Three methods are described in this protocol:collecting corbicular pollen using pollen traps, sorting pollen by color, and acetolysis. These methods are commonly used in studies related to pollinators, such as pollination efficiency, pollinator foraging dynamics, and diet quality and diversity. Pollen trapping is an easy way to collect bulk quantities of pollen from honeybee colonies.
Sorting pollen pellets by color is an affordable method to classify pollen that requires low expertise, and acetolysis permits identifying the plant taxon associated with each pellet color. Demonstrating the procedure will be Ellen Topitzhofer, faculty research assistant, and Emily Carson, a graduate research assistant from my lab. To begin, determine when to trap pollen from the desired apiary location and select the optimal honeybee colonies for trapping pollen.
Assess colony strength by counting foragers returning to the colony entrance for two minutes and then select colonies with the highest total number of returning foragers. Select hives with woodenware that is in good condition, preferably without extra entrances and warped lids. Use colonies with fewer alternative entrances, as forager bees returning to such colonies are more likely to reorient to the trap entrance.
Select south-facing hive entrances whenever possible. If hives are palletized, install pollen traps on every colony facing the same direction on a given pallet to avoid the drift of foragers into neighboring hive entrances. If desired, assess the brood nest of the colony by inspecting frames for the presence of larvae and select colonies with relatively large amounts of larvae.
Next, install pollen traps on the selected honeybee colonies. For front-mount traps, attach the trap in front of the entrance with staples, screws, and tape, or connect the trap to bungee cords wrapped around the hive. Seal all other possible entrances into the colony with a non-adhesive and moldable material, such as latex or polyurethane foam or a number eight hardware cloth for auger holes.
Use adhesive tape for small cracks. If using front-mount traps that are on ground-level hives, place a barrier such as a rubber mat between the collection basket and grass to avoid moisture damage from the dew. Engage the trapping mechanism of the pollen trap 24 hours after installation and before the day's foraging flight commences.
Collect corbicular particular pollen from the collection tray and place it into plastic bags or centrifuge tubes, then store it in a cooler with ice. Use the scooper or a large spoon to scoop out 10 grams of pollen as a representative sub-sample. Remove all bee parts and debris from the sub-sample, then sort each pollen pellet from the 10-gram sub-sample into a color group using both pollen color and texture to differentiate between groups.
To ensure at least 0.25 grams of each color group for downstream steps, place any pellets that are not plentiful enough to form a color group of at least 0.25 grams into a miscellaneous group. Name each individual color group using the Pantone Color Guide. Weigh out 0.25 grams of pollen pellets from each color group.
Don the appropriate PPE. Then, slowly add 500 microliters of glacial acidic acid to each microcentrifuge tube containing 0.25 grams of color group pollen. While visually inspecting the tube, stir the pollen with a clean toothpick for 10 to 15 seconds.
Centrifuge the sample for three minutes at 1, 100 times G, then decant the supernatant into the acid waste beaker. To prepare the acetolysis mixture, add 10.8 milliliters of glacial acidic acid to the beaker labeled acetolysis mixture. Then, slowly add 1, 200 microliters of concentrated sulfuric acid from the stock solution.
Slowly add 1, 000 microliters of acetolysis mixture to each tube and stir with a clean toothpick for 10 to 15 seconds. Place the samples on a heat block preheated to 80 degrees Celsius. Incubate the tubes for five minutes, stirring each tube thoroughly with a clean toothpick halfway through incubation.
After the incubation, slowly add 500 microliters of glacial acetic acid to each tube and stir with a clean toothpick for 10 to 15 seconds. Centrifuge the samples, then decant the supernatant from each tube into the acid waste beaker. Rinse each sample three times with water by adding 1, 000 microliters of distilled water to each tube and stirring with a clean toothpick.
Centrifuge the samples and decant the supernatant from the tubes into the beaker of sodium bicarbonate. Then, repeat the water rinses two more times. Add 1, 000 microliters of 95%ethanol from the ethanol beaker to each tube and stir with a clean toothpick.
Then, centrifuge the samples and decant the supernatant from the tubes into the ethanol waste beaker. Mix the safranin-O stain stock solution and add 5 to 10 drops to each tube. Stir with a toothpick for 10 to 15 seconds and leave the toothpick in the tube.
Add 1, 000 microliters of 95%ethanol from the ethanol beaker to each tube and stir. After centrifuging the samples, decant the supernatant into the ethanol waste beaker. Add 10 to 15 drops of glycerin to each tube and stir the contents for 10 to 15 seconds.
Leave the tubes open in the fume hood to evaporate the ethanol for at least two hours at ambient room temperature. Label microscope slides for pollen identification. Remove one drop of pollen residue from a tube and place it in the center of its labeled microscope slide, allowing the drop to spread slightly.
Place a clean cover slip over the drop on the slide and seal the cover slip to the slide with clear nail polish. Using the described methods, corbicular pollen was collected, sorted by color, and the plant sources of each pellet color group were identified to assess pollen diversity. Every pollen collection sample had multiple distinguishable color groups.
In some samples, pollen color groups contained as few as four to five pellets. However, most groups had significantly more, and served as their own labeled color group for acetolysis. After acetolysis, bright-field light microscopy was used to effectively identify each color group to its lowest-possible taxonomic rank by confirming the morphological characteristics with those of voucher specimens collected from the area surrounding each study site.
The pollen collected from almond crop locations had relatively lower pollen diversity than pollen collected from other crops, with an average of three pellet colors in 3.2 plant taxa per site Sorted pollen can be tested for pesticides to elucidate the agrochemicals found on honeybee forage plants, or analyzed for chemical composition to determine its nutritional value to honeybees. The results from this research provided valuable insights on the species diversity observed in pollen collected by colonies, highlighting the substantially low diversity of pollen collected in almond cropping systems.
