Progress 02/15/18 to 07/14/22
Outputs
Target Audience:Our primary has been the research community, whom we have sought to share our work into the sublethal effects of neonicotinoid pesticides on bee behavior, introducing new perpsectives on the consequences of this exposure for bee health, sensory performance, and plant-pollinator interactions. We reached this audience in two ways. First, we attended and presented talks and posters at scientiic conferences regionally (e.g. Pacific Branch Meeting of the Entomological Society of America), as well as nationally and internationally (e.g. International Conference on Pollinator Health, Biology and Policy; International Congress of Entomology; Entomological Society of America Meeting). Likewise, we have reached this audience by publishing research in peer-reviewed journals (e.g. Functional Ecology, Ecology Letters, Oecologia), and promoting this work on our social media accounts (Twitter, Facebook) and via up-to-date public websites (www.anneleonard.com, ResearchGate). A secondary audience has been members of the public, who often have an interest in learning about "how to help bees", whether they are gardeners, hobby beekeepers, wildlife enthusiasts, or simply community members who want to learn more about research at UNR. Because consumers who apply pesticides in gardening or landscaping contexts are a major route of neonicotinoid exposure for bees, we sought to educate members of our local community about how pestcide over-use can affect bees in a variety of formal and informal outreach settings. For example, we have used "plain language" blog posts on journal websites (fesummaries.wordpress.com) to highlight our Functional Ecology paper on nectar chemistry, podcast interviews (via UNR's Reynolds School of Journalism), and interviews with local journalists (Discovery) to reach a wide audience via social media and media exposure. While the pandemic affected our ability to participate in many in-person events, we nonetheless managed to give public lectures on pollinator health (e.g. "Operation Pollination" lecture series at the Great Basin Brewing), and to participate in two community open-houses held at the Nevada Musuem of Natural History/UNR Pollinator Garden, where we exhibited live bees and specimens. The museum open houses targeted families with children, an audience that we also reached via our involvement with the Daugherty Summer Science Exploration. For this program, that brings middle-schoolers from low-income schools to the UNR campus for hands-on science-related activities, we developed a two day workshop for students and teachers focusing on bee biology and plant-pollinator interactions. Finally, we have also reached beekeepers via talks at both the Nevada Beekeepers' Association Meeting, as well as at a meeting of the Marin County Beekeepers' Association. Beekeepers have an interest in both understanding the sublethal effects of neonicotinoid pesticides on bee physiology and behavior, as well as in understanding how these chemicals may combine with natural phytochemicals found in plants or used in hive treatments (e.g. thymol) to affect bee health (e.g. Richman et al. 2022 Functional Ecology). Interfacing with UNR's Desert Farming Initiative (a demonstration, educational and outreach farm on campus), PD Leonard organzied a seminar on Bee Identification and Natural History that DFI staff participated in; with growing interest in diversified urban farming and local produce, over the course of the project we realized there was much interest among local growers in learning about and providing habitat for native pollinators. Changes/Problems:Pandemic-associated shut downs and constraints on conducting in-person research slowed our progress on the electrophysioloical components of the project. In response, we placed more of an emphasis on behavioral assays, fieldwork, and conceptual contributions (e.g. a review/conceptual framework in Current Opinion in Insect Science; a meta-analysis in Ecology Letters). Additionally, we broadened our scope to include a project on how microbial communities might be perturbed by pesticide exposure, giving rise to altered reward composition. This project led to a productive collaboration with a UNR Biochemist, Dr. Dylan Kosma, who has helped us develop and refine a new analytical pathway for analyzing pollen nutrition. Similarly, when the chance to collaborate with an insect ecoimmunologist (Dr. Angela Smilanich, UNR Biology Dept.) to collect data on how diet and pesticide exposure affected bee immune function, we included this measure in our work (Richman et al. 2022). In sum, all the major changes were in response to pandemic (and PD Leonard's parental leave timing), as well as opportunistically