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Method Article

Assessing Agrochemical Risk to Mated Honey Bee Queens

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DOI:

10.3791/62316

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March 3rd, 2021

 ,  ,  , 
1Invasive Species and Pollinator Health Research Unit, USDA-ARS, 2Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 3Neuroscience Program, University of Illinois at Urbana-Champaign, 4Department of Entomology, University of Illinois at Urbana-Champaign

In This Article

Summary

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.

Abstract

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'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.

Introduction

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

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Protocol

1. QMC assembly

  1. 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.
  2. 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.32 for further assembly details.
  3. Collect the wax comb frames containing the capped worker brood from honeybee colonies 24 h prior to adult eclosion and place them in an incubator (34.5 °C) inside a brood box. 24 h later, brush the eclosed bees off the frames and into an open container that has been lined with an insect barrier paint (e.g., Fluon) to prevent the bees from crawling out.
  4. Add at least 50 bees by weight (5 g ≈ 50 bees44,45) to the egg laying chamber of each QMC. To ensure that a diverse genetic pool of workers is represented in the experiment, obtain an approximately equal number of worker bees from at least three colonies and mix them prior to adding them to the QMCs.
    NOTE: Newly eclosed worker bees less than 1 day old cannot fly or sting due to their underdeveloped flight muscles and unhardened cuticle. If they are added at this age, there is no need to anesthetize them prior to handling. They can be weighed by gently scooping bees from the container using a small ¼ cup volume measuring cup and placing them into a second container (lined with insect barrier paint e.g., Fluon) that has been tared on a scale. The area of the frames covered by capped brood should be roughly equal to ensure that source colonies are equally represented in the QMC worker populations. Homogenization of worker bees can be achieved by brushing newly eclosed bees from frames taken from all colonies into the same container and allowing them to mix for 5 min prior to adding them to QMCs.
  5. Add the feeders containing sucrose solution, water, and pollen supplement (See section 2).
  6. Expose the individual mated queens to CO2 gas to stimulate egg laying46 and to ease transfer into QMC.
    1. Use queens purchased from a commercial breeder within 48 hours of receipt. While the queen is still inside the shipping cage, place it in a clear plastic bag. Place one end of a plastic tube connected to a CO2 gas cannister inside the bag and gently open the cannister valve to allow the CO2 gas to flow.
    2. When the bag has been inflated with gas, simultaneously close the cannister valve and hold the bag closed to trap the gas inside. Keep the bag closed for 30 s or until the queen has stopped moving. Remove the queen and open the shipment cage once she is observed to be unconscious.
  7. Partially open the door to the egg laying chamber, gently place the unconscious queen inside and close the lid, taking care not to crush the queen or workers inside. Add the second egg laying plate to each QMC as shown in Figure 1C. Place a piece of laboratory tape across the top of the two ELPs to keep them from separating from the QMC frame and prevent workers from exiting the cage.
  8. Place the cages in a dark incubator with stable environmental conditions of 34 ± 0.5 °C and 60% ± 10% relative humidity, like the conditions inside a normal colony.

Bee colony simulation setup, diagrams showing feeding chamber, queen excluder, and egg laying chamber.
Figure 1. A: Disassembled QMC. B: Partially assembled QMC with 1 ELP inserted. C: Fully assembled QMC with 2 ELPs. Please click here to view a larger version of this figure.

