Identifying Mechanisms behind Interacting Stressors on Wild Bees to Inform Pollinator-Friendly Agricultural Landscapes

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 [1].

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 [4].

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 3^rd and 4^th 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.