pursuing collaborations which we believe enhanced the project scope as a whole, and provided training opportunities for the graduate students and postdoctoral researchers involved. What opportunities for training and professional development has the project provided?New technical skills PhD student Anna Tatarko had previously no experience with electrophysiological assays, and has had the chance to train extensively under Dr. Mathew's mentorship. Likewise, Dr. Sarah Richman joined the lab with limited behavioral/lab-based experience, and has had the chance to run two lab-based projects on physiology and behavior. More specifically, she sat in on a graduate seminar on ecoimmunology, and developed a collaboration with UNR Biology Associate Professor Angela Smilanich to design and complete the immune component of her study into the combined effects of imidacloprid and nectar chemicals on bee health (Richman et al. 2022, Functional Ecology). Through this, she has gained hands-on experience in insect physiology techniques. Dr. Richman's co-authoring a meta-analysis (Siviter et al, 2020 Ecology Letters) was another opportunity to gain a specific new set of analytic skills, and to network with other early career researchers doing cutting-edge work on the sublethal effects of pesticides on bees. Scientific Presentations and Networking Overall, work from this project has been presented by graduate students or postdoctoral researchers 8 times at regional, national, and international conferences: PhD student Anna Tatarko presented at the: 2019 Entomological Society of America meeting (St. Louis, awarded first prize in Student Competition for the President's Prize in her session), 2029 International Conference on Pollinator Biology Health and Policy (Davis, CA); 2022 Pacific Branch meeting of the Entomological Society for America (Santa Rosa); 2022 International Congress of Entomology (Helsinki). Postdoctoral researcher Dr. Sarah Richman presented at the: 2019 International Conference on Pollinator Biology Health and Policy (Davis, CA); 2019 American Society of Naturalists meeting (Asilomar, CA) and 2019 Evolution (Providence, RI, where she was an invited speaker at a symposium on mutualisms in a changing world); 2020 Botany meeting (virtual); 2020 Ecological Society of America meeting (virtual); 2020 Entomological Society of America meeting (virtual). PI Leonard and Postdoctoral Researcher Sarah Richman travelled to the 2018 Entomological Society of America Meeting in Vancouver, Canada. At this meeting, we attended (among others) a symposium on "Understanding and Mitigating the Risks of Pesticide Exposure for Pollinators and Other Beneficial Insects" which drew international leaders working on the intersection of bee pesticide exposure, nutrition, and behavior. Likewise, PD Leonard's participation in a symposium on the sensory basis of insect foraging at the 2022 IUSSI meeting was an important chance to network with international colleagues after pandemic-related lack of professional conference travel. Mentoring Dr. Richman engaged in a pedagogy workshop during Spring semester 2019, and participated in UNR's "Mentoring Mentors" workshop this year as well (these workshops are co-led annually by PD Leonard). This project has involved seven undergraduates in data collection (all female), and ultimately three of these students became co-authors on resulting manuscripts. Two of these students are members of groups traditionally underrepresented in the sciences (Hispanic and African American). Through their involvement, students and recent graduates have gotten hands-on experience with troubleshooting protocols, interpreting results, and preparing results for publication. Six of these students were mentored by Postdoctoral researcher Sarah Richman, who gained feedback on her mentoring efforts and best practices through regular meetings with PD Leonard. PhD student Anna Tatarko mentored one undergraduate student who was in the early stages of preparing a poster for the UNR Undergraduate Poster Session, when it was unfortunately cancelled due to COVID. Career advancement Across the 6 publications that have so far resulted from this project, 3 undergraduate students, 2 graduate students and 2 postdocs have been co-authors. Dr. Richman presented a departmental seminar on her research to the UNR Ecology, Evolution, and Conservation Biology program which served as practice for a future job talk. Dr. Richman completed her 3-year tenure on the grant, and accepted a postdoctoral position at ETH Zurich. PhD student Anna Tatarko prepared a dissertation proposal and a successful NIFA Predoctoral Fellowship application that builds on some of the themes of the grant, exploring how agricultural chemicals alter