Progress 01/01/22 to 12/31/24
Outputs
Target Audience:We disseminated findings to the scientific community viapresentations and peer-reviewed publications. Once our final datasets are analyzed and we better understand the impact of antibiotic field spraying on managed bee microbiomes, we will translate scientific findings into publications accessible to growers. Changes/Problems:The laboratory objective of this project relied on evolving antibiotic-resistant microbes. However, this aspect proved challenging. Generating GFP-labeled antibiotic-resistant transconjugants of bumble bee symbionts was not possible. We then focused on inducing de-novo antibiotic resistance via serial passaging in liquid selective media (amended with antibiotics). We monitored the growth (OD600) of bumble bee symbionts in 96-well plates to determine whether they could grow at increasing antibiotic concentrations.Still, despite many changes to our methodologies, we experienced the same issues: very slow growth rates inliquid media and contamination during this growth phase. The contamination was not evident when cultures were plated in solid selective media, but it was detectable via metagenomic sequencing. This made it impossible to attribute growth at increasing antibiotic concentrations in liquid media to de-novo mutations in the target bumble bee symbionts. What opportunities for training and professional development has the project provided?During the course of this project, we provided one-to-one research mentoring to five undergraduate students in the areas of bee behavioral assays, microbiology, and molecular techniques used to study bee microbiomes. We alsotrained one technical and one professional staff member on pollination and field bee biology techniques andone professional staff memberon techniques related to bee microbiome research. How have the results been disseminated to communities of interest?Through presentations and a peer-reviewed publication. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. We successfully conducted a field study in pear orchards in central Washington State. We were able to set up four colonies of Bombus vosnesenskii per siteat five oxytetracycline-sprayed sites, five sites sprayed with a biological antagonist, and three control sites (unsprayed). We collected bees at these sites pre- and post-sprays. In addition, we collected and monitored the foraging of honey bees at these sites. We also released and monitored the fitness of Osmia lignaria bees at the oxytetracycline and biological-sprayed sites plus a single control site. In collaboration with a Research Toxicologist at the Center for Environmental Measurement and Modeling Ecosystem Processes Division-Office of Research and Development-EPA, we developed a protocol for assessing oxytetracycline residues in our samples. We found oxytetracycline in pollen recovered from honey bee's bodies24 hrs after application, ranging from 0.02 to 35 ppm. We successfully dissected andextracted the DNAfrom the guts ofhoney bees (Apis mellifera) andbumble bees collected (Bombus vosnesenskii) in our field experiments. Via quantitative polymerase chain reaction, we detected an increase in the gut bacterial abundance forbees placed within fields sprayed with the biological antagonist. Although not statistically significant, we detected a decline in the gut bacterial abundance for bees placed in oxytetracycline-sprayed sites. These results are relative to those ofbees collected at the same sites pre-sprayand those collected at control sites without any sprays. We sequenced the extracted gut microbial DNA from honey bees and bumble bees collectedand are analyzing the results to understand changes to the bee microbiome after exposure to one application of oxytetracycline and one application of thebiological antagonist (Aureobasidium pullulans). We published the field-level data on antibiotic sprays' impacts on honey bee foraging and pear pollination. The oxytetracycline spray negatively impacts honey bee visitation to pear flowers, while the application of the biological antagonist increasedbee visitation. However, due to the abundant presence of honey bees in fields and the parthenocarpic nature of the pear varieties studied, we did not detect negative impacts on pear pollination (pollen deposition and seed count) and yield (fruit count, weight, and size). We analyzed fitness data from Osmia lignaria bees released in pear orchards. Osmia are known to be effective pollinators of pears. The preliminary data showed a potential negative impact of fireblight sprays (antibiotics and the biological alternative) on Osmia egg laying and/or egg hatching. There were almost no eggs or larvae in provisions, even though we recovered released adult bees from