Progress 07/01/21 to 06/30/24
Outputs
Target Audience:Results from project objectives have been written up and are in various stages of the publication process (see publications from reporting period section). This work with reach other researchers and practitioners in the form of peer-reviewed publications in scientific journals. Changes/Problems:In nectar, we saw no effects of neonicotinoid treatment on microbe community metrics. Rather, microbe abundance and diversity responded to inherent plant qualities like nectar volume, with certain taxa growing inherently more than others in canola nectar. In summary, found no evidence microbe respond to field-relevant neonicotinoid levels in nectar within a 24 h time span, but this study suggests that context, specifically assay methods, time, and plant traits and identity, is important in assaying effects of neonicotinoids on microbial communities. What opportunities for training and professional development has the project provided? Training: This project provided the PD with numerous new technical and scientific skills. In the past reporting period, these specifically include microbial inoculation and culturing, plant identification, pesticide quantification in samples using LC-MS, and new multivariate statistical techniques in the R programming language, especially analyses incorporating data on phylogenetic relatedness of species. The PD has also gained further experience writing scientific manuscripts and submitting them for publication. Professional development: In the past year, the PD has formed professional relationships with several of the mentor's collaborators. This collaboration has led to a new project which is currently being written up as a manuscript. Also, in summer 2023, the PD had the opportunity to informally mentor a high school student who joined the lab through a summer research experience program. The student assisted with field and laboratory work related to the floral inoculation project (Obj. 3). How have the results been disseminated to communities of interest?Results from project objectives have been written up and are in various stages of the publication process (see publications from reporting period section). This work with reach other researchers and practitioners in the form of peer-reviewed publications in scientific journals. The findings from this project will contribute to our general knowledge of how floral microbes and pollinators like solitary bees are impacted by agricultural practices, particularly irrigation and systemic insecticide use. This knowledge can eventually help develop more sustainable agricultural practices that take into account the impacts of chemical use on both plant microbiomes and pollinators, enhacing food security for the broader public. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Obj. 1: We employed two approaches to assess impacts of neonicotinoid exposure on nectar microbe taxa. First, we assayed in vitro effects of six neonicotinoid compounds on microbe growth using plate assays. Second, we inoculated a standardized synthetic microbe community into nectar of neonicotinoid-treated canola and assessed survival and growth after 24 hours. With few exceptions, in vitro neonicotinoid exposure tended to decrease bacterial growth metrics. However, the magnitude of decrease and the neonicotinoid concentrations at which effects were observed varied substantially across bacteria. Yeasts showed no consistent in vitro response to neonicotinoid. In nectar, we saw no effects of neonicotinoid treatment on microbe community metrics. Rather, microbe abundance and diversity responded to inherent plant qualities like nectar volume, with certain taxa growing inherently more than others in canola nectar. In summary, found no evidence microbe respond to field-relevant neonicotinoid levels in nectar within a 24 h time span, but this study suggests that context, specifically assay methods, time, and plant traits and identity, is important in assaying effects of neonicotinoids on microbial communities. Obj. 2: Using a laboratory capillary tube feeding assay, we assessed female alfalfa leafcutter bee (Megachile rotundata) feeding preferences for sucrose solutions containing different nectar microbes. Microbes were assayed one-by-one in pure cultures, with different batches of individual bees allowed to feed from control and/or microbe-treatment tubes over the course of three hours. We also used the same assay to test the response of leafcutter bees to solutions containing different concentrations (0, 10, or 100 ppb) of either of two neonicotinoid compounds (imidacloprid or dinotefuran). There appears to be some variation in how bees respond to different nectar microbe species. Some species of microbes appear to be aversive, while others elicit no response in feeding behavior, while none appeared to be attractive over the control solution. There also did not appear to be a response by bees to neonicotinoid compounds at the concentrations tested. Obj. 3: We inoculated a synthetic community of 5 nectar-inhabiting microbes into 31 phylogenetically diverse plant species, excluding pollinator visitation, and monitored community trajectory after 24 hours. We found that plant species, plant phylogeny, and the concentration of peroxide in nectar, but not floral morphological traits, influenced microbial abundance and composition in nectar. Higher ambient maximum and minimum temperatures promoted overall growth and the growth of specific microbe species.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Cecala, J. M., & Vannette, R. L. (2024). Nontarget impacts of neonicotinoids on nectar?inhabiting microbes. Environmental Microbiology, 26(3), e16603.