floral and bee microbial communities. A former undergraduate who assisted with the Penstemon research was hired for a post-baccalaureate program to work on a separate line of research in PD Leonard's lab, and an undergraduate co-author was accepted to a PhD program in Entomology at Michigan State, where she currently conducts pollination research. How have the results been disseminated to communities of interest?The pandemic forced us to rethink some of the traditional ways in which we would normally otherwise disseminate our research, as it curtailed travel to in-person conferences, and largely shut down public events in our state (like our annual Pollinator Festival, held as part of UNR's Museum Day community open house). When we found opportunities to connect with our target audiences, we took them: Media PI Leonard and Co-PI Mathew were interviewed about this research for a podcast produced by students participating in UNR's Hitchcock Project for Visualizing Science at the Reynolds School of Journalism. PD Leonard was interviewed about this project for UNR's Discovery magazine. PhD student Anna Tatarko was interviewed for a science-themed podcast, "Science! With Friends" Postdoc Sarah Richman prepared a blog post summarizing our accompanying journal article about nectar chemistry and neonicotinoids: https://fesummaries.wordpress.com/2022/02/08/when-humans-alter-nectar-chemistry-what-happens-to-bees/ Scientific audiences Including the eight conference presentations by graduate students and postdocs, PD Leonard presented this work at the 2022 IUSSI (International Union for the Study of Social Insects) conference, and traveled to Penn State University to give a seminar which described some of the project research. We have published six manuscripts in peer-reviewed journals so far resulting from this project, and have three more manuscripts in preparation for submission. We promoted findings on social media (Twitter, Facebook) as well as on our lab website, and on sites such as ResearchGate. Beekeepers PD Leonard was an invited speaker at the 2020 Nevada Beekeepers Association annual meeting (Yerington, NV), and shared basic information about the complex interaction between nutrition and pesticide exposure on bee health. PD Leonard shared research as an invited speaker for the Marin County Beekeepers Association in May 2020 (audience ~60). Community members and K-12 outreach In 2019, we shared our work with the public via an exhibit at the UNR Natural History Museum's Museum Day event in the UNR Pollinator Garden which PI Leonard helped create. Grant personnel exhibited a live bumblebee colony, museum specimens, and facts about bee behavior and nutrition to families (total attendance ~750) attending this community open house event. Typically this is an annual event, but due to pandemic restrictions, it has not happened since. We also contributed to a Valentine's Day exhibit at the UNR Natural History Museum, which focused on threats to the stability of interactions between plants and pollinators. PD Leonard participated in a public lecture series held at the Great Basin Brewing Company, "Operation Pollination" during which she highlighted the lab's current work on pesticides. PD Leonard and PhD student Anna Tatarko developed a hands-on lesson plan for middle school students involving plant-pollinator interactions and pesticides, as part of the 2022 Daugherty Summer Science Exploration, a program that brings students and science teachers from local schools to campus. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The most significant findings of the project include: Neonicotinoid pesticides disrupt insect olfactory function, and do so particularly strongly as compared to other sensory modalities. Given how important the sense of smell is to bees, we identified a mechanism that could impact their ability to acquire high quality nectar and pollen, both at the individual and colony level. Neonicotinoid exposure changes the costs and benefits of nectar phytochemicals. Phytochemicals in nectar can be nutritionally beneficial for bees, and support their health in working lands. Our work offers the caveat that pesticide exposure changes this, a finding relevant to the health of both managed and wild bees. Pesticides alter the nutritional value of nectar and pollen, but concerningly can also result in plants that are more attractive to bees. This issue should be considered in planning pollinator-friendly plantings in areas where pesticide exposure may be inevitable. Two important "negative results": in contrast to other work, we failed to find evidence that the major managed bumble bee pollinator species in the US prefers neonicotinoid-laced nectar. Additionally, we found no evidence that