Progress report for GNE19-200
Original Objective 1: We will document variation in gut microbiomes of multiple bee species along a floral diversity gradient in Amherst, Massachusetts. We have selected six farms that range in floral composition from highly diverse (>10 co- flowering species) to sunflower-only plantings. We will collect solitary leaf-cutter bees (Megachile spp.) and bumble bee workers (Bombus spp.) at each site and sequence their gut microbiomes and will assess patterns and differences between solitary and social taxa and between landscapes. If bees within the same site host similar communities of bacteria or patterns of microbial diversity, this would suggest that the floral community has an important role influencing the gut microbiome. Alternatively, we may find that host genus or sociality plays a more important role than landscape in structuring gut microbial communities. This study may also allow us to identify specific bacterial taxa and community patterns driven by sunflower pollen in an agricultural context, shedding light on the underlying mechanism of sunflower’s medicinal effect.
Modified Objective 1: Objective 1 samples were successfully collected from farms in Amherst during summer 2020. Because Megachile spp. were not found in high abundance at all sites, we collected Halictus ligatus as the solitary species.
Hypotheses: Gut microbiome diversity will increase with floral diversity, but gut microbiomes of solitary species will reflect site differences more than social species because solitary bees acquire gut microbes primarily from their environment, while social bees acquire microbes primarily from their nest .
Original Objective 2: To complement findings from the field survey, we will experimentally test how diet diversity and quality affect the gut microbiomes and immune responses of two bee species, Bombus impatiens and Megachile rotundata. If diet diversity increases immune response, then bees would be more susceptible to pathogens in low diversity habitats. On the other hand, if diet diversity reduces immune response, then bees in less diverse habitats may have increased performance in the presence of pathogens. However, an active immune response is energetically costly in the absence of pathogens, requiring increased food intake and decreasing bee performance. Thus, floral diversity and pathogens could synergistically affect bee performance.
Modified Objective 2 (2021): : Instead of collecting the immune responses of B. impatiens and M. rotundata, I will infect them with the gut pathogen, Crithidia bombi, and measure the pathogen loads after the experiment, as a proxy for immune function. The pandemic has led to difficult logistics in the lab and after learning the immune function methods at ISU last year, I realized that it would require more materials than I had anticipated. Therefore my advisor and I have agreed to take this modified approach, which will be more feasible at UMass given the constraints in how many people can occupy the lab at one time. This change will simplify my experimental design to make it more feasible under the constraints of COVID.
Modified Objective 2 (2022): Due to loosening COVID restrictions, we ultimately decided it was possible to test the bee immune response directly, albeit with a simpler and more feasible protocol than originally planned, rather than infecting bees with a pathogen (the previous modified plan for Objective 2). We originally proposed to measure the immune enzyme, phenoloxidase, which is the pre-cursor to the encapsulation response. Instead of measuring phenoloxidase activity, we decided to measure the downstream outcome, the intensity of the encapsulation response. The purpose of the encapsulation response is to recognize, surround, and kill a foreign object inside the body by depositing melanin around it. To estimate encapsulation response, I inserted a foreign object into the bee’s abdomen, allowed the bee to mount an immune response against it, and then removed the object to estimate how much melanin was deposited around the object. This approach has been used to estimate immunity in bees in previous studies [2,3] and encapsulation responses have been shown to differ between bee species .
On day 7 of the experiment, I anesthetized each bee by placing it in a -10C freezer for 3-6 min, and then inserted a sterile nylon microfilament (1mm long piece of clear fishing line) in the bee’s abdomen. To insert the microfilament, I first punctured the ventral membrane between the bee’s 3rd and 4th abdominal segments using a sterile needle. Using forceps, I then passed the implant through the membrane to fully enter the body cavity. Each bee was then placed back in its container to recover at room temperature, with access to sugar water and its diet treatment. After 2 hours, each bee was anesthetized in -10C and then frozen in -80C, where they are currently stored. This spring (2022), I will dissect out the implants and mount them on a glass slide. I will then photograph each implant and estimate the amount of melanin deposited. To estimate the darkness of the implant, I will use methods described in Davis et al. 2015. Briefly, I will place slides on an illumination table under a dissecting microscope and take a photograph. Each photo will contain a black reference point so that all photos contain the same range of darkness values. Using ImageJ software, I will obtain a mean gray-scale value for each implant. These values will be later used for statistical analyses of the immune response based on diet treatment.
We successfully ran the experiment with bumble bees, but ran into issues with the M. rotundata bees. We had enough bees to perform the gut microbiome experiment, which was conducted successfully, but the group of bees planned for the immune response experiment did not emerge from the refrigerator, possibly due to temperature fluctuations during shipment. Thus, I was unable to conduct the immune function experiment with M. rotundata. I did, however, conduct both the gut microbiome and immune response experiments with B. impatiens workers. I used wild-caught B. impatiens for the gut microbiome experiment and used commercial B. impatiens for the immune response experiment.