Published 17 March 2024. https://doi.org/10.1111/1462-2920.16603. This publication corresponds to Objective 1: Determine how neonicotinoid treatment and irrigation level interact to affect microbes in floral nectar.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Cecala, J., Landucci, L., & Vannette, R. (2024). Seasonal assembly of nectar microbial communities across angiosperm plant species: assessing contributions of climate and plant traits. ESS Open Archive eprints, 119, 11940961.
Preprinted 16 July 2024. https://doi.org/10.22541/au.172115321.11940961/v1. This manuscript corresponds to the re-focused Objective 3. It is currently in a second round of review at Ecology Letters (ISSN: 1461-0248).
- Type:
Journal Articles
Status:
Other
Year Published:
2024
Citation:
Microbial effects on pollinator feeding preference. Co-authors: Amber Crowley-Gall, Alexia Martin, Genevieve Olavarri, and Rachel L. Vannette. This manuscript corresponds to Objective 2: Determine the feeding responses of solitary bees to artificial nectar inoculated with the different microbial communities from Obj. 1 . This manuscript will be submitted shortly.
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Progress 07/01/22 to 06/30/23
Outputs
Target Audience:During this reporting period (July 1, 2022 to June 30, 2023), this project has enabled me to share my research with other scientists and pesticide industry professionals by presenting my results so far at the 2023 annual Pesticide Stewardship Alliance conference in Reno, Nevada (2/7/2023-2/9/2023). I have also spoken about my research to the Research Scholars Program in Insect Biology at UC Davis. This journal club is attended by undergraduate students participating in entomology research opportunities on campus. Changes/Problems:Objective 1 -- No major changes. Objective 2 -- No major changes. Objective 3 -- We have refocused Objective 3 based on the results of Objective 1 and preliminary results from Objective 2. We know from the plate readers experiments (Obj. 1A) that none of the screened microbes were completely inhibited by neonicotinoids, so it is unlikely any microbes would be prevented from growing entirely in floral nectar via neonicotinoid application to the plant. It is evident from greenhouse results with canola plants (Obj. 1B) that plant irrigation level and neonicotinoid application do not significantly alter which microbes grow in floral nectar or the density or abundance these microbes reach in the nectar. Rather, it appears that the canola nectar itself, irrespective of treatment, is acting as a filter determining which microbes can grow and which don't: Metschnikowia reukaufii, the nectar-specialist yeast species included alongside the three bacteria in our floral inoculum, did not grow well in canola nectar, even in flowers from control (no neonicotinoid treatment) plants. This suggests that an inherent biological quality of the nectar, perhaps antifungal peptides or some other antimicrobial metabolites secreted by the plant, inhibit the growth of Metschnikowia relative to the bacteria, which each displayed growth in all treatments. The above result indicates two things: (1) Conducting Obj. 3, as originally described in the fellowship proposal, would be largely meaningless. The original goal of Obj. 3 was to determine if and how neonicotinoid treatment and irrigation level affect crop pollination and seed set under semi-field conditions, via these factors' effects on microbes. Since we know that neonicotinoid treatment and irrigation level do not influence microbial growth in canola flowers, we would not expect there to be effects on bee visitation to flowers or pollination. Secondly (2), results so far raise an interesting question about how different plant species essentially "filter" certain types of microbes from growing in their nectar. This question is particularly relevant because of the wide variety of nectar-producing plant species cultivated in agriculture. Learning what species of plants filter certain microbes and how they do so biologically is directly relevant to biological control and other efforts involving inoculating crops with specific microbes to achieve a desired result (increased pollination, reduced damage from pathogenic microbes, etc.). This project is still relevant to the original objective of my fellowship in that it will clarify the factors and processes in an agricultural setting that contribute to the assembly of microbial communities in floral nectar of crop flowers, with implications for insect visitation and pollination. To address this topic, we have begun to conduct an experiment on the University of California, Davis campus in which we are inoculating a wide variety of plant species, including both ornamentals and food crop species grown at the campus' Student Farm, with a standardized microbial community. We are also collecting extra samples of nectar from these plant species for subsequent metabolomics analyses, to determine whether they contain any known antimicrobial compounds which would explain