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The Absence of Nosema bombi in Bumblebees (Bombus spp.) on Farms in Michigan.


Pollinator species, such as honeybees (Apis spp.) and bumblebees (Bombus spp.), provide pollination for over one-third of the food we eat and the plants that grow around us (Klein et al., 2007). Annual pollination services account for 206 billion dollars worldwide and 1.2 billion dollars in the United States alone (Otii and Schmid-Hempel, 2007; Partap and Ya, 2012). The worldwide population decline of bumblebees has made apparent both their ecological and economic importance. It was found there was a 23% to 87% reduction of the Bombus species throughout North America within the last 20 y (Cameron et al., 2011).

There are many suggested causes for the population decline of bumblebees, including habitat loss and reduced floral abundance and diversity (Goulson et al., 2008). Emerging pathogens, such as microsporidia in bumblebees, have also been shown to contribute. Bumblebees that are commercially-reared have higher pathogen prevalence than wild bees. Pathogens, such as microorganisms (Apicystis bombi, Crithidia bombi, Nosema bombi), and mites (Locustacarus buchneri), are thought to be carried by commercial bumblebees and transmitted to wild bees (Colla et al, 2006). These pathogens were found in bumblebees that were commercially reared by three main producers in Europe, which claimed to be parasite-free (Graystock et al, 2013). Crithidia bombi was also found lo infect 27% of bumblebees near greenhouses with commercially reared bees in Ontario, Canada as compared to an absence of infected bees found al sites lacking a greenhouse and commercially reared bees (Colla et al., 2006). Pathogen spillover of commercially reared bumblebees within North American may cause an epidemic in the wild species (Meeus et at., 2011). These population declines are believed to lead to an overall lower genetic diversity and a higher susceptibility to infection by microsporidia and other pathogens (Brown, 2017).

Nosema bombi is a fungal pathogen that can be found in the gut lumen and the Malpighian tubules of its infected host. The parasite can then spread within the host during its life cycle. Because N. bombi is a microsporidian, fresh spores are released into the environment through feces or a decaying host. The microsporidian goes through a proliferative phase (merogony) during which injected sporoplasm develops into meronts, spores are then produced (sporogony), and the host cell becomes distended with mature spores, ruptures, and releases infectious spores into the environment (Han and Weiss, 2017). Infection leads to reduced fitness of bees; for example, the mortality rate of infected worker bees is approximately 55% compared to a 15% mortality rate of uninfected control bees (Otti and Schmid-Hempel, 2007). Sperm number in the infected male bees is also significantly lower than in noninfected bees. Many of the bumblebees that were infected, but still reproduced, had offspring with distended abdomens or crippled wings, demonstrating N. bombi has a multi-generational fitness impact (Otti and Schmid-Hempel, 2007). This pathogen can be found throughout the United Stales with variable infection rates, (e.g., 5.48% in Maine (Buslunann et al., 2012) and 2.9% in Illinois (Cordes, 2010)). The ease of pathogen spread may contribute to a significant role in bumblebee population declines.

Another possible cause for bumblebee decline is pesticide use on farms (Gill et al, 2012). Bees are vulnerable to pesticide exposure as they gather pollen from plants and transfer pesticides unintentionally. The toxins are then carried into their nests, poisoning the colony (James and Xu, 2012). Pesticides can affect an insect's innate immune system in many ways, one being through the cellular immune response. Pesticides have also been found to increase stress in insects and decrease the abundance and variation in both phagocytes and nodules (James and Xu, 2012), causing an insect's immune system to be less responsive, which allows for more pathogens to infect the insect.

Studies involving bumblebees in Michigan are scarce, as are studies involving the microsporidian Nosema bombi. This fungus, however, has been found in bumblebees throughout the east and west coast of North America, such as Maine, California, Oregon, and Washington (Cordes et al., 2012). Nosema bombi is found in areas of Canada bordering the upper Midwest as well as in Minnesota, Wisconsin, Illinois, and Ohio (Colla et al, 2006).

With pesticide usage, pathogens that infect bees may become more prevalent. Pathogens, such as N. bombi, may be more successful at infecting a bumblebee with a weaker immune system resulting in a larger overall effect on pollinating insects than suspected. The purpose of this study is to examine if Nosema bombi, in its sporulated form, is present in wild bumblebees in Michigan, and if infection rates vary in bees collected on farms that use pesticides as compared to those that do not.