Hypotheses: Gut microbiome diversity and immune response will increase with diet diversity in both species, but B. impatiens will have lower gut microbial diversity across treatments than M. rotundata due to coevolution with a small group of bacterial taxa. Furthermore, M. rotundata will have overall higher immune responses than B. impatiens because solitary insects tend to exhibit higher physiological immune responses than social insects due to the lack of hygienic behaviors.
 McFrederick QS, Wcislo WT, Taylor DR, Ishak HD, Dowd SE, Mueller UG. 2012. Environment or kin: whence do bees obtain acidophilic bacteria? Molecular Ecology. 21:1754-1768.
 Doums C & Schmid-Hempel P. 2000. Immunocompetence in workers of a social insect, Bombus terrestris L., in relation to foraging activity and parasitic infection. Canadian Journal of Zoology, 78:1060-1066.
 Allander K & Schmid-Hempel P. 2000. Immune defence reaction in bumble-bee workers after a previous challenge and parasitic coinfection. Functional Ecology, 14:711-717.
 Davis S, Malfi R, Roulston T. 2015. Species differences in bumblebee immune response predict developmental success of parasitoid fly. Oecologia, 178:1017-1032.
Two aspects of modern agriculture impede sustainable practices: (A) lack of floral diversity in and around farms and (B) reliance on managed bees. Agricultural land is often dominated by one or few flowering species, resulting in a monotonous diet for pollinators, which can negatively affect bee fitness, especially when combined with other stressors such as pathogens . Studies on the effects of nutrition on bee health have been focused almost exclusively on the social and often managed taxa, honey bees  and bumble bees . However, solitary bees comprise 85% of bee species, provide essential pollination services to many natural and agricultural ecosystems , and are vulnerable to pathogens . The adoption of social behavior (i.e., a colony with a queen and workers) is often accompanied by other differences in life history and physiology, and bees with different levels of social contact likely differ in their responses to diets and pathogens. Therefore, we can cannot apply what is known about social bees to solitary species. Relying on one species of managed bee agriculturally is not sustainable  and managed bees can negatively impact native ecosystems by disrupting plant-pollinator networks  and transmitting diseases to wild congeners . Understanding and protecting wild bees is critical to minimize reliance on managed bee colonies. Improving our strategies to promote wild bee populations will require more studies on the factors and mechanisms underlying solitary bee health.
To conserve wild bee populations, we need a better understanding of how both social and solitary bees respond to pathogens under reduced floral resource diversity. Research on social bees has found that diet impacts not only growth and development, but also the immune response  and gut microbiome . Since the structure of the bee gut microbiome is dictated by evolutionary history of sociality , the microbiome could be an important factor determining species-specific responses to diet and pathogens. Therefore, I propose to test the effect of floral diversity and consumption of medicinal pollen on gut microbiomes and immune response in social and solitary bee species. Our lab recently found that pollen from sunflowers reduced infection levels of a common gut pathogen in one bumble bee species and that worker bees from farms with higher acreage of sunflower have lower pathogen prevalence . This study will identify the impact of sunflower pollen on the gut microbiome and immune response in multiple native bee species in the field and lab to better understand this pattern. These results will provide insight on how reduced floral diversity impacts performance of multiple bee species, which will shed light on how wild populations and communities will respond to landscape simplification.
 Goulson D, Nicholls E, Botias C, Rotheray EL. 2015. Bee declines driven by combined stress from parasites, pesticides, and lack of flowers. Science. 347(6229): 1255957.
 Dolezal AG, Toth AL. 2018. Feedbacks between nutrition and disease in honey bee health. Current Opinion on Insect Science. 26:114-119.
 Roger N, Michez D, Wattiez R, Sheridan C, Vanderplanck M. 2017. Diet effects on bumblebee health. Journal of Insect Physiology. 96:128-133.
 Cane JH. 2008. Pollinating bees crucial to farming wildflower seed for U.S. habitat restoration. In Bee Pollination in Agricultural Ecosystems, ed. James RR, Pitts-Singer TL, 4:48–64. New York: Oxford University Press. 232 pp.
 Potts SG, Biesmeijer JC, Kremen C, Neumann P, Schweiger O, Kunin WE. 2010. Global pollinator declines: trends, impacts and drivers. Trends in Ecology & Evolution 25:345-353.
 Kearns CA, Inouye DW, Waser NM. 1998. Endangered mutualisms: The conservation of plant-pollinator interactions. Annual Review of Ecology, Evolution, and Systematics. 29:83-112.
 Valido A, Rodriguez-Rodriguez MC, Jordano P. 2019. Honeybees disrupt the structure and functionality of plant-pollinator networks. Scientific Reports. 9:4711.
 Graystock P, Blane EJ, McFrederick QS, Goulson D, Hughes WOH. 2016. Do managed bees drive parasite spread and emergence in wild bees? International Journal for Parasitology: Parasites and Wildlife. 5:64-75.
 Alaux C, Ducloz F, Crauser D, Le Conte Y. 2010. Diet effects on honeybee immunocompetence. Biology Letters. 6:562-565.
 Billiet A, Meeus I, Van Nieuwerburgh F, Deforce D, Wackers F, Smagghe G. 2016. Impact of sugar syrup and pollen diet on the bacterial diversity in the gut of indoor-reared bumblebees (Bombus terrestris). Apidologie 47:548-560.
