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

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.