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.

Agrochemical exposure can have major impacts on all members of a honeybee colony, including the most important member, queens. But until recently, few tools have been available to study these effects. This technique allows researchers to expose queens to agrochemicals under controlled conditions that simulate conditions they might experience in the field.

It's quite easy to disturb honeybee queen egg-laying behaviors, so it's important to take care when handling workers and queens before and during the experiment. Demonstrating these procedures will be, Amy Floyd and Eliza Litsey, laboratory technicians at the Invasive Species and Pollinator Health Research Unit, Honeybee Research Lab. To begin, assemble queen monitoring cages, or QMCs, by inserting the door panels, then insert the queen excluder in the feeding chamber door over the feeding chamber, to keep the queen from entering and contacting the treated diet.

Insert a single ELP. Do not add feeder tubes until after the workers have been added to the cage, and temporarily cover the four feeder holes with laboratory grade tape. After collecting and incubating the wax comb frames containing the capped worker brood from honeybee colonies, brush the closed bees off into an open container lined with an insect barrier paint, to prevent the bees from crawling out.

Add at least 50 bees by weight to the egg-laying chamber of each QMC. To ensure that a diverse genetic pool is introduced, obtain similar number of worker bees from at least three colonies, and allow them to mix prior to adding bees to the QMCs. Add the feeders containing sucrose solution, water and pollen supplement.

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. Then place one end of a plastic tube connected to a carbon dioxide gas canister inside the bag, and gently opened the canister valve to allow the carbon dioxide gas to flow.

When the bag has been inflated with gas, simultaneously close the canister valve and hold the bag closed to trap the gas inside. Keep the bag closed for 30 seconds or until the queen has stopped moving. Remove the queen by opening the shipment cage once the queen is observed to be unconscious.

Partially open the door to the egg-laying chamber, gently place the unconscious queen inside and close the lid. Add the second ELP to each QMC. 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.

Place the cages in a dark incubator with stable environmental conditions of 34 to 35 degrees celsius, and 50-70%relative humidity. To administer the diet laced with agrochemicals, prepare the stock solutions of agrochemicals in an appropriate solvent, such as acetone, at a concentration that can be added to the diet to achieve the desired final concentration of the agrochemical of interest. Administer the agrochemical treatments in a sucrose solution, a commercial pollen supplement, or both.

Prepare the experimental diet for use on the same day, by adding an appropriate amount of stock solution to chilled or room temperature, 50%sucrose solution. Mix thoroughly by vortexing at a medium speed. For pollen supplements, add a calculated amount of agrochemicals stock solution to sucrose solution, and vortex thoroughly before adding it to powdered supplement.

Add diet to feeder tubes and record the feeder weights prior to placing them in the QMC. To quantify the egg laying, begin by removing QMCs from the incubator. Examine the backs of the clear ELPs, if eggs are present, remove the door panel in front of the plate of interest.

Remove the tape from across the ELPs, and slide the door panel between the ELP and the bees inside the QMC. 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.

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.

To monitor the food consumption, replace the food with a freshly prepared diet every two days. Prepare new feeder tubes and weigh them before starting the egg production monitoring activities. Swap old tubes with new ones, and weigh old tubes before disposing of them.

Compare the final weight of the feeder tube and unconsumed diet to the weight of the same tube, to estimate diet consumption. Check once per day 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 two-day diet consumption total for the QMC.

At a selected point during a QMC experiment, remove the ELPs containing freshly laid eggs from the QMC as described previously, but do not dislodge the eggs from the ELP. Cover the ELP first with a PCR plate seal, and then a universal microplate lid, and place it inside a desiccator with a saturated potassium sulfate solution. Assess hatching rates approximately 78 hours after removing ELPs from the QMC.

A C-shaped larva in the bottom of the cell is indicative of a successful hatching event. If the QMCs had been populated with excess workers, sample the worker bees at a selected timepoint during the experiment to assess the treatment-induced changes in their physiology. 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 centimeters from the base of the cage, and remove a worker bee from inside the QMC using featherweight tweezers. For gene expression analysis, snap freeze the bees in liquid nitrogen and store them at 80 degrees Celsius. Preserve the collected bee for follow up analysis, and repeat this process until the desired number of samples have been collected.

Dietary imidacloprid treatment-related changes in daily egg counts were evaluated by implementing a Poisson log linear generalized estimating equation, with an auto-regressive correlation matrix. Daily egg production was significantly lower in QMCs given pollen supplement and sucrose containing 50 parts-per-billion imidacloprid. A dose-dependent effect was observed on the total number of eggs produced in each QMC for imidacloprid treatments administered in both sucrose and pollen supplement.

With the largest reduction observed in QMCs fed 50 parts-per-billion imidacloprid, followed by 10 parts-per-billion in pollen supplement plus sucrose. No difference in total eggs produced was observed between control and QMCs fed 10 parts-per-billion imidacloprid and pollen supplement alone. Pollen supplement, water and sucrose consumption were recorded every 48 hours for 10 or 12 days.

Daily rates of sucrose consumption significantly increased as the experiment progressed, but rates of pollen supplement consumption decreased. Higher rates of pollen consumption were observed when 10 parts-per-billion imidacloprid were administered in pollen supplement alone. And the rate decreased when 10 or 50 parts-per-billion was administered in pollen supplement and sucrose solution together.

Using day old bees is critical to ensure the acceptance of the queen and to reduce worker over reactivation. Also critical, is the careful handling of the queen and to keep the cages in a stable environment.