 Martinson VS, Danforth BN, Minckley RL, Rueppell O, Tingek S, Moran NA. 2011. A simple and distinctive microbiota associated with honey bees and bumble bees. Molecularly Ecology. 20:619-628.
 Giacomini JJ, Leslie J, Tarpy DR, Palmer-Young EC, Irwin RE, Adler LS. 2018. Medicinal value of sunflower pollen against bee pathogens. Scientific Reports. 8:14394.
During summer 2019, my colleagues conducted bee community surveys and collected specimens at farms varying in sunflower plantings. Although this was before the official start date for this grant, the original plan was to coordinate and collect specimens at some of the same farms. However, bees in the genus Megachile were not abundant and were not found at most farms. We therefore decided to postpone the field collections until summer 2020 and postpone the sequencing until after both Objectives 1 and 2 are complete, according to the updated timeline below.
During fall 2019, I conducted an experiment for a USDA-funded grant funded with similar methods to the proposed Objective 2. The goal of the USDA experiment was to test the effect of sunflower pollen and infection status on the gut microbial community in commercial bumble bee workers. Individual bees were either infected with a gut parasite (Crithidia bombi) or a sham inoculum and fed either sunflower pollen or a wildflower pollen mix. Bees were sacrificed at the end of one week and their guts were dissected out in a sterile environment and stored in -80oC. I will follow a similar protocol when dissecting bees in Objective 2 after the immune challenge, and so this fall experience has provided valuable training in learning new protocols. The USDA samples will be processed in April 2020, where I will continue to learn methods that I will implement in Objective 2.
During spring 2020, I successfully completed the USDA-funded experiment at Illinois State University on the bumble bee immune system, through which I learned the methods that I proposed to use to complete Objective 2. I was planning to travel University of California Riverside in April 2020 to process the gut microbiome samples, and learn the methods for extracting DNA and preparing samples for sequencing. However, this trip was cancelled due to the COVID-19 pandemic and our collaborators there are processing some of the samples for us. This is unfortunate because I was planning to have guidance learning these new methods, which I will need to know in order to do my SARE projects. While that was a setback, I still plan to prepare the samples for sequencing here at UMass. With the modification to Objective 2, I could use those expenses to get training from the UMass Genomics Core to prepare the samples for sequencing.
Update: As the COVID-19 pandemic changed over the course of 2021, travel restrictions lifted, and I was able to travel to UC Riverside. I am now at UC Riverside processing the gut microbiome samples for both Obj 1 and 2.
January – March 2021: Continue processing Objective 1 data and write up first USDA project for publication. Completed.
After I collected bees from the farms this summer, I collected pollen from the bodies and legs. I put a sample of this pollen on a microscope slide for each bee and am in the process of analyzing these slides to estimate the diversity of pollen species on each bee. I hope to then compare this diversity metric with the landscape diversity information to see if the landscape diversity is reflected in the bees’ foraging preferences, as detected by the pollen on the bee. I plan to finish analyzing the slides and the landscape data before starting the summer experiment. In addition to processing those data, I am also analyzing and writing up the immune function project that I did at ISU last year. I am waiting to receive the data from our collaborators at UC Riverside, so I will analyze those data when it is ready.
April – December 2021: Conduct experiment for Objective 2. Completed.
In April, I will order commercial Megachile rotundata bees. The bees will arrive as pupae and I will keep them in the refrigerator (7oC) until the experiment, the timing of which will depend on the phenology of the wild bumble bees. In late April and early May, I will collect bumble bee (Bombus impatiens) queens and rear them in the Adler lab at UMass Amherst. Once enough worker bees have emerged (approximately July), I will remove workers for the experiment. I will induce emergence of M. rotundata by placing cocoons in an incubator at 27oC, which will induce adult development. I will then place M. rotundata individuals in the experiment at the same time as the B. impatiens workers to have both species represented simultaneously, controlling for environment and season. I will then perform Objective 2 with both M. rotundata individuals and B. impatiens workers and store their guts in the -80oC freezer to be sequenced.
January - March 2022: Sequence all samples and start analyses. Currently underway.
I am currently working on extracting DNA to prepare the 16S libraries, and sequence ALL the samples from both Objective 1 and 2 at the McFrederick Lab at UC Riverside.
April - September 2022: Finish bioinformatics analyses for the gut microbiome data and assess melanization of implants.
I will spend the spring and summer analyzing the data from Objectives 1 and 2.
October 2022 - January 2023: Write up the results and submit for publication.
After all data has been processed and analyzed, I will spend the fall and winter assembling the results and writing up the paper for publication.
Education & Outreach Activities and Participation Summary
I created a website to document and disseminate the research activities associated with this grant. I have shared this website with the farmers involved and have linked to this website on the UMass Pollinator Extension Page as well as the Adler Lab home page. Additionally, I plan to communicate with the South Deerfield Growers Association. Since in-person gatherings are currently suspended, I hope to communicate the lab’s findings with this group via email or video call. My research website can be found here: https://aefowler.github.io/website/