screening assays involving a nutritionally simplified nectar returns different results than assays using more chemically realistic nectar surrogates. Problem: Neonicotinoids may have sublethal effects on insect sensory systems and foraging, impairing their ability to recognize high quality nectar and pollen. Obj.1: Establish how neonicotinoids affect foragers' ability to discriminate among floral rewards that vary in composition using taste and scent. Project 1: To assess whether neonicotinoids impact bees' ability to discriminate among nectar solutions varying in composition. We (Richman et al, Oecologia 2021) asked whether neonicotinoid consumption altered bees' preferences for artificial nectars of varying quality. We compared two common preference assays (free-flying vs. ad libitum feeding), and quantified the efficiency of each. We developed a novel assay (the Restricted Volume Preference Assay RVPA) that returns results that, compared to ad libitum feeding assays more closely matches the more realistic (but inefficient) free-flying preference assay. We used RVPA to compare the sucrose preferences of bees dosed with the neonicotinoids imidacloprid vs. control, but found no effect. Project 2: Research on other bee species had suggested that bees might prefer to consume nectar laced with neonicotinoids. We wanted to determine whether 1) these preferences were based on taste or post- ingestive feedback, and 2) whether nectar sugar concentration matters. We asked whether the US commercial bumble bee species, Bombus impatiens had such preferences across a varying range of nectar sugar concentrations (Muth et al. 2020, Royal Society Open Science), We failed to detect any preference for neonicotinoids suggesting that the previous findings relate either to methodological issues, or that there are species-specific responses. Project 3: Assessing how acute exposure to neonicotinoids alters sensory performance in bees, we found (Muth et al. 2019 Biology Letters) that when we compared bees' ability to learn color vs. scent from artificial "flowers", olfactory, but not visual, learning was impaired. This is consistent with olfactory processing being disrupted. Project 4: To use electrophysiology to explore the impact of neonicotinoids on olfaction, we asked this question in Co-PD Dr. Mathew's system of expertise, Drosophila. This system offered the possibility of fine-scale insight into the mechanisms of olfactory disruption. We collected single-neuron recordings from the antennae of flies exposed to the neonicotinoid imidacloprid and compared whole- antennal performance. Imidacloprid altered performance in single neurons and antennal function as a whole. A behavioral assay showed these impairments further scale to impact olfactory preferences. This manuscript will be submitted to Scientific Reports. Project 5: How do nectar phytochemicals impact the performance of bees given neonicotinoid exposure? We explored how nutrition interacts with pesticides to impact behavior and health (Richman et al. 2022 Functional Ecology). We maintained bees on plain sucrose or sucrose containing a nectar chemical: caffeine (in Citrus); thymol, (in Thymus and used vs. mites in honeybees); or digoxin (in Digitalis). Half of the bees within a diet treatment were given an acute exposure to imidacloprid, and we measured several outcomes to explore how pesticide exposure works alongside nutrition to affect health. By adding in realistic nectar chemicals, we assessed how well using sugar water as "nectar" (the common approach) estimates the impacts of these pesticides on bees. We further showed that a single exposure to a neonicotinoid eliminates some of the beneficial effects of these phytochemicals. Problem: Most research on the sublethal effects of neonicotinoids on bees have focused on individual behavior. Yet, chemically-mediated communication can be important for nutritional regulation at the colony level, and there are many gaps in our understanding of how pesticide exposure impacts social communication in foraging. Obj. 2 and 3a: Exploring the physiological and behavioral effects of neonicotinoids on the social regulation of foraging. Progress on this objective was most affected by the pandemic, combined with PD Leonard's parental leave. Project 6: Adapting to pandemic work-at-home conditions, Postdoc Sarah Richman co-authored a meta-analysis (Siviter et al, 2021 Ecology Letters), on the effects of neonicotinoids on non-Apis bees, in line broadly with these objectives. This involved data extracted from 53 papers (212 effect sizes) focusing on Bombus (a social genera) and Osmia (a solitary genera). Key findings include a pattern whereby neonicotinoid exposure negatively affected reproductive output across all