Identification and cultivation of probiotic bacteria that improve the health and performance of managed bumble bees

IDENTIFICATION AND CULTIVATION OF PROBIOTIC BACTERIA THAT IMPROVE THE HEALTH AND PERFORMANCE OF MANAGED BUMBLE BEES

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

ACTIVE

Funding Source

AFRI COMPETITIVE GRANT

Reporting Frequency

Annual

Accession No.

1032122

Grant No.

2024-67013-42275

Cumulative Award Amt.

$300,000.00

Proposal No.

2023-08413

Multistate No.

(N/A)

Project Start Date

May 1, 2024

Project End Date

Apr 30, 2026

Grant Year

2024

Program Code

[A1113]- Pollinator Health: Research and Application

Recipient Organization
UNIVERSITY OF CALIFORNIA, IRVINE
(N/A)
IRVINE,CA 92697

Performing Department
(N/A)

Non Technical Summary
Managed bumble bees (Bombus impatiens) and various species of wild bumble bees are vital pollinators in U.S. agriculture. However, disease threatens the sustainability of their pollination services. Moreover, efforts to optimize the health and pollination performance of commercially produced B. impatiens colonies are still in their infancy. Recent research shows that bacterial probiotics are a highly promising tool for bolstering disease resistance and performance of honey bees. However, as honey bee bacteria generally fail to establish in bumble bees, there is a need to identify bumble bee-specific bacteria that could be used to support their health and pollination.The overall goal of this project is to develop bacterial probiotics specific to B. impatiens that improve performance and sustain pollination services in the face of disease and other stressors. The supporting objectives are: 1) identify gut bacteria from bumble bees that promote resistance to Crithidia bombi, a common parasite in managed colonies that can spread to wild bumble bees; 2) identify gut bacteria that improve pollination-relevant aspects of bumble bee performance (cognition, lifespan, and reproduction); 3) build a culture collection targeted to promising bacterial species. A "rewilding" approach will be used to discover beneficial probiotics. From cryopreserved gut material derived from wild B. impatiens populations, bacterial strains will be isolated, identified, and experimentally inoculated into bumble bees under controlled laboratory conditions. Strains that promote one or more aspects of bee health will be prioritized as probiotic candidates for further development. Overall, this work will accomplish the first key steps toward naturally derived probiotics for managed B. impatiens that support bumble bee health and production of bumble bee-pollinated crops.

Animal Health Component

(N/A)

Research Effort Categories

Basic

85%

Applied

(N/A)

Developmental

15%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3085	1100	100%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3085 - Other bees (non-Apis bees) - All bees except Apis cerana; includes Bombas, Alfalfa leaf-cutter;

Field Of Science
1100 - Bacteriology;

Keywords

Goals / Objectives
The overall goal of the project is to develop probiotics that will ultimately improve disease resistance and performance of managed bumble bees (Bombus impatiens) in the United States. Managed bumble bees are agriculturally important for crop pollination, but often suffer from disease or other factors that reduce their productivity. Further, they have been linked to pathogen spillover into wild and declining bumble bee species. One potential strategy for addressing this problem is based in the gut microbiome, which is known to improve bees' pathogen resistance and nutrition. The project will take the first steps toward microbiome-targeted bee management by identifying and culturing probiotic bacterial strains that support bumble bee health.The supporting objectives are:1. Identify gut bacterial strains and species that promote bumble bee resistance to parasites.2. Identify gut bacterial strains and species that improve pollination-relevant aspects of bee performance.3. Build a culture collection, targeted to the most promising bacterial species, for evaluation as probiotics for managed Bombus impatiens.

Project Methods
1. The first experiments will evaluate which bacteria improve the parasite resistance of bumble bees (Bombus impatiens).Different strains and species of bacteria, isolated from wild Bombus impatiens, will be cultured and inoculated individually into the diet of germ-free (sterile) bumble bees, following established techniques. Subsequently, bees will be exposed to a defined dose of parasite (Crithidia bombi) cells. After two weeks, Crithidia infection levels will be measured. Experiments will be replicated across bee colonies and across microcolonies (small cages with three worker bees each). Generalized linear models will be used to identify strains showingstatistically significant reductions in C. bombi load relative to controls, accounting for microcolony and colony identity as random effects. 16S rRNA gene sequencing will be used to determine colonization success of the focal bacterial strains. Strains that both colonize the bees, and reduce parasite infection, will be prioritized for the culture collection as candidate probiotics. Success will be evaluated as production of robust data on Crithidia loads across bacterial strain treatments.2. The second experiments will adopt the same design as above, but with different focal phenotypes. Specifically, lifespan, reproductive output, and cognitive ability will be used as metrics of overall performance. Lifespan will be measured by checking survival every 2 days. Reproduction will be measured as the number and size of brood produced over the lifespan of each microcolony. Cognition will be assessed through a collaboration with Dr. Felicity Muth. Dr. Muth's free-moving proboscis extension response assay will be applied to age-controlled bees to measure associative learning and memory retention. Similar statistical analyses will be used as above to assess relationships between bacterial treatment and performance metrics, but modified to account for differences in data structure. 16S rRNA gene sequencing will again be used to determine colonization success of the focal bacterial strains. Strains that both colonize the bees, and improve one or more of the performance metrics, will be prioritized for the culture collection as candidate probiotics. Success will be evaluated as production of robust data on bee performance traits across bacterial strain treatments.3. Strains identified from the above experiments as having one or more positive effects on bumble bees will be used to establish a culture collection. The strains, which will be first identified by 16S rRNA gene sequencing, will also be submitted for whole-genome sequencing. After locating an appropriate repository that can provide it upon request (such as the USDA ARS Culture Collection), and after creating backup glycerol stocks of all strains, the culture collection will be deposited for use by the research community. Success of this objective will be evaluated as production of a collection of bacterial strains, with known positive effects on bumble bees, and deposition of this collection in an accessible repository.