the nests. We followed up on these findings by designing a pilot laboratory study that provisioned Osmia eggs with pollen amended with several doses of oxytetracyclineand the biological antagonist. We did not find that antibiotic exposure increased egg or larval mortality in the lab, but it delayed molting into cocoons. We also found that exposure to the biological product increased mortality at the larval stage. First, this indicates that the application of oxytetracycline might be repellent to the foraging and provisioning of nests by Osmia bees, which parallels the behavioral impacts we recorded for honey bees.Second, the residues of the biological antagonist on pollen provisions could directly hinder thehatching of Osmia eggs. These field and laboratory findings were used as pilot data for a Washington State Departmentof Agriculture Specialty Crop Block Grant. All the Osmia work has been carried out in partnership with researchers at the Pollinating Insect-Biology, Management Systematics Research Unit at the USDA ARS. Objective 2. We carried out a detailed dose-response assay to study changes in bumble bee (Bombus impatiens) associative learning (associating a color with a reward), weight change, and survival with increased streptomycin concentration. We did not find an increase in mortality nor a decrease in weight with increasing streptomycin concentrations, although bees decreased sucrose solution consumption as the streptomycin concentration increased.Gut samples from this experiment were dissected and extracted, but the gutmicrobiome sequencing is ongoing. We are still working on dissecting the brains to identify metabolomic changes in the bee brain, emphasizing neurotransmitters. This work is in collaboration with a Research Toxicologist at the Center forEnvironmental Measurement and Modeling Ecosystems Processes Division-Office of Research and Development-EPA.
Publications
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Progress 01/01/24 to 12/31/24
Outputs
Target Audience:Findings were disseminated as a peer-reviewed publication, formally published in 2024. Changes/Problems:The laboratory objective of this project relied on evolvingantibiotic-resistant microbes. However, this aspect proved challenging. GeneratingGFP-labeledantibiotic-resistant transconjugants of bumble bee symbionts was not possible. We then focused on inducing de-novo antibiotic resistance via serial passagingin liquid selective media (amended with antibiotics). Still, despite many changes to our methodologies,we experienced the same issues:very slow growth rates in liquid mediaandcontamination during thisgrowth phase. The contamination was not evident when cultures were plated in solidselective media, but it was detectable via metagenomic sequencing. This made it impossible to attribute growth at increasing antibiotic concentrations to de-novo mutationsinthe target bumble bee symbionts. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Peer-reviewed publication. What do you plan to do during the next reporting period to accomplish the goals?Thisis the final reporting period for this seed grant. Still, we plan to finalize all data analysis of the microbiome datasets for peer-reviewed publications in the next six months. After this time, we will prepare extension publications to disseminate information to growers with the help of our co-authors, who are extension researchers at Washington State University.
Impacts
What was accomplished under these goals?
We successfully extracted the DNA from the gut microbesof bumble bees (Bombus vosnesenskii) collected in our field experiment(n=550 bees). We performed amplicon (300 pb paired-end reads of the V4 region of the 16s rRNA gene) Illumina sequencing of the gut microbes froma subsample (n = 384 bees)to determine if theB.vosnesenskiimicrobiome is affected byfield exposure to antibiotics and a biological antagonist (Aureobasidium pullulans) used to treat fireblight in pomme fruit orchards. We are in the process of analyzing this dataset. In addition, we carried out quantitative polymerase chain reactions (qPCR)in triplicate of the microbial DNA extracted from B. vosnesenskiiguts. Preliminary analysis indicates a decline in the microbial abundance of bees placed within oxytetracycline-sprayed fields relative to those placed at biological sprayed sites. We followed up on findings thatOsmia lignariadid not lay eggs in fields sprayed with antibiotics and biological products by designing a pilot laboratory study where we provisioned Osmia eggs with pollen amended withseveral doses of an antibiotic (oxytetracycline) orthe biological antagonist.We did not find that antibiotic exposure increased egg or larval mortality in the lab, but itdelayed molting into cocoons.We also found that exposure to the biological product increased mortality at the larval stage. These preliminary laboratory findings were used as pilot data in a Washington State Department of Agriculture Specialty Crop Block Grant. All the Osmia work has been carried out with the support and collaboration ofresearchers atthe Pollinating Insect-Biology, Management Systematics Research Unit atthe USDA ARS.