differences in microbial growth across plant species. Field work and analyses are currently ongoing. What opportunities for training and professional development has the project provided?Training activities: In terms of my scientific development, I have gained my first experience with preparing and submitting samples for LC-MS chemical analysis. I have also continued to enhance my proficiency in field and laboratory microbial ecology techniques by working closely with my advisor, Dr. Rachel Vannette, and other postdoctoral scholars in her lab (sterile hood techniques; pipetting techniques; microscopy techniques and using hemocytometers; preparing media for selective growth of microbes; agar media streaking techniques; preparing and storing ultracold stock cultures of microbe strains; using spectrophotometer plate readers to monitor optical density of microbial cultures; identification of bacterial unknowns using single-gene PCR and Sanger sequencing; floral inoculation and nectar extraction). I have also gained further experience in generating presentations and scientific writing. Finally, this fellowship has also allowed me to informally mentor graduate students and visiting scholars in the Vannette Lab. Professional development: As a PD, this fellowship has further improved my leadership and organizational skills. While I first gained experience as a NIFA predoctoral scholar at UCR executing a project of my own design as well as managing a budget, I have been able to experience this at a higher level of research. I also regularly meet with my advisor who provides constant feedback on experimental design, troubleshooting, manuscript writing, job opportunities, job application strategies, and more. How have the results been disseminated to communities of interest?This fellowship has allowed me to attend and present the results of this project so far at the 2023 Pesticide Stewardship Alliance conference in Reno, Nevada (2/7/2023-2/9/2023). At this meeting, I discussed the results from Objective 1. I was able to reach both my peers and network with potential collaborators and future employers at academic institutions and in the pesticide industry. What do you plan to do during the next reporting period to accomplish the goals?I will continue to write the manuscript for Objective 1 - only the Introduction, Discussion, and Abstract sections remain to be written. I plan to submit this manuscript for publication in a peer-reviewed scientific journal near the end of summer 2023. Data collection for Objective 2 (laboratory feeding assays with alfalfa leafcutter bees) will continue through the end of summer. Objective 3 has been modified (see Changes/Problems section).
Impacts
What was accomplished under these goals?
Impact Statement Issue: Nectar-inhabiting microbes (NIMs) have garnered attention due to their potential to alter interactions between bees and flowers, and thus influence pollination. These bacteria and yeasts, just like macroorganisms, can be subjected to non-target impacts of agricultural pesticides, potentially affecting bee behavior and crop pollination. Neonicotinoids are the most widely used class of insecticides worldwide and can alter certain microbial communities, yet we know nothing of how they impact NIMs. Furthermore, irrigation (i.e., plant water availability) influences neonicotinoid translocation through plant tissues and nectar properties, yet the impacts of this agricultural practice on NIMs also remain unstudied. Activities and Results: During this reporting period (July 1, 2022 to June 30, 2023), I have been conducting capillary feeding (CaFe) assays with alfalfa leafcutter bees (Megachile rotundata), testing their feeding responses to artificial nectar laced with neonicotinoids (imidacloprid and dinotefuran, the same compounds used in the Obj. 1B canola greenhouse experiment described in the last reporting period) as well as artificial nectar inoculated with one of various species of NIMs. I have also been conducting floral inoculation experiments on the UC Davis campus examining how plant species identity, along with nectar volume and chemistry, impact the trajectory of inoculated NIM communities (see Changes/Problems). Data so far suggest that leafcutter bees show little if any response to neonicotinoid compounds in artificial nectar relative to control nectar, but I am still running additional experiments and collecting more data for an adequate sample size for statistical power. On the other hand, nectar inoculated with certain species of microbes appears slightly aversive to bees, while some other microbes elicit no difference in feeding response relative to a sterile artificial nectar solution (30% sucrose m/m). Preliminary data from the floral inoculation experiments on campus are currently being analyzed, but preliminary trends in data suggest that there are large differences in how inoculated NIM communities develop in different plant species. In certain plant species, some NIM taxa grow quite well, while the growth of others is almost entirely