bees and impaired bumblebee colony growth and foraging. Neonicotinoids also reduced bumblebee individual development but had no effect on Osmia development. Problem: Pesticide exposure could impact both floral displays and the nutritional value of the nectar and pollen bees collect, either via effects on plants, or indirectly by altering microbial communities that shape these traits. Objective 3b: Establish how neonicotinoid exposure scales up to affect the nutritional value of wildflowers in agroecosystems: Project 7: We completed a project on how pesticide exposure impacted floral traits in two species of Penstemon. Analysis has so far yielded intriguing connections between pesticide exposure, floral display production, and visitation: pesticide-treated plants treated produced more flowers, and received more bee visits. This raises concerns about how exposure to pesticides may result in wildflowers that both contain pesticides and are more attractive to bees. This manuscript is next in line to be submitted. Project 8: We sought to characterize how neonicotinoids in pollen and nectar may alter the microbial communities found there, in turn potentially changing the nutritional composition of pollen. We collected pollen loads from bumble bees, treated them with a neonicotinoid, and saved samples for both microbial and nutritional analyses. Although we found no effect on microbial abundance, we did note that imidacloprid altered the frequency of common fungal morphotypes; analysis to determine whether this is nutritionally meaningful for bees is ongoing. This latter effort involves a collaboration with Dr. Dylan Kosma (UNR Biochemistry) who is helping develop a new analytical pipeline for pollen chemistry. Project 9: In a special issue of Current Opinion in Insect Science (Francis et al. 2020), we shared our thoughts regarding how nectar and pollen microbes might alter the nutritional value of floral rewards, with possible consequences for bee foraging and plant performance.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Leonard, A.S. "Sensory Ecology of Pollination and Anthropogenic Change" IUSSI 2022 (XIX International Congress of the International Union for the Study of Social Insects) Talk presented as part of Symposium, "The sensory ecology of social insect foraging". San Diego, CA (July 6 2022).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Tatarko, A., Richman, S. and Leonard A.S. "Do flowers containing pesticides, fungicides, and their combination alter bee-microbe-plant interactions?" Pacific Branch Meeting of the Entomological Society of America meeting. Santa Rosa, CA (April 13 2022).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Tatarko, A., Richman, S. and Leonard A.S. "Do flowers containing pesticides, fungicides, and their combination alter bee-microbe-plant interactions?" XXVI International Congress of Entomology. Helsinki, Finland (July 2022).
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Progress 02/15/21 to 02/14/22
Outputs
Target Audience:Our primary has been the research community, whom we interfaced with through attending conferences and preparing research for submission to peer-reviewed journals. A secondary audience has been members of the public whom we have reached via a blog post highlighting our work on pesticides and nectar chemistry (fesummaries.wordpress.com). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has involved numerous undergraduates in data collection, and ultimately two of these students (Denyse Marquez Sanchez and Sharon Miller) became co-authors on the resulting manuscript. Both of these students are members of groups traditionally underrepresented in the sciences, and through their involvement, they have gotten hands-on experience with troubleshooting protocols, interpreting results, and preparing results for publication. Sharon Miller is now enrolled in a graduate program in Entomology at Michigan State. A former undergraduate who assisted with the Penstemon research, Jillian Luthy, was hired for a post-baccalaureate program to work on a separate line of reseach in PI Leonard's lab. More broadly, new PhD student Anna Tatarko had previously no experience with electrophysiological assays, and has had the chance to train extensively under Dr. Mathew's mentorship. Likewise, Dr. Sarah Richman joined the lab with limited behavioral/lab-based experience, and has had the chance to run two lab-based projects involving physiology and behavior. Additionally, she developed a collaboration with UNR Biology Professor Angela Smilanich to design and complete the immune component of her study on the combined effects of imidacloprid and nectar secondary compounds on bee performance. Through this, she has gained hands-on experience