Pollen Removal and Quantification
Pollen was removed from the exterior of insect bodies following the
protocol by Lucas et al. (2018). Each specimen was washed in 500 μL of
wash solution containing 2% PVP and 1% SDS (buffer solution) using a
1.5 mL Eppendorf tube. For larger specimens (>8 mm)
additional wash solution was added until they were submerged. The
specimens and controls were agitated by hand for 1 minute and then
centrifuged at 15800 g for 20 seconds. Afterwards they were incubated
for 5 minutes and shaken for an additional 20 seconds to resuspend
pollen. The insects were then removed from the tube and stored in 95%
ethanol. The remaining washing solution and suspended pollen were
centrifuged at 15800 g for 5 minutes. The supernatant was discarded, and
samples stored at -20°C.
The pollen pellet was resuspended in 250 μL of 95% ethanol by vortexing
for 4 minutes. Samples that were difficult to homogenize were heated at
56°C for 5 minutes and vortexed for an additional 4 minutes. An aliquot
of 50 μL was taken and dried in a sterile incubation oven for
quantification and the remainder was used for metabarcoding. Pollen
counts were determined for each sample using a Multisizer 3 Coulter
Counter (Beckman Instruments, Fullerton, CA, USA). A blank of 10 mL of
Isoton II diluent was measured in a 30 mL cuvette and used to calibrate
the machine. The pollen sample was suspended in 300 μL of diluent by
vortexing for 10-20 seconds. This pollen suspended diluent was added to
the measuring cuvette. Additional diluent was added to reach 11 mL of
liquid. The cuvette was gently vortexed for 3-5 seconds to homogenize
the sample. The Coulter counter was then used to quantify the number of
particles in the size range 10-120 μm for three 1 mL samples.