2. Preparing and administering diets laced with agrochemicals

  1. To prepare 1000 g of 50% (g/g) sucrose solution, place a stir bar in the bottom of a clean 1 L glass reagent bottle. Add 500 g sucrose and 500 mL of deionized water. Unscrew the lid of the bottle and use a heated stir plate set to low heat to mix the solution until all the sucrose has dissolved. Allow the solution to cool to room temperature before adding the agrochemical stock solutions.
  2. Prepare the stock solutions of agrochemicals in an appropriate solvent, such as acetone, at a concentration that can be added to diet to achieve the desired final concentration of the agrochemical of interest.
    NOTE: When using acetone as a vehicle solvent, the Organization for Economic Cooperation and Development (OECD) guidelines stipulate that the final concentration of acetone in diet must be ≤ 5% for chronic oral toxicity tests on adult honey bees47. However, some solvents such as n-methyl-2-pyrrolidone5,31 and dimethyl sulfoxide25 can exert toxic effects below this concentration, so it is recommended to keep solvent concentrations as low as possible in treatment diet. Depending on the volume and type of solvent used, it may be necessary to include both a solvent control group and a negative control group to ensure that potential effects due to solvent toxicity are detected. When using formulated products, the amount of the product used must be adjusted based on the concentration present in the formulation. Depending on the stability of the agrochemical of interest in the solvent, stock solutions can be kept for up to 2 weeks at -20 °C.
  3. Select sublethal doses based on the results of OECD Test No. 245: Honey Bee (Apis mellifera L.), Chronic Oral Toxicity Test (10-Day Feeding)47, and identify the relevant literature by querying the Ecotox knowledgebase48.
  4. Administer the agrochemical treatments in a sucrose solution, a commercial pollen supplement (if available as a powder), or both. Prepare the experimental diet for use the same day by adding an appropriate amount of stock solution to chilled/room temperature 50% sucrose solution (w/w). Mix thoroughly by vortexing or with a stir bar set to medium speed. For pollen supplements, add the agrochemical laced sucrose solution to the powdered supplement instead of the syrup according to manufacturer protocols, making sure to adjust the volume of stock solution used according to the final weight of the pollen diet. See Table 1 for example calculations.
  5. Prepare the feeder tubes from 2 mL microcentrifuge tubes.
    1. For liquid diet feeders, heat the tip of a 20-gauge needle on a hot plate/stovetop and puncture the bottom of the tube twice. Close the tube lid and pipette approximately 1.5 mL of sucrose solution or water through one of the puncture holes. Set the tube down with the punctured side up until it is added to the QMC.
    2. For pollen supplement feeders, use a razor blade to slice off the bottom of the tube. Close the lid and a push a 1-2 g ball of pollen supplement into the tube until it touches the lid.
  6. Record the feeder weights prior to placing them in the QMCs. Do not keep unused diet at 4 °C for over 48 hours.
Desired ConcentrationDesired Solvent Vehicle ConcentrationDesired final volume/mass of sucrose solutionVoume of stock solution Imidacloprid in stock solutionSuggested stock solution recipe
Sucrose solution10 ppb (w/w)0.05% (v/v)81.45 mL/100 g*40.7 µL0.001 mg/40.7 µL0.02 mg/814 µL
Pollen supplement10 ppb (w/w)10 g**4.07 µL0.0001 mg/4.07 µL0.02 mg/814 µL
Sucrose solution50 ppb (w/w)0.05% (v/v)78.5 mL/100 g*40.7 µL0.005 mg/40.7 µL0.1 mg/814 µL
Pollen supplement50 ppb (w/w)10 g**4.07 µL0.0005 mg/4.07 µL0.1 mg/814 µL

Table 1: Example recipes for treated sucrose solution, pollen supplement, and stock solution. *Volume based on the density of 50% (w/w) sucrose solution (1.228 g/mL). **The density of the pollen supplement will vary depending on what product is used, but if this solvent volume is used, the final solvent concentration in pollen supplement will be within the desired range of ≤ 5% by volume.

3. Monitoring - Egg Production Rate

  1. Quantify the egg laying 1 to 2 times per day in the morning and/or evening. Begin by removing QMCs from the incubator to check for eggs.
    NOTE: In a successful experiment, egg production will commence in most of the control QMCs within 3 days of initial cage assembly. Only take as many QMCs out of the incubator at one time that can be checked and fed within 10 min. Longer periods outside the incubator may disrupt egg production.
  2. Examine the backs of the clear ELPs for eggs. If eggs are present, remove the door panel in front of the plate of interest. Remove the tape from across the ELPs and carefully slide the door panel between the ELP and the bees inside the QMC, taking care not to crush any bees that might be cleaning the cells in the ELPs.
  3. With the door panel in place, remove the ELP, and count and record the number of eggs inside the ELP cells. Remove the eggs by tapping the edge of the ELP, open cell-side down, on a hard surface (such as the lip of a waste receptacle). Once the eggs fall out, replace the empty ELP in the QMC. Gently remove and replace the door panel behind the ELP on the outside of the QMC. Repeat as necessary with the second ELP and replace the tape across the QMC when finished.
    ​NOTE: Egg production generally declines and mortality increases in QMCs after 2 weeks32,33, therefore it is recommended to conclude experiments after 14 days.