halted. Broader Outcomes: This project will help us to understand how commonplace agricultural practices (i.e., pesticide use, irrigation, and plant identity) may influence pollination via the floral microbiome, in hopes of further integrating sustainable pest and pollinator management in North American agriculture. Objective 1. Determine how neonicotinoid treatment and irrigation level interact to affect microbes in floral nectar. Activities Completed: Data collection and analysis for Objective 1, parts A and B, is mostly complete. As detailed in the last reporting period, I conducted two separate experiments examining how NIMs are impacted by neonicotinoids: a laboratory plate reader experiment and a greenhouse experiment using canola (Brassica napus). During this reporting period, we analyzed nectar samples collected from experimental plants in the greenhouse that I stored at -20 °C since extraction. Samples were tested for residues of imidacloprid or dinotefuran, depending on which was applied to that plant. Briefly, I diluted nectar samples in ultrapure water and analyzed them via electrospray ionization (ESI) liquid chromatography-mass spectrometry (LC-MS) on an Orbitrap machine (5 µL injection volume) at the UC Davis Campus Mass Spectrometry Facilities (CMSF). Data Collected: For LC-MS samples, we created standard curves and back-calculated concentrations of imidacloprid or dinotefuran from sample peak areas using dilutions of 5 ppm analytical standards. Results: Data are still being analyzed, but all treated plants contained detectable levels of the compound they were treated with. Concentrations of neonicotinoid residues are roughly proportionate with the amount (mass) of the formulation delivered to the plant during the beginning of the experiment back in spring 2022. Key Outcomes: The LC-MS results are crucial to the interpretation of the results of this experiment. These results show that the experimental pesticide treatments applied to plants were successful in creating nectar containing roughly the desired range of neonicotinoid concentrations in canola plants. Therefore, we can be sure that the microbes inoculated into canola nectar experienced the intended range of exposure to neonicotinoid residues while they were growing. Objective 2. Determine the feeding responses of solitary bees to artificial nectar inoculated with the different microbial communities from Obj. 1. Activities Completed: I am currently in the process of collecting data for this objective. Last year, I worked on troubleshooting methods for refining the laboratory assay that is being used to assess female alfalfa leafcutter bee (Megachile rotundata) feeding preferences. This capillary feeding (CaFe) assay can detect differences in consumption by bees between treatments down to several microliters (µL). Microbes are being tested one-by-one in pure cultures, with different batches of individual bees. I am also using the same assay set-up to test the response of leafcutter bees to solutions containing different concentrations (0, 10, or 100 ppb) of either of two neonicotinoid compounds (imidacloprid or dinotefuran). Data Collected: I have run trials with several microbe species so far (including Metschnikowia reukaufii, Neokomagataea thailandica, etc.), and separate trials with neonicotinoid compounds. Results: There appears to be some variation in how bees respond to different microbes. Some species of microbes appear to be aversive, while others elicit no response in feeding behavior. There does not appear to be a large response to neonicotinoid compounds so far. Key Outcomes: Objective 3. Determine how neonicotinoid treatment and irrigation level affect crop pollination and seed set under semi-field conditions. Activities Completed: Objective 3 has been modified (see Changes/Problems section). Data collected: Results: Key Outcomes:
Publications
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Progress 07/01/21 to 06/30/22
Outputs
Target Audience:During this reporting period (September 20, 2022* to June 30, 2022), this project has enabled me to reach other scientists, extension professionals, agricultural industries, and stakeholders by presenting project results so far at the 2021 Pacific Branch meeting of the Entomological Society of America in April 2022. I have also reached undergraduate biology students by discussing my postdoctoral research so far during a virtual seminar for Sonoma State University in February 2022. *While this reporting period is shown to begin on July 1, 2021, my actual start date at UC Davis was September 20, 2021, roughly twoand a half months later. