What’s killing the buzz? Investigating the multiple stressor hypothesis for bumble bee health

Featured Scientist: Austin C. Calhoun, M.S. (he/him/his), PhD student (Anticipated: Spring 2025), School of Biological Sciences, Illinois State University

Austin posing on tree stumps at a riverbed.
“Act like a bluebell and the bees will come.” – Austin Calhoun

Birthplace: Danville, IL, USA

My Research: I am interested in how bumble bees defend against parasites and pathogens.

Research Goals: I would like to continue to study disease. I am interested in how diseases arise, how individuals can defend against them, and what environmental contexts amplify the effects of disease. I would also like to study methods that can be used to predict the occurrence of disease in the future.

Career Goals: Ideally, I see myself as a professor at a university, where I could continue to conduct research and to teach. Then, maybe I’ll retire after running a field research station somewhere.

Hobbies: Cooking on a cast iron, building things out of wood, and getting deep into YouTube holes about jiu-jitsu.

Favorite Thing About Science: My favorite thing is the freedom of creativity. Being interested in some question about nature, designing a project to answer that question, and then telling the world about something new.

Organism of Study: The common eastern bumble bee and its pathogen, Nosema bombi (N. bombi).

A close-up picture of a bee on a brood of developing juveniles and a microscope image of N. bombi.
Bumble Bee (left) and N. bombi (right). Photos by Dr. Benjamin Sadd, Illinois State University

Field of Study: Disease Ecology

What is Disease Ecology? Disease Ecology is the study of how organisms interact with their pathogens. A pathogen is a disease-carrying microorganism, like a bacterium or a virus. When an organism is infected with a pathogen, it becomes a host for that pathogen. People who work in disease ecology also study other factors that affect how the host and the pathogen interact. For example, a disease ecologist might study the environment, pesticides, or climate change and how they impact the host or the pathogen.

Check Out My Original Paper: “Testing the multiple stressor hypothesis: chlorothalonil exposure alters transmission potential of a bumblebee pathogen but not individual host health”

 A QR code that links to the original publication.
QR code to the original publication

Citation: Calhoun A.C., Harrod A.E., Bassingthwaite T.A., Sadd B.M. 2021 Testing the multiple stressor hypothesis: chlorothalonil exposure alters transmission potential of a bumblebee pathogen but not individual host health. Proc. R. Soc. B 288: 20202922. (doi: 10.1098/rspb.2020.2922)

Research At A Glance: Bees serve important roles as pollinators, but they deal with many stressors that can negatively affect their health. Climate change, habitat loss, pesticide exposure, and pathogens are types of stressors that can negatively impact bee populations. Although each of these are individually harmful, bees likely experience more than one at a time. When bees are exposed to multiple stressors at once, they may experience worse outcomes. We call this concept the multiple stressor hypothesis. In this research, we test the multiple stressor hypothesis using a species of bumble bee, Bombus impatiens (B. impatiens).

Several species of bumble bee are in decline across North America. One possible reason could be a pathogen called Nosema bombi (N. bombi). N. bombi is a stressor found in many of the bumble bee species that are experiencing population declines. After infecting the bee, it will produce a spore as part of its reproductive cycle. The spores have a hard outer layer that makes them environmentally resistant and these N. bombi spores can infect bees. At the same time, many bumble bees are also exposed to a fungicide called chlorothalonil. A fungicide is a special type of pesticide used to kill fungal pathogens. In 2017, a study found a relationship between the use of chlorothalonil, the presence of N. bombi, and declining bee populations. We wanted to explore this relationship further.