4. Monitoring - Food Consumption

  1. Replace all the food remaining in QMC feeders with freshly prepared diet every two days. Prepare new feeder tubes (including water) and weigh them before removing QMCs from the incubator for monitoring. Swap all old tubes with new ones and weigh old tubes before disposing of unconsumed diet. Compare the final weight of the feeder tube and unconsumed diet to the weight of the same feeder tube prior to placing it in the QMC to estimate diet consumption.
  2. Between days when feeders are scheduled to be replaced, check diet consumption once per day (at the same time when QMCs are monitored for egg production) to ensure that feeders are never empty. If a feeder tube is empty or near empty, remove it, refill it, record the weight of the tube before and after and add the difference to the 2-day diet consumption total for the QMC.

5. Monitoring - Embryo Viability

  1. At a selected point during a QMC experiment, remove ELPs containing freshly laid eggs from the QMC according to step 3, but do not dislodge eggs from the ELP.
  2. Cover the ELP with a universal microplate lid and place it inside a desiccator with a saturated K2SO4 solution (150 g K2SO4 in 1 L of water, kept in a shallow dish).
    NOTE: Some salt should be visible on the bottom of the dish after the mixture comes to temperature in the incubator.
  3. Keep the desiccator in an incubator set to 34.5 °C, resulting in a relative humidity of 95% inside the desiccator, similar to the conditions used by Collins49.
    NOTE: Almost all eggs will hatch within 72 ± 6 hours of when they were laid49, hence hatching rates can be assessed as early as 78 hours after the ELPs were removed from the QMC. A "C" shape larva in the bottom of the cell is indicative of a successful hatching event. Some variation in this timing is possible if, for example, the eggs are drones and not workers50.

6. Worker Sampling

  1. If the QMCs have been populated with excess workers, sample the worker bees at a selected time point during the experiment for assessment of treatment induced changes in their physiology. Perform the collections in conjunction with daily feeding and egg counting activities to minimize the time for which the QMCs are outside of the incubator.
  2. Before sampling, place a door panel between an ELP and the interior of the QMC, and remove the ELP. Carefully lift the door panel approximately 0.5 cm from the base of the cage and remove a worker bee from inside the QMC using featherweight tweezers. To prevent bees from escaping, cover portions of the 0.5 cm opening with a gloved finger or piece of cotton as necessary.
  3. Preserve the collected bee for follow-up analysis and repeat this process until the desired number of samples have been collected. For gene expression analysis, snap freezing bees in liquid nitrogen and immediate storage at -80 °C is strongly recommended51.

7. Worker Mortality

  1. Assess worker mortality during the experiment by counting the number of dead bees at the bottom of the feeding chamber and the egg laying chamber. Perform this assessment in conjunction with daily egg laying quantification.
  2. Using forceps, carefully remove the dead bees through the feeder holes, covering the hole with a gloved finger or piece of cotton while the forceps are not inserted.
  3. Remove the dead bees from the egg laying chamber by carefully lifting the door panel approximately 0.5 cm from the base of the cage and inserting forceps. To prevent bees from escaping, cover portions of the 0.5 cm opening with a gloved finger or piece of cotton as necessary.
  4. Assess the worker mortality at the conclusion of the experiment by removing and counting all the dead bees from the QMCs using the previously described methods prior to euthanizing the remaining bees.
    NOTE: In the absence of worker bees, queens will not produce eggs and will starve within 24 hours. Therefore, if all the workers in a QMC are observed to be dead, the QMC should be removed from the experiment. Likewise, if a queen dies during the experiment, the QMC should be removed, and the data should be appropriately censored.

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Results

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...

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Discussion

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...

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Disclosures

The authors have no conflicts of interest to declare.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
FluonBioQuip, Rancho Dominguez, CA2871A
Honey bee queensOlivarez Honey Bees, Orland, CA
ImidaclopridSigma-Aldritch, St. Louis, MO37894
MegaBee PowderMegaBee, San Dieago, CA
Microcentrifuge tubes 2 mLThermoFisher Scientific, Waltham, MA02-682-004
Needles 20 gaugeW. W. Grainger, Lake Forest, IL5FVK4
Potassium SulfateSigma-Aldritch, St. Louis, MOP0772
Queen Monitoring CagesUniversity of Illinois Urbana-ChampaignPatent application number: 20190350175
SucroseSigma-Aldritch, St. Louis, MOS8501
Universal Microplate LidsThermoFisher Scientific, Waltham, MA5500

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Agrochemical ExposureQueen Monitoring CageEgg Production TrackingImidacloprid TreatmentWorker Queen AttendantsPollen SupplementSucrose SolutionGene Expression AnalysisRisk Assessment
Assessing Agrochemical Risk to Mated Honey Bee Queens
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