Pathogen Prevalence in Wild Bumblebees (Bombus spp.)
across the Fraser Valley

Kennedy Zwarych with Supervisor Dr. Sandra Gillespie

Introduction

Results

Wild bumblebee populations (Bombus spp.) worldwide
have declined over the past twenty years. [1] [3]
A common microsporidia pathogen that infects bumblebees
is Nosema bombi. Another pathogen species, Nosema
ceranae, typically affects honeybees (Apis spp.), but has
sporadically been observed in some Bombus species. [4]
Managed commercial bees are used to pollinate crops
grown on farms and greenhouses. [5]
Commercially reared bumblebees are likely to have higher
rates of infection. [5]

Please note that results are still being collected and 2020 data is
preliminary.
Analysis suggests there are no significant relationships between
Nosema infections and the percentage of farmland or the presence of
greenhouses at collection sites.
No N. ceranae has been detected yet in the bumblebees we have
found.
The E.Z.N.A. Fungal DNA Mini Kit provided the cleanest-looking PCR
gels, though the Chelex method used less toxic reagents and had a
shorter incubation period; therefore, we used this protocol most
frequently.
1 2 3 4 5 6 7 8

Bombus
flavifrons

Bombus
Bombus
Bombus
Bombus
impatiens
melanopygus
mixtus
vosnesenskii
The five most common bumblebee species observed in our sampling.

PCR products separated by gel
electrophoresis. Lane 1 is a DNA ladder (#'s
= base pairs), lanes 2-4 are empty, lane 5
has the N. ceranae primer, lane 6 has a
Bombus ribosomal protein primer, lane 7 has
an Apis ribosomal protein primer and lane 8
has an N. bombi primer. These results show
this bee was positive for N. bombi and DNA
was successfully extracted.
Nosema sp. observed at 40x magnification.
Our seven collection sites across the Lower Mainland.
Pathogen Spillover: "[a process] occurring when pathogens spread
from a heavily infected 'reservoir’ host population to a sympatric ‘nonreservoir’ host population." [2]

Methods

Analysis suggests that
bumblebee collection
sites (left) and different
species (not pictured)
may influence the rates of
Nosema infection
(p<0.01).

Fieldwork & Dissections

Purpose
We are investigating if there is evidence for
pathogen spillover from commercial bumblebees to
wild bumblebees in the Fraser Valley. Specifically,
1. Do infection rates of N. bombi change with the
site of each bumblebee collection?
2. Do bumblebees collected from sites with more
surrounding farmland and greenhouses have
higher rates of N. bombi?
3. Is N. ceranae present in the Fraser Valley?
Tangent Purpose: Test different protocols of fungal DNA isolation is there a method that overall provides more coherent results?

We collected Bombus spp. from seven different sites in the Fraser Valley over 2018-2020.
We dissected the bee's gut and hindgut and used a compound microscope at 40x
magnification to check for the presence of Nosema spores.
A subset of bees that were Nosema positive underwent molecular analysis.

DNA Extraction & Amplification
We tested three protocols to find the most efficient way to isolate fungal DNA.
1. Chelex 100 resin to mechanically break cell walls; the same protocol used in 200
level UFV Biology labs.
2. E.Z.N.A. Fungal DNA Mini Kit. This included using RNaseA and betamercaptoethanol to chemically extract fungal DNA.
3. Quiagen DNeasy Blood & Tissue Kit. Similar to the above method, though with
different chemicals and a long incubation period.
We then ran PCR to amplify the DNA and separated products via gel electrophoresis.

Acknowldegements
To everybody who helped make this project possible; specifically
the UFV Biology Department for allowing me the freedom to
undertake this study and the Fraser Valley Regional District & Metro
Vancouver Regional Parks for permitting us to use their property. I
am very grateful to Avril Alfred, Fabiola Rojas and Valentina
Jovanovic for letting me use their equipment and always being
willing to help, to Dr. Justin Lee for his expertise in molecular
analysis and to Dr. Sandra Gillespie for her support and guidance
from the beginning. Thank you so very much.
References

GIS Data & Statistical Analysis

Bumblebee pictured on a lavender plant.

Male Bombus mixtus viewed
under a dissection microscope
at 10x magnification.

For GIS data, we used Google Earth Pro to plot a 1500m and a 2000m radius from the
centre of each site, then calculated the percent cover of farmland cover and distance from
the nearest greenhouse.
Type III ANOVA's were performed in R for comparing Nosema infection rates between sites,
species, sex, collection date, percentage of farmland cover and distance from greenhouses.

[1] Cameron, S. A., Lozier, J. D., Strange, J. P., Koch, J. B., Cordes, N., Solter, L. F., & Griswol, T. L. (2011). Patterns of
widespread decline in North American bumble bees. Proceedings of the National Academy of Sciences of the United
States of America, 108(2), 662–667. https://doi-org.proxy.ufv.ca:2443/10.1073/pnas.1014743108
[2] Colla, S. R., Otterstatter, M. C., Gegear, R. J., & Thomson, J. D. (2006). Plight of the bumble bee: Pathogen spillover
from commercial to wild populations. Biological Conservation, 129(4), 461–467. https://doiorg.proxy.ufv.ca:2443/10.1016/j.biocon.2005.11.013
[3] Gillespie, S. (2010). Factors affecting parasite prevalence among wild bumblebees Sandra Gillespie. Ecological
Entomology, 35(6), 737–747. https://doi-org.proxy.ufv.ca:2443/10.1111/j.1365-2311.2010.01234.x
[4] Imhoof, B., Schmid-Hempel, P. (1999). Colony success of the bumble bee, Bombus terrestris, in relation to infections
by two protozoan parasites, Crithidia bombi and Nosema bombi. Insectes soc. 46, 233–238
https://doi.org/10.1007/s000400050139
[5] Murray, T. E., Coffey, M. F., Kehoe, E., & Horgan, F. G. (2013). Pathogen prevalence in commercially reared bumble
bees and evidence of spillover in conspecific populations. Biological Conservation, 159, 269–276. https://doiorg.proxy.ufv.ca:2443/10.1016/j.biocon.2012.10.021