To do this, we infected bumble bees with N. bombi and exposed them to chlorothalonil. Our goal was to test how the combination of these stressors might impact bumble bee health. We measured health by looking at how long the bees survived, how big they were, and how much protein was in their bodies. To make sure that exposure to N. bombi resulted in infection, we calculated how much N. bombi DNA was in each bee as a measure of total infection intensity. We also counted the number of N. bombi spores present in the gut of each bee as a separate measure of infection. We found that chlorothalonil exposure did not increase the total infection intensity or worsen bumble bee health. But we did find that bees exposed to chlorothalonil had more N. bombi spores in their bodies. This meant that bees infected with N. bombi had a higher potential to transmit the environmentally resistant form of the pathogen to other bees. In this research, we did not find solid support for the multiple stressor hypothesis because the bees did not have worse health outcomes when exposed to both N. bombi and chlorothalonil. Instead, we found that chlorothalonil exposure enhances the potential for N. bombi to transmit to new bees.

Highlights: In this research, we exposed bumble bee larvae to chlorothalonil, N. bombi, or both. Larvae are immature bees. Nosema only infects bees during the larval stage of development, which is why we infected them at this stage. We kept the larvae in the lab and allowed nursing bees to take care of them until they grew into adults. At that point, we measured bee health, quantified total infection intensity for each bee, and counted the number of N. bombi spores each bee had. To quantify total infection intensity, we used a technique called quantitative polymerase chain reaction (qPCR). qPCR is a molecular technique that allows a researcher to count how much DNA is in a sample. We used this technique to measure how much N. bombi DNA was inside adult bees that had been exposed to chlorothalonil as larvae and in those that had not been exposed to chlorothalonil. Figure 1 shows the results of this test. The dots in Figure 1 show the average N. bombi infection for bees exposed to chlorothalonil and for those not exposed to chlorothalonil. The “ns” above the dots in Figure 1 show the results of the statistical test that we did to see if the total infection intensity was different between the two groups. Here, “ns” stands for “not significant.” This indicates that the average infection was not significantly different when bees were exposed to both chlorothalonil and N. bombi.

A graph that shows total infection intensity data from two groups of bees, those that were exposed to chlorothalonil as larvae and those that were not. There is no significant difference in total infection intensity between the two groups.
Figure 1. The effect of the chlorothalonil treatment on the total N. bombi infection intensity in adult bees. The y-axis shows the relative amount of N. bombi DNA measured in the bees using qPCR. The x-axis shows whether bees were exposed to the chlorothalonil treatment as larvae.

To count the number of N. bombi spores each bee had, we euthanized each bee, pulverized their abdomens, looked at it under a microscope (see the N. bombi picture in the “Organism of Study” section), and recorded the number of spores present in each bee. We did this because the number of N. bombi spores inside each bee should tell us how ready the pathogen is to transmit to a new host. Figure 2 shows the results of this test. The dots in Figure 2 show the average number of N. bombi spores in bees exposed to chlorothalonil and in those not exposed to chlorothalonil. The two stars (**) above the dots in Figure 2 show the results of the statistical test that we did to see if the number of N. bombi spores was different between the two groups. The stars indicate that there was a significant difference between the two groups. These results show that bees exposed to both N. bombi and chlorothalonil as larvae had significantly more spores per bee when compared to bees that were not exposed to chlorothalonil. This is important because bees that have more spores are more likely to transmit N. bombi pathogens to other bees.

A graph depicts data from counting the number of N. bombi spores in bees exposed to chlorothalonil and those not exposed to chlorothalonil. Bees exposed to chlorothalonil had more N. bombi spores in their abdomens.
Figure 2. The effect of the chlorothalonil treatment on the number of N. bombi spores. The y-axis shows how many N. bombi spores were in each bee. The x-axis shows whether bees were “exposed” to the chlorothalonil treatment as larvae.

What My Science Looks Like: To conduct this experiment, we needed to infect bumble bees with N. bombi. The image below shows the exact method of delivery. N. bombi can only infect bees when they are larvae, so each larva had to be hand-fed the spores. We did this by peeling open the wax casing that protects the larvae and placing food infected with spores at the mouth of each larva. After feeding, they were returned to their homes, the nursing bees would re-seal the wax, and the bees would continue to develop. Nursing bees were bees randomly selected from the colonies to raise the larvae. They are used to maintain the brood until adulthood.