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Laura Avila, Christopher McCullough, Annie Schiffer, JoMari Moreno, Neha Ganjur, Zachary Ofenloch, Tianna DuPont, Louis Nottingham, Nicole M. Gerardo, Berry J. Brosi, Effects of a field-sprayed antibiotic on bee foraging behavior and pollination in pear orchards, Agriculture, Ecosystems & Environment, Volume 359, 2024, 108757, ISSN 0167-8809
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Progress 01/01/23 to 12/31/23
Outputs
Target Audience:During the second year of the project we disseminated findings to the scientific community in the form of a contributedonline presentation at the Pollinator Health: Research and Application (A1113) ProjectDirectors Meeting, one peer-reviewed publication, and one undergraduate honors thesis. Findings will be shared with growers at the Tree Fruit Days organized by Washington State University in Spring 2024. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We have provide training and one-to-one research mentoring to three undergraduate students in the second year of the project. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In the last year of the project we plan to: 1) finish processing (e.g., DNA extractions and sequencing) bumble bee samples collected in the field; 2) submit a manuscipt for peer-review related to thechanges in the gut microbiome of honey bee and bumble bees exposed to field-sprayed oxytetracycline; 3) quantify antibiotic residues from pear flowers; 4)screen for metabolomic changes on bees exposed to antibiotics in the lab; 5) evolve resistance to antibiotics in three other bee gut core microbes, and carry out bee behavioral assays related to the project's laboratory objective; 6) analyze and write up results of laboratory assays; 7) submit honor's thesisfor peer-review.
Impacts
What was accomplished under these goals?
Impact Antibiotics are essential for treating crop pathogens in US fruit tree orchards. Nevertheless, not much is known about the impact of these applications on beneficial organisms. This is concerning because short-term exposure to high antibiotic doses can change the microbiome composition of beneficial insects, which is known to modulate behaviors associated with insect foraging. Moreover, long-term exposure to low concentrations of antibiotics may drive the development of antibiotic-resistant microbiomes. Our overarching goal is to characterize the impact of crop antibiotic applications on the beneficial organisms and foraging behavior of bees. Thus far, our results indicate that after oxytetracycline application (a widely used antibiotic), there is a decline in the number of pear flowers visited by honeybees. In turn, the spray of a biological product (alternative to antibiotics)increased honey beefloral visitation.However, due to the parthenocarpic nature of the pear cultivars, these changes did notscale up to impact pollination and fruit set. In the lab, bumble bee foraging is affected by oxytetracycline and a biological alternative, relative to controls.We are working to dissentangle whether changes in foraging behavior are due to the disruption of the bee microbiome.Our preliminary microbiome data indicates that honey bee's gut microbiome is highly disrupted (low abundance of beneficial microbial taxa), even prior to antibiotic exposure.Through this project, we have engaged collaborators who have expanded our research's field and laboratory outputs. Additionally, in the second year we trained three undergraduate students, one of which completed a honors-thesis. We look forward to disseminating our findings to growers and other stakeholders in the project's final year. Specific Accomplishments Related to Objectives Our overarching goal is to characterize the impact of antibiotic applications to pear orchards on the microbiomes, communities of symbiotic bacteria living within animals, and the foraging behavior of bee pollinators. Specifically, we have one field and one laboratory objective: 1. To measure the extent to which bees are exposed to antibiotics in pear orchards, their associated impact on colony-level gut microbiome composition, and individual and colony foraging in the field. We published the field-level data related to the impacts of antibiotic sprays on honey bee foraging and pear pollination. We successfully processed all honey bees(Apis