Using 7-dram vials, foraging bumblebees were collected from nine farm sites in Michigan during late July and early August of 2017 (Table 1). All farm sites were at least 5 km apart to lake into account the maximum distance of bumblebee foraging (Osborne et al., 2008). The farm sites were categorized based on pesticide usage (Table 1). At each site 25 or 26 bumblebees were collected for a total of 232 bumblebees (Table 2). At all sites foraging bumblebees were collected during the morning or evening by walking the span of the fields for approximately 2 h. Bees were placed individually in labeled vials and transported in a cooler before being frozen and stored in a -80[degrees] C freezer.


Each bee was first identified to species level (Evans, n.d.; Colla et al., 2008). Bees were dissected lo determine the presence of N. bombi spores using a dissection protocol adopted from Kalyn Bickerman-Martens, M.A. (pers. comm.). Bees were pinned ventral side-up and the abdomen was cut and completely opened. The entire digestive tract was removed from each bee and placed into individually labeled microcentrifuge tubes to which 200 [micro]L of Insect Ringer Solution was added. Tubes were then vortexed for 1 min and frozen for later use (Whitehorn et al., 2011).


Two different spore identification procedures were utilized. The first method used Giemsa stain. After microcentrifuge tubes were thawed, slides were smeared with 10 [micro]L of the solution, fixed with methanol and stained with Giemsa stain (McIvor and Malone, 1995). The Giemsa-stained smears were then observed under a compound light microscope at 1000x. The second procedure used phase contrast microscopy. After the samples were thawed, 200 [micro]L of distilled water was added to each and vortexed. 10 [micro]L of this mixture was then pipetted onto a slide. A coverslip was added and the slides were all observed under 400x magnification on a phase contrast microscope. Approximately 5 min was spent on each slide looking for spores in both procedures. A sample was scored as showing no infection, if fewer than two spores were present; the presence of only a single spore could be the result of incidental contamination or evidence of a non-infectious presence (Bickerman-Martens, pers. comm.).

These protocols were used to detect actual infections rather than a possibly non-infectious presence of fungus. By focusing on sporulating Nosema bombi infections rather than focusing on non/low sporulating N. bombi infections, a more accurate infection rate can be determined. Some studies have used a more sensitive method involving molecular screening for detection of N. bombi, such as POR (Blaker et ai, 2014), which would detect all samples containing N. bombi, instead of bumblebees that are being directly impacted by a sporulating infection.


Of the 232 total bumblebees collected, nine different species were identified (Table 2). Across all nine farm sites, no Nosema bombi infections were detected. Due to the absence of any infections, no analysis could be conducted on the hypothesis of pesticide usage impacts on infection.


The bumblebees in this study were found not to be infected with Nosema bombi. By conducting two different methods in spore identification, we can confidently conclude that the bumblebees were free of fungus. This fungus is, however, found in surrounding states, including Minnesota, Wisconsin, Illinois, and Ohio, and across the border in Canada (Cordes et al, 2012). Overall 2 to 4% of bumblebees are infected in North America, but infection rate is highly variable among species (Cordes et al, 2012). Although we could not find in the literature other published results documenting infection rates of N. bombi in live, wild-caught bumblebees from Michigan, Cameron et al (2016) did test museum specimens from Michigan using PCR and detected N. bombi DNA in about 13% of the B. affinis and 4.6% of the B. terricola specimens tested. However, most of these specimens were over two decades old and did not lest positive in all PCR replicates. In addition, as stated earlier, detecting the presence of some pathogen DNA does not necessarily indicate the bee itself was actually infected by a sporulating N. bombi.

The most common bumblebee species to be infected with N. bombi are Bombus occidentalis and Bombus pensylvanicus (Cordes et al, 2012), neither of which were collected in this study. Of these two species, only B. pensylvanicus bas a home range that extends into part of Michigan (Tuell et at.. 2009). Bombus occidentalis has an infection rate of 37.2% in the west (Cordes et al., 2012) and was the most commonly collected and infected species in one study (Koch and Strange, 2012). Bombus pensylvanicus has an infection rate of 15.2% in midwestern slates, such as Illinois, Indiana, Minnesota, and Iowa, and across the east coast (Cordes et al., 2012). The bumblebee species most commonly found in Michigan, Bombus impatiens, has one of the lower infection rates across the United States (0.73%; Cordes et al, 2012). Sokolova et al. (2010) showed that out of the 68 bumblebees classified as B. impatiens, only one individual was found lo be infected with N, bombi. This could be one explanation for the absence of N. bombi in our study samples.

