A) with a single egg laying plate (ELP) inserted as shown in Figure 1B. Do not add feeder tubes until after the workers have been added to the cage. Temporarily cover the 4 feeder holes with laboratory grade tape.</li>
	<li>Insert the queen excluder and the feeding chamber door over the feeding chamber to keep the queen from entering the feeding chamber and contacting the treated diet. See Fine et al....

The fecundity of female solitary insects as well as queens in eusocial insect colonies can be influenced by abiotic stressors such as agrochemicals25,28,29,30,33. In honey bees, the effects of agrochemicals on queens may be indirect, as they can occur via changes in their care and feeding by worker bees. Our representative results, which are similar to those r...

Due to increased global demand for agricultural products, modern farming practices often require the use of agrochemicals to control numerous pests known to reduce or harm crop yields1. Simultaneously, the growers of many fruit, vegetable, and nut crops rely on the pollination services provided by commercial honey bee colonies to ensure abundant crop yields2. These practices may result in pollinators, including honey bees (Apis mellifera), being exposed to harmful levels of pesticide residues3. At the same time, the widespread presence of parasitic Varroa destructor mite infestations in honey bee colonies frequently require beekeepers to treat their hives with miticides, which may also exert negative effects on the health and longevity of the colony4,5,6. To reduce and mitigate harmful effects of agrochemical products, it is necessary to fully evaluate their safety to honey bees prior to their implementation so that recommendations for their use can be made to protect beneficial insects.
Currently, the Environmental Protection Agency (EPA) relies upon a tiered risk-assessment strategy for honey bee pesticide exposure, which involves laboratory tests on adult bees and sometimes honey bee larvae7. If lower tier laboratory tests fail to alleviate concerns of toxicity, higher tier field and semi-field testing may be recommended. While these laboratory tests provide valuable insight into the potential effects of agrochemicals on worker longevity, they are not necessarily predictive of their effects on queens, which differ significantly from workers biologically8 and behaviorally9. Furthermore, there are numerous potential effects of agrochemicals on insects beyond mortality, which can have considerable consequences for social insects that rely on coordinated behaviors to function as a colony unit10,11.
Although mortality is the most commonly considered effect of agrochemical pesticides12, these products can have a wide range of effects on both target and non-target arthropods including altered behavior13,14,15,16, repellency or attractancy17,18,19, changes in feeding patterns20,21,22, and increased or decreased fecundity20,21,22,23,24,25. For social insects, these effects can systemically disrupt colony interactions and functions11. Of these functions, reproduction, which is heavily reliant on a single egg-laying queen supported by the rest of the of the colony unit9, may be particularly vulnerable to perturbation due to pesticide exposure.
Studies performed on immature queens have demonstrated that developmental exposure to miticides can affect adult queen behavior, physiology, survival26,27. Similarly, studies using full or reduced sized colonies have demonstrated that agrochemicals can affect adult honey bee queens by decreasing mating success28, decreasing oviposition29, and decreasing the viability of the eggs produced25,30,31. These phenomena have previously been difficult to observe without the use of whole colonies, due largely to a lack of available laboratory methods. However, a method to study queen oviposition under tightly controlled laboratory conditions using Queen Monitoring Cages (QMC)32 has recently been adapted to examine the effects of agrochemicals on queen fecundity33. Here, these techniques are described in detail along with additional methods to measure and track worker diet consumption in QMCs.
These methods are more advantageous than experiments requiring full sized colonies because they allow for the administration of precise doses of agrochemicals to a greatly reduced number of workers relative to the tens of thousands typically present inside a colony34, which then provision the queen. This exposure technique mirrors the second-hand exposure that queens would experience in real-world scenarios because, within a colony, queens do not feed themselves and rely upon workers to provision them with diet9. Similarly, queens do not generally leave the hive except during colony reproduction (swarming) for mating flights35. Mated honey bee queens can be purchased from commercial queen breeders and shipped overnight. Typically, queen breeders sell queens directly after confirming that they have started to lay eggs, which is taken as an indication of successful mating. If more precise information on queen age or relatedness is needed, researchers may consult with the queen breeder before placing an order.
QMCs allow for precise observation and quantification of honey bee queen oviposition and egg hatching rates32,33, yielding valuable data related to the effects of agrochemical exposure on queen fecundity. The representative results presented here describe an experiment quantifying oviposition, diet consumption, and embryo viability in QMCs under chronic exposure to field relevant concentrations of the systemic neurotoxicant neonicotinoid pesticide imidacloprid36. Once applied, imidacloprid translocates to plant tissues37, and residues have been detected the pollen and nectar of numerous bee pollinated plants38,39,40. Exposure to imidacloprid can have a broad range of detrimental effects on honey bees including impaired foraging performance16, impaired immune function41, and decreased rates of colony expansion and survival42,43. Here, imidacloprid was selected for use as a test substance because field experiments have shown that it can affect honey bee queen oviposition29