in insect physiology techniques. Collaborating on a meta-analysis was another opportunity to gain a specific new set of analytic skills, and to network with other early career researchers doing cutting-edge work on the sublethal effects of pesticides on bees. Dr. Richman completed her 3-year tenure on the grant, and accepted a postdoctoral position at ETH Zurich, during this reporting period. How have the results been disseminated to communities of interest?When we found opportunities to connect with interested audiences we did so--PI Leonard and Co-PI Mathew were interviewed about this research for a podcast produced by students participating in UNR's Hitchcock Project for Visualizing Science, and PI Leonard and Postdoc Sarah Richman prepared a blog post summarizing our accompanying journal article about nectar chemistry and neonicotinoids: https://fesummaries.wordpress.com/2022/02/08/when-humans-alter-nectar-chemistry-what-happens-to-bees/ What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Establish how neonicotinoids affect foragers' ability to discriminate among floral rewards that vary in composition using taste and scent. We are currently working to get this paper submitted by July 15th, as all data collection is complete and we are finalizing analyses and figures (including results of a new behavioral experiment quantifying how pesticide exposure affected olfactory preferences and responsiveness). Objective 2: Explore whether neonicotinoids disrupt perception of chemical signals used to regulate colony-level foraging effort. As noted above, this effort rests upon getting the electrophysiology rig up and modified for bees. Using the time granted by our extension, we will fund a postdoctoral researcher already in the Leonard Lab (Dr. Anthony Vaudo) to work with Co-PI Mathew to collect data on this point. Objective 3: Establish how neonicotinoid exposure scales up to affect: a) Bombus response to nutritional perturbations: The pandemic made this objective a logistical challenge, given the safety requirements for multiple people to work in close proximity. We are still thinking about how we might address this question on a smaller scale. b) The nutritional value of wildflowers in agroecosystems. In contrast, this ended up being a pandemic-friendly line of research where we think we have a chance to make a relatively large impact. We are currently analyzing plant samples, pollen nutritional profiles, and microbial communities associated with pollen and nectar. We expect at a minimum data analysis to be complete by July 15th, with submission of 2-3 associated manuscripts to follow. Likewise, we will share the results of this work at three conferences spring-summer 2022 (Pacific Branch Meeting of the Entomological Society of America; IUSSI; and the International Congress of Entomology).
Impacts
What was accomplished under these goals?
Objective 1: Establish how neonicotinoids affect foragers' ability to discriminate among floral rewards that vary in composition using taste and scent. Postdoctoral Researcher, Sarah Richman, and PhD student Anna Tatarko have focused on this question using both behavioral assays and electrophysiology. Project 1: Assessing how exposure to neonicotinoid pesticides impacts bees' ability to discriminate among nectar solutions varying in composition. Status: paper published during this reporting period at Oecologia. Postdoctoral Researcher Sarah Richman led this project, in collaboration with PI Leonard and collaborator Dr. Felicity Muth. In this paper, Dr. Richman compared three different preference assays and explored whether neonicotinoid consumption altered bees' sucrose preferences. We began this project after realizing it was essential to validate the assumption that our tube-based preference assays would return results qualitatively similar to bees in free-flying assays (bolstering any potential findings of the effects of neonicotinoids on the expression of preferences). We compared two designs commonly used to assay bee foraging preferences (free-flying assays and ad libitum feeding assays in preference tubes), and quantified the relative efficiency of each (e.g. by comparing person-hours per data point). We also developed a novel high throughput assay (the Restricted Volume Preference Assay RVPA) whereby bees in preference chambers are given repeated small amounts of each solution to sample, mimicking floral volumes. This technique represents an advance over ad libitum feeding assays because bees do not satiate before having the opportunity to sample options-- instead, RVPA returns results that, compared to ad libitum feeding assays more closely matches the more realistic (but inefficient) free-flying preference assay. We used RVPA to compare