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: In our pilot study in summer 2019 we found very few Megachile at farms. We will proceed with the proposed approach except with a potentially different solitary bee genus. We will collect individuals of the same genera across all sites, determined by what’s available. In 2019, Halictus spp. individuals were common and would be a good candidate since they are generalist, solitary bees that frequently forage on sunflowers. If there is another generalist solitary species that is common at the sites in 2020, such as Megachile or Osmia, we will collect individuals from that genus instead.
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 .
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.
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.
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.
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 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.
In 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.
January – July 2020: USDA projects to learn methods
I am working on two projects funded by the USDA that use similar methods as I have proposed in the SARE projects. In Jan 2020, I am working at Illinois State University learning how to measure the immune system of bees. I will use these skills in the immune assays in Objective 2. In April 2020, I will travel to the University of California Riverside to learn how to extract and sequence DNA from bee guts from experimental samples. During this trip, I will learn the protocols and methods for these analyses, which I will later implement at UMass for Objectives 1 and 2. We originally proposed to bring samples from Objective 1 to UC Riverside during this time in April 2020, but since we did not collect bees in 2019 and have postponed this until summer 2020, I will not sequence Objective 1 bees until after I’ve also completed Objective 2 and do them all together at UMass.
August – September 2020: Collect bees for Objective 1
I will collect bees (Bombus and a solitary generalist bee, probably Halictus) on sites with varying acreage of sunflower plants in Amherst, MA and measure floral resources. I will store the specimens in a -80oC freezer on the UMass campus until DNA extraction and sequencing.
October 2020 – March 2021: Write up USDA projects for publication
April – September 2021: Conduct experiment for Objective 2
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.
October – December 2021: Sequence all samples and start analyses
After the experiment of Objective 2 is complete, I will extract DNA, prepare the 16S libraries, and sequence ALL the samples from both Objective 1 and 2 at the Genomics Core facility at UMass.
January – May 2022: Finish analyses and write up the data for publication
I will spend the spring analyzing the data from Objectives 1 and 2. Both data sets will be submitted for publication.