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fluon</td>
			<td>BioQuip, Rancho Dominguez, CA</td>
			<td>2871A</td>
			<td></td>
		</tr>
		<tr>
			<td>Honey bee queens</td>
			<td>Olivarez Honey Bees, Orland, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imidacloprid</td>
			<td>Sigma-Aldritch, St. Louis, MO</td>
			<td>37894</td>
			<td></td>
		</tr>
		<tr>
			<td>MegaBee Powder</td>
			<td>MegaBee, San Dieago, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 2 mL</td>
			<td>ThermoFisher Scientific, Waltham, MA</td>
			<td>02-682-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles 20 gauge</td>
			<td>W. W. Grainger, Lake Forest, IL</td>
			<td>5FVK4</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Sulfate</td>
			<td>Sigma-Aldritch, St. Louis, MO</td>
			<td>P0772</td>
			<td></td>
		</tr>
		<tr>
			<td>Queen Monitoring Cages</td>
			<td>University of Illinois Urbana-Champaign</td>
			<td></td>
			<td>Patent application number: 20190350175</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldritch, St. Louis, MO</td>
			<td>S8501</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Microplate Lids</td>
			<td>ThermoFisher Scientific, Waltham, MA</td>
			<td>5500</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing agrochemical risk to mated honey bee queens

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not accurately capture the effects of agrochemical exposure on honey bee queens. As the sole producer of fertilized eggs inside a honeybee colony, the queen is arguably the most important single member of a functioning colony unit. Therefore, understanding how agrochemicals affect queen health and productivity should be considered a critical aspect of pesticide risk assessment. Here, an adapted method is presented to expose honey bee queens and worker queen attendants to agrochemical stressors administered through a worker diet, followed by tracking egg production in the laboratory and assessing first instar eclosion using a specialized cage, referred to as a Queen Monitoring Cage. To illustrate the method&#39;s intended use, results of an experiment in which worker queen attendants were fed diet containing sublethal doses of imidacloprid and effects on queens were monitored are described.

The production of eggs was monitored in QMCs assembled and maintained as described above with once daily observations of egg production and 15 cages per treatment group. Newly mated queens of primarily Carniolan stock were purchased and shipped overnight from a queen breeder, and honey bee workers were obtained from 3 colonies maintained according to standard commercial methods at The Bee Research Facility at the University of Illinois Urbana-Champaign. Here, 4 dietary treatment groups were used: 1) 50 ppb (g/g) imidaclo...

Watch this Scientific Journal Video about Assessing Agrochemical Risk to Mated Honey Bee Queens at JoVE.com

Assessing Agrochemical Risk to Mated Honey Bee Queens

This protocol was developed to enhance the understanding of how agrochemicals affect honey bee (Apis mellifera) reproduction by establishing methods to expose honey bee queens and their worker caretakers to agrochemicals in a controlled, laboratory setting and carefully monitoring their relevant responses.

Current risk assessment strategies for honey bees rely heavily upon laboratory tests performed on adult or immature worker bees, but these methods may not ...

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