A picture of bumble bee larvae being hand-fed N. bombi spores with a pipette.
B. impatiens larvae receiving a dose of N. bombi spores.

The Big Picture: Bumble bees are especially valuable insects. They increase agricultural production and provide many services that improve ecosystem health. However, some species of bumble bees are in decline. To help them, we must understand how known stressors such as pathogens may impact their health. In our research, we found that a commonly used fungicide may increase the potential for a pathogen to be transmitted to other bees. One possible explanation for our results is that the fungicide chlorothalonil makes the current host less suitable for the pathogen. In response, N. bombi produces more spores to transmit to a new, more suitable host. While higher spore production was the only negative effect that we found related to chlorothalonil, it is important to keep in mind that we only used one bee species, B. impatiens. B. impatiens populations are stable in nature, so it is possible that other bumble bees are more sensitive to the dual effects of chlorothalonil and N. bombi. Regardless, researchers should continue to look at multiple stressors and how they impact bee health. Our research is important because if we can identify factors that increase pathogen transmission or virulence, we can make predictions about how future diseases may impact bee populations. This type of research helps us to understand patterns of disease, prevent outbreaks, and preserve important native pollinators.

Decoding the Language:

Bombus impatiens (B. impatiens): B. impatiens is the scientific name for the common eastern bumble bee.

Chlorothalonil: Chlorothalonil is a type of fungicide that is often sprayed onto crops and used in household gardens. When bees pollinate a plant that has been treated with this type of fungicide, it is easy for bees to pick up chlorothalonil and bring it back to the colony because it stays on the surface of the plant.

Fungicide: A fungicide is a pesticide used to kill fungal pathogens. They are generally used to protect crops from pathogens that may hinder crop development.

Host: A host is the organism infected by a pathogen. The pathogen will extract energy from the host.

Larvae (plural), larva (singular): A larva is the immature stage of an insect’s life, well before it reaches adulthood. The stages of growth go from egg to larva, to pupa, then to adulthood.     

Multiple stressor hypothesis: The multiple stressor hypothesis is the concept that exposure to many stressors will have a more negative impact on the organism than exposure to one alone.

Nosema bombi (N. bombi): N. bombi is the scientific name for the pathogen used in this study, which is known to harm bumble bees.

Nursing bees: A nursing bee is one type of worker bee in a colony. The nursing bees are responsible for taking care of the developing larvae. In the context of this study, our nursing bees were those that were picked to raise the larvae used in our research.  

Pathogen: A pathogen is a disease-causing microorganism, like a bacterium or a parasite.               

Quantitative polymerase chain reaction (qPCR): qPCR is a molecular technique that is used to quantify the amount of DNA within a sample. In this process, the DNA of interest has a fluorescent tag added to it and the DNA is amplified inside a machine. The fluorescent tag glows each time a new DNA strand is made, and the machine counts how many times the DNA glows.

Spores: In the context of this study, a spore is the environmentally resistant stage of Nosema bombi. The spores have a tough, thick outer coating, which allows them to be environmentally resistant.

Stressor: A stressor is any identifiable factor that can negatively affect an individual’s health.

Total infection intensity: Total infection intensity refers to our measure of how infected the adult bees were, after they had been exposed to N. bombi as larvae. We quantified the intensity of infection using qPCR.

Virulence: Virulence refers to theharmfulness of a disease. If it is more virulent, it is more harmful.

Learn More:

A book on how to create good pollinator habitat

Information from the United States Department of Agriculture (USDA) on how to gardeners can help pollinators

Synopsis edited by Emily Kerns (she/her/hers), PhD student as of September 2021, University of Wisconsin-Madison, Integrative Biology, and Maisam Yousef, B.S. 2019, Illinois State University.

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