Although the underlying reason infection rates vary among species is unknown, there may be a connection between genetic variation and the infection rate of a species. One study found that the most commonly infected bumblebee species (B. occidentalis and B. pensylvanicus) had lower genetic diversity compared to other species (Lozier et al, 2011). Host genetic diversity does offer protection against the spread of biological pathogens (King and Lively, 2012) and may offer some explanation for the varying rates of infection of N. bombi between Bombus species. The two species of bumblebee in which N. bombi DNA was not detected in Michigan museum samples (Cameron et al, 2016) were not collected in this study, given recent population declines throughout their ranges (Evans et al, 2008); in fact, II. affinis has not been documented in Michigan since 1999 (Michigan Natural Features Inventory, n.d.). To continue with this research, greater sample sizes over wider areas, with a longer timeframe, and a more concerted effort to collect B. pennsylvanicus may help confirm the possible absence or low levels of N. bombi infection in Michigan's wild bumblebees.

Acknowledgments.--We would like lo acknowledge The Unity College Student Activity Engagement Fund for providing funds to support this project. A special thank you goes to Kalyn Bickerman-Martens from the University of Maine for her guidance and expertise in ibis field. A huge thank you to everyone who helped with bee and data collection: Kelsev Diamond, Kathv Voughl, Mike Skuse, Marisol Ontiveros, and Morgan Dubois. We also thank the farms that made this research possible: AJ's Garden and Produce, Grandpa Tiny's Farm, Hooper's Farm Garden, Kendall Sumerix Farm, Loma Farm, The Rack and Track Club, Steyma Potato Farm, and Weinkauf Family Farm. Finally, we thank two anonymous reviewers for their thoughtful suggestions.

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BRITTON SKUSE (1), AIMEE PHILLIPPI and ELLEN BATCHELDER, School of Biodiversity Conservation, Unity College, 90 Quaker Hill Road, Unity, Maine 04988. Submitted 4 March 2019; Accepted 21 June 2019

(1) Corresponding author: E-mail:
TABLE 1.--Description of each farm site collected
at during the summer of 2017 in Michigan

Code   County       GPS coordinates         Main crops

O1     Alpena       45.059120, -83.804680   Beans, rye,
                                              wheat, clover
O2     Saginaw      43.310661, -83.734016   Pumpkins
O3     Alpena       45.031101, -83.489883   Rye, rape seed
OP     Grand        44.842800, -85.700401   Vegetables
P1     Alpena       45.061913, -83.757988   Strawberries,
P2     Grand        44.921135, -85.527748   Flowers, herbs
P3     Alpena       44.999428, -83.523895   Rye

P4     Alpena       45.164150, -83.598106   Potatoes

H      Alpena       45.036518, -83.778748   Alfalfa,

Code    Wildflower/     Pesticide usage   Date of
       bee pollinated                     collection

O1          Yes         None              8/8/2017

O2           No         None              8/18/2017
O3           No         None              8/16/2017
OP          Yes         Organic           8/9/2017
P1          Yes         Conventional      7/28/2017
P2          Yes         Conventional      8/9/2017
P3           No         Conventional      8/23/2017
P4           No         Conventional      8/13/2017
H           Yes         Herbicides        7/31/2017

TABLE 2.--The observed species abundance of bumblebees
at each farm in Michigan site N = 232; H = herbicides,
O = no pesticides, OP = organic pesticides,
P = conventional pesticides

                  O1    O2    O3    OP    P1    P2    P3    P4     H

B. impatiens      18    26    24    20     9    13    23    18     8
B. bimaculatus     3     0     0     3     8     5     1     1    16
B. borealis        1     0     0     2     1     0     0     1     1
B. fervidus        1     0     0     0     2     0     0     0     0
B. ternarias       1     0     0     0     4     0     2     5     1
B. rufocinctus     0     0     0     0     2     0     0     0     0
B. griseocollis    1     0     2     0     0     7     0     0     0
B. variabilis      0     0     0     0     0     1     0     0     0
B. perplexas       0     0     0     1     0     0     0     0     0
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Title Annotation:Notes and Discussion Piece
Author:Skuse, Britton; Phillippi, Aimee; Batchelder, Ellen
Publication:The American Midland Naturalist
Geographic Code:100NA
Date:Oct 1, 2019
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