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training activities: I have greatly enhanced my proficiency in field and laboratory microbial ecology techniques by working closely with my advisor, Dr. Rachel Vannette, and other postdoctoral scholars in her lab. These techniques include, but are not limited to: sterile hood techniques; pipetting techniques; microscopy techniques and using hemocytometers; preparing agar and broth media for selective growth of yeast and bacteria; agar media streaking techniques; preparing and storing ultracold (-80°C) stock cultures of microbe strains; using spectrophotometer plate readers to monitor optical density (OD600) of microbial cultures; identification of bacterial unknowns using single-gene PCR and Sanger sequencing; floral inoculation and nectar extraction. I have also gained further experience in generating presentations and scientific writing. Finally, this fellowship has also allowed me to informally mentor graduate students in the Vannette Lab. Professional development: As a PD, this fellowship has further improved my leadership and organizational skills. While I first gained experience as a NIFA predoctoral scholar at UCR executing a project of my own design as well as managing a budget, I have been able to experience this at a higher level of research. I also regularly meeting with my advisor who provides constant feedback on experimental design, troubleshooting, manuscript writing, job opportunities, job application strategies, and more. How have the results been disseminated to communities of interest?The fellowship allowed me attend and present the results of this project so far at the 2021 Pacific Branch Meeting of the Entomological Society of America meeting in Santa Clara, CA in April 2022. At this meeting, I co-organized a symposium session with a PhD student at University of Nevada, Reno on factors influencing plant-pollinator interactions. I have been able to reach both my peers and network with potential collaborators and future employers. What do you plan to do during the next reporting period to accomplish the goals?I have almost completed all data collection for Objective 1. I am beginning to write up a draft manuscript of the experiments comprising Objective 1 and I plan to submit it for publication in a scientific journal this fall. As outlined above, data collection for Objective 2 (laboratory feeding assays with alfalfa leafcutter bees) will occur this fall 2022, and Objective 3 (outdoor flight cage experiments with leafcutter bees) in spring 2023.
Impacts
What was accomplished under these goals?
Impact Statement Issue: Nectar-inhabiting microbes (NIMs) have garnered attention due to their potential to alter interactions between bees and flowers, and thus influence pollination. These bacteria and yeasts, just like macroorganisms, can be subjected to non-target impacts of agricultural pesticides, potentially affecting bee behavior and crop pollination. Neonicotinoids are the most widely used class of insecticides worldwide and can alter certain microbial communities, yet we know nothing of how they impact NIMs. Furthermore, irrigation (i.e., plant water availability) influences neonicotinoid translocation through plant tissues and nectar properties, yet the impacts of this agricultural practice on NIMs also remain unstudied. Activities and Results: During this reporting period (September 20, 2022* to June 30, 2022), I have gained insights into how exposure to neonicotinoids influences the growth of NIMs when exposed to different types of these compounds in vitro, as well as how NIM communities are impacted by neonicotinoid treatment and differential irrigation of the plant they are inhabiting. Data so far suggest that there is little variation across the different types of neonicotinoid compounds in terms of the their effects on growth of NIMs in vitro. Overall, neonicotinoids appear to have negative effects on growth of NIMs in vitro, though the magnitude of this effect varies greatly across microbe taxa and does not always change linearly with neonicotinoid concentration. Data from in plantae experiments with canola are currently being analyzed, but preliminary trends in data suggest that, as in in vitro experiments, there are large differences in how different microbe species respond to neonicotinoid exposure. Environmental filters may also be impacting the trajectory of NIM community composition within a flower. Broader Outcomes: This project will help us to understand how commonplace agricultural practices (i.e., pesticide use and irrigation) may influence pollination via the floral microbiome, in hopes of further integrating sustainable pest and pollinator management in North American agriculture. Objective 1. Determine how neonicotinoid treatment and irrigation level interact to affect microbes in floral nectar. Activities Completed: I conducted two separate experiments examining how NIMs are impacted by neonicotinoids: a laboratory plate reader experiment and a greenhouse experiment using canola. In the plate reader experiment, I measured the growth of seven different species of NIMs (the yeasts Metschnikowia and Aureobasidium, and the bacteria Pantoea, Rosenbergiella, Acinetobacter, Neokomagataea, and Apilactobacillus) in artificial nutrient broths, placed in the wells of 96-well plates. In these wells, microbes were exposed to either (1) a control broth containing no neonicotinoids or (2) broth containing either 10 ppb, 100 ppb, or 1000 