the sucrose preferences of bees dosed with the neonicotioid imidacloprid vs. control, but found no strong effect. Project 2: Using electrophysiological techniques to explore the impact of neonicotinoids on olfaction. Status: Data collection on electrophysiological portion involving Drosphila complete, planned extension to bees interrupted by pandemic. Co-PI Dennis Mathew has led this effort, which also comprises a chapter in PhD student Anna Tatarko's dissertation research. In order to learn the electrophysiological techniques required to address this question in bees, Anna completed a project to ask this question in Dr. Mathew's system of expertise, Drosophila. Starting on this model system made the most sense for training purposes; however, since it also offered the possibility of achieving much more fine-scale insight into the potential mechanisms of olfactory disruption (e.g. single neuron recordings) this work will stand on its own as a publication (currently in prep). Anna first developed a protocol to dose Drosophila with neonicotinoids, then collected single-neuron recordings from the antennae of flies exposed to a sublethal dose of the neonicotinoid imidacloprid. She then scaled up to look at whole-antenna activity in dosed vs. control flies using electroantennagrams (the technique she plans to also use on bees). We found that imidacloprid alters performance in single olfactory neurons, but may not disrupt antennal function as a whole. During this reporting period, Anna completed a behavioral preference assay, demonstrating that these impairments may scale up to impact olfactory-guided preferences. We are currently working on the manuscript (methods and results are complete) with a goal of submitting it by July 15, 2022. Project 3: How does a diet of realistic secondary nectar metabolites impact the performance of bees given an acute neonicotinoid exposure? Status: Manuscript submitted to a few top journals and ultimately in review at Functional Ecology during the reporting period. Inspired by her work exploring how neonicotinoid exposure impacts bees' preferences for nectar solutions (Project 1) Dr. Sarah Richman completed a second lab-based project looking at how nutrition interacts with neonicotinoids to impact behavior and health. She maintained bees on plain sucrose or sucrose containing a naturally-occurring nectar chemical: caffeine (in Citrus nectar); thymol, (in Thymus nectar and used against varroa mites in honeybees); or digoxin (in Digitalis nectar). Half of the bees within a diet treatment were given an acute exposure to the neonicotinoid imidacloprid, and we measured several outcomes to explore how pesticide exposure works alongside nutrition to affect survival and health (sucrose consumption; activity levels; immune function). This paper will be impactful because nearly all work exploring the impacts of pesticides on bee health present them in a simple sucrose solution. By adding in nutritionally realistic dietary compounds, we gain insight into how well using nutritionally simplified sugar water as "nectar" estimates the impacts of these pesticides on bees. We submitted this manuscript to several high impact journals sequentially, and it went out to peer review at PNAS; ultimately, it was well-received with minor revisions at Functional Ecology. Objective 2 and 3a: Progress depends upon the modification to Co-PI Dennis Mathew's electrophysiology setup, both of which have been delayed by the pandemic. However, adapting to pandemic work-at-home conditions, Postdoctoral Sarah Richman was invited to co-author a meta-analysis regarding the effects of neonicotinoid pesticides on non-Apis bees, which is in line broadly with these objectives. During the reporting period, this meta-analysis was published in the top journal Ecology Letters, and involves data extracted from 53 papers (212 effects sizes) focusing on two genera: bumblebees (Bombus spp.)and mason bees (Osmia spp.). Key findings include a pattern whereby neonicotinoid exposure negatively affected reproductive output across all bees and impaired bumblebee colony growth and foraging. Neonicotinoids also reduced bumblebee individual development (growth and body size) but we found no effect onOsmiadevelopment. Objective 3b: Establish how neonicotinoid exposure scales up to affect the nutritional value of wildflowers in agroecosystems. PI Leonard took on this research objective as a sabbatical project, and completed an experiment intended to explore how pesticide exposure alters reward composition and ecological interactions with herbivores and pollinators in two species of Penstemon. Analysis of this data is ongoing; due to unexpectedly low rates of flowering, the clearest signal relates to interactions with