mellifera) and 2/3 of the bumble bees collected (Bombus vosnesenskii) in our field experiments. We sequenced all the honey bee gut samplesand will do so for the bumble bee samples in Winter-Spring 2024. We analyzed fitness data fromOsmia lignariabees released and nests placed in orchards. The preliminary data shows a potential negative impact of fireblight sprays (antibiotics and the biological alternative)onegg hatching. We are following up on these findings by carrying out palynological assessment of pollen provisions. This assessment will allow us to assess if the fitnessimpacts were due to differences in floral diversity across sites vs. fireblight treatment. This work is done incollaboration with a Research Entomologist at the Pollinating Insect-Biology, Management Systematics Research Unit at the USDA ARS. In collaboration with a Research Toxicologist at the Center for Environmental Measurement and Modeling Ecosystems Processes Division-Office of Research and Development-EPA, we were able to develop a protocol for assessing residues of oxytetracycline from pollen carried by bees. We found oxytetraclyne in bees pollen 24 hrs after application, ranging from 0.02 to 35 ppm. 2. Test if microbiome changes alone can drive individualchanges in bee learning, memory, and foraging, in the laboratory. In 2023 an undergraduatestudent completed her honors thesis developing a protocol for evolving beecore bee symbionts via sequential passaging at increased antibiotic concentrations. Shefound that Gilliamella sp (a core member of the bee microbiome)is capable of evolving de novo mutations in-vitro that increase itsstreptomycin minimum inhibitory concentration (MIC) up to 39 ppm. We expect to continue evolving three other core symbionts, using this protocol, in Spring 2024. We carried out detailed dose-response assays to study changes in bumble bee behavior, weight, and survivalwith increased streptomycin concentration. Samples from this experiment will be used to develop a protocol to screen for metabolomic changes, with emphasis in neurotransmitters,in collaboration with aResearch Toxicologist at the Center for Environmental Measurement and Modeling Ecosystems Processes Division-Office of Research and Development-EPA.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Laura Avila, Christopher McCullough, Annie Schiffer, JoMari Moreno, Neha Ganjur, Zachary Ofenloch, Tianna DuPont, Louis Nottingham, Nicole M. Gerardo, Berry J. Bros. 2023. Effects of a field-sprayed antibiotic on bee foraging behavior and pollination in pear orchards. Agriculture, Ecosystems & Environment, 359:108757. https://doi.org/10.1016/j.agee.2023.108757.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2023
Citation:
Sherry Tsui. 2023. Evolution of Antibiotic Resistance in Bumblebee Gut Symbionts. Undergraduate Honors Thesis. https://etd.library.emory.edu/concern/etds/td96k3947?locale=en
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:Target Audience: During the first year of the project, we mainly reached participating growers. As initially proposed, we are on target to present our findings at the Tree Fruit Days organized by Washington State University in Spring 2023. We also disseminated fieldwork findings to other entomologists through a national conference. Efforts:In the Fall of 2022, we shared our fieldwork findings through an on-demand talk at the Entomological Society of America Meeting. Over 30 entomology researchers viewed the recorded presentation. Changes/Problems:Changes: We have encountered some delays in attaining our laboratoryobjective. A crucial step to accomplish this objective is engineering antibiotic-resistant bee gut microbiomes. However, we have been unsuccessful in selecting GFP-tagged antibiotic-resistant bee symbionts under microaerophilic conditions at our lab. We are pursuing two approaches to move this experiment forward: 1) we have partnered with scientists at Illinois State University who are also trying to develop the resistant symbionts, and 2) at our lab, we are inducing antibiotic-resistance via passage at sub-MIC antibiotic concentrations (basically relaying on mutations). What opportunities for training and professional development has the project provided?We have provide training and one-to-one research mentoring to five undergraduate students in the first year of the project. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In the second year of the project we plan to finish processing (e.g., DNA extractions and sequencing) thebeesamples collected in the field. We will also measure fitness impacts of the field treatments on blue orchard bees. We quantify antibiotic residues on pear flowers. Finally, we will carry out bee behavioral assays related to the project's laboratory objective.