Progress 05/01/24 to 04/30/25

Outputs
Target Audience:During the reporting period, the primary focal audiencewas other scientists working on insect (and particularly bee) microbiomes.This audience was reached through the Project Director's participation in three conferences. The second audience wasbee conservation practitioners, with whom the PDcommunicated directly about the potential utility of probiotics. Third, members of the local public (in Orange County, CA) were reached through an outreach talk by the PD, and a talk and workshop by Kristal Watrous (a Professionalfunded on this grant). Changes/Problems:The original goal of this project was to first identifybeneficial probiotic bacteria for bumble bees (Bombus impatiens), and next,build a culture collection ofthe most promising species for further evaluation. In an initial trial, the approach intended to complete the first step--transplanting cryopreserved gut material into commercial Bombus impatiens bees in the laboratory--was not as reliable as anticipated based on published work from bumble bees and other insects. Specifically, freezing and thawing the original gut homogenateappears to reduce the efficacy of bacterial transplantation into the gut of recipient bees. Hence, it was decided to reverse the order of the steps: first build a culture collection of diverse gut bacterial strains from wildBombus impatiens (Objective 3), and then test a subset in commercial Bombus impatiens in the laboratory, in order to find beneficial probiotic candidates (Objectives 1 and 2). What opportunities for training and professional development has the project provided?Kristal Watrous, a technician funded by this project, has been provided with skills development in microbiology,molecular methods, and data analysis in the software platform "R". Further, she gained experience with teaching and community outreach.Ozichi Ikegbu, an undergraduate student at UC Irvine, has also been trained in laboratory techniques (particularly bacterial culturing) scientific methods. How have the results been disseminated to communities of interest?Although final results are not yet ready for dissemination, the motivation and plans for the project have been communicated to researchers and the public in three ways: Direct communication with entomologists and bee conservation practitioners (including Xerces Society, USDA Bee Lab) Participation in the International Congress of Entomology (August 2024), Entomological Society of America Annual meeting (November 2024), and Gordon Conference on Animal-Microbe Symbiosis (June 2025). A lecture for the Laguna Beach Garden Club (October 2024). What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, the following activities will be carried out: Submit a Microbiology Resource Announcement to the American Society of Microbiology's journal, describing the culture collection andand publicizing its availability to other researchers (Objective 3). Complete nutrition assays for the experiment evaluating probiotic effects on bumble bee performance (Objective 2). Conduct an experiment assessing the effect ofArsenophonus, a probiotic candidate that we have determined can establish in treated commercial Bombus impatiens colonies, on beeresistance to parasites(Objective 1). Write and submit a manuscript for peer review and publication, in whichthe results of Objective 1 and Objective 2 are described. Present findings at the Entomology Society of America Annual Meeting (November 2025), and TBD conferences in 2026.

Impacts
What was accomplished under these goals? The culture collection, comprising 50 bacterial strains, has been developed and will soon be submitted for publication as a Microbiology Resource Announcement (Objective 3). Strains were sourced from wild bees of the same species (Bombus impatiens) that is commercially producedfor crop pollination. The collection includes: i) pure cultures, cryopreserved in glycerol in an ultralow freezer, ii) high-quality, full-length 16S rRNA gene sequences (Sanger sequencing), which are used to classify strains to the species level. The strains will be made publicly available for use by the bee research community. A major experiment has been completed using five bacterial strains from the culture collection, chosen based on their high prevalence in wild Bombus impatiens, addressing Objective 2. The strainswere tested for i) their ability to persistently establish in commercial Bombus impatiens colonies, ii) their potential effects on bee performance, measured as a change in bee survival rates or reproductive output, iii) their effects on bee nutrition. DNA sequencing was used to determine establishment. No effects on bee survival or reproduction were observed, indicating that the strains are not pathogenic--a necessary precondition for probiotic development. One of the strains, Arsenophonus, was able to successfully establish in the gut of treated bees. This bacterium is now the focus of follow-up experiments. Nutrition assays are currently underway. Please see the "Changes/Problems" statement for why the order of the objectives, as described in the proposal, has been reversed.

Publications