ppb of each of six major neonicotinoid compounds: acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, or thiamethoxam. These concentrations were chosen to represent field-realistic low and high concentrations of neonicotinoids found in floral nectar of treated plants, as well as a field-unrealistic concentration to inform dose-response curves. In the greenhouse experiment on the UC Davis campus, I grew a commercial cultivar of canola from seed in 3-gallon pots in a fully crossed, randomized block experiment, testing the effects of three different factors: (1) low or high irrigation, controlled using irrigation flow spikes inserted into soil of pots, (2) low or high neonicotinoid application, and (3) two types of neonicotinoids - imidacloprid and dinotefuran. These two neonicotinoids were chosen due to their popularity of use in agriculture as well as their somewhat dissimilar effects on microbes in the plate reader experiment. When canola plants finally were in bloom (April-May 2022), I inoculated numerous flowers with a previously-prepared stock culture of microbes constituting an artificial community consisting of Metschnikowia, Neokomagataea, Actinetobacter, and Apilactobacillus), each at roughly 10,000 cells per µL. After 24 hours, I returned and extracted nectar from inoculated flowers. Nectar was then aliquoted in the lab. Half of samples were plated on agar media to encourage the growth of fungi and bacteria colonies. After incubating media for a week, the number of colony-forming units (CFUs) of each microbial morphotype were tallied and identified using 16S barcoding to estimate relative abundance of microbes in nectar samples. The other half of nectar samples were frozen at -80°C, for later identification of relative abundance of microbe taxa using a novel molecular sequencing method. Data Collected: For the laboratory plate reader experiment, I collected data on optical density (OD600) of the seven microbe taxa in well plates over a 72 hour period at 25 °C (or 30 °C for Apilactobacillus, since this microbe grows better at higher temperatures). From these readings, I calculated the maximum optical density (K) and maximum population growth rate (r) by fitting the data to a classical logistic growth curve using the package growthcurver in R. For the greenhouse experiment, I collected data on nectar volume and flower mass of plants in different treatments, the abundance and diversity of CFUs on agar media from plated nectar samples, and am in the process of sequencing microbial DNA in frozen nectar samples. Results: For the laboratory plate reader experiment, responses to neonicotinoid compounds varied across microbe taxa. Within a single microbe taxon, responses to different types of neonicotinoids (e.g., imidacloprid versus clothianidin, etc.) were generally not statistically different. The two yeast species showed little if any responses to neonicotinoid exposure, while bacteria showed greater responses. Impacts on bacterial growth were consistently negative, but varied in magnitude across bacterial taxa. For the greenhouse experiment, plants responded strongly to the irrigation treatment. Plants in the higher irrigation treatment produced both more nectar per flower and larger flowers. Microbial data is still being analyzing, but data from CFUs on agar media so far suggests that responses to our treatments varied across the four inoculated microbe taxa. Some of our inoculated microbe taxa, particularly Metschnikowia, did not grow well in canola nectar regardless of treatment, suggesting plants may exert background filtering on microbial communities in addition to neonicotinoid exposure and water availability. How this impacts community dynamics in the face of our experimental treatments is still being assessed. Key Outcomes: I have gained knowledge on methods and techniques cultivating and analyzing microbes. I have also improved various research-related skills (keeping a lab notebook, analyzing data, etc.). Objective 2. Determine the feeding responses of solitary bees to artificial nectar inoculated with the different microbial communities from Obj. 1. Activities Completed: Most of the data collection for this objective will be undertaken during the next reporting period (year 2 of the project). So far, I have worked on troubleshooting methods for refining the laboratory assay that will be used to assess alfalfa leafcutter bee feeding preferences. This assay should be able to detect differences in consumption by bees down to several microliters (µL). During a validation run comparing 5% versus 45% sucrose solutions, female leafcutter bees appear to preferentially feed from the higher sugar solution, suggesting this assay is capable of representing leafcutter bee feeding preferences. Objective 3. Determine how neonicotinoid treatment and irrigation level affect crop pollination and seed set under semi-field conditions. Activities Completed: This objective will be undertaken during the next reporting period.
Publications
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