Impacts
What was accomplished under these goals?
Impact Antibiotics are essential for treating crop pathogens in US fruit tree orchards. Nevertheless, not much is known about the impact of these applications on beneficial organisms. This is concerning because short-term exposure to high antibiotic doses can change the microbiome composition of beneficial insects, which is known to modulate behaviors associated with insect foraging. Moreover, long-term exposure to low concentrations of antibiotics may drive the development of antibiotic-resistant microbiomes. Our overarching goal is to characterize the impact of crop antibiotic applications on the beneficial organisms and foraging behavior of bees. Thus far, our results indicate that after oxytetracycline application (a widely used antibiotic), there is a decline in the number of flowers visited per bee. However, at least in pears, these changes do not scale up to impact pollination and fruit set. Through this project, we have engaged new collaborators who have expanded our research's field and laboratory outputs. Additionally, we have trained five undergraduate students, some of whom belong to groups underrepresented in the sciences. We look forward to disseminating our findings to growers and other stakeholders in the project's final year. Specific Accomplishments Related to Objectives Our overarching goal is to characterize the impact of antibiotic applications to pear orchards on the microbiomes--communities of symbiotic bacteria living within animals--and the foraging behavior of bee pollinators. Specifically, we have onefield and onelaboratory objetive: Measure the extent to which bees are exposed to antibiotics in pear orchards, their associated impact on colony-level gut microbiome composition, and individual and colony foraging in the field. In the Spring of 2022, we successfully: Set up sentinel bumble bee (Bombus vosnesenskii) colonies and collected honey bee (Apis mellifera) foragers at thirteen pear orchards in central Washington State. The samplings occurred before and after the antibiotic (oxytetracycline) or biological (Aureobasidium pullulans) product sprays. We have processed and sequenced the microbiome of a third of the bees collected. The remaining bees will be processed and sequenced in the project's second year. Monitored and analyzed individual honey bee foraging behavior and colony-level bumble bee foraging (via videos and weight change tracking). In addition, we collected pear flowers to quantify antibiotic residues, but the analysis of those samples will take place in the project's second year. Thanks to the forging of new collaborations with researchers at the Wenatchee Tree Fruit Research and Extension Center of Washington State University and the Pollinating Insects Research Unit of the USDA ARS, we expanded the original scope of our proposed work. We: Measured pear pollination and agronomic outcomes (seed set and fruit quality). Tested the impact of the oxytetracycline field dose and the biological product on bumble bee brood emergence and foraging behavior in the lab. Set up blue orchard bee (Osmia lignaria) nests at the orchards to test the impact of sprays on bee fitness. So far, we have analyzed the foraging, pollination, and agronomic data. In the field, we found that honey bees visited fewer flowers after oxytetracycline applications, while the opposite was true for the biological spray. However, we did not detect any impacts on pear pollination or agronomic outcomes. Test if microbiome changes alone can drive individual changes in bee learning, memory, and foraging, in the laboratory. We are?currently working on critical aspects of the experiments related to this goal. We have found that in-vitro, some bumble bee symbionts are more sensitive than others (e.g., slower to grow) at sub-minimum inhibitory concentrations (sub-MIC) of antibiotics. Overall, the tested bumble bee symbionts do not grow at or above 12.5 ppm of streptomycin, which is about an order of magnitude lower than the concentrations sprayed in the field. We have established a collaboration with bee microbiome scientists at Illinois State University to complete this objective. We expect to move on with the behavioral aspects of this project in the second year of the project.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Avila, L., McCullough, C., Schiffer, A., Ganjur, N., Moreno, J., Nottingham, L., Gerardo M.M., and B.J. Brosi. (2022, Nov 28). Effects of field antibiotics on bee foraging behavior and pear pollination. Entomological Society of America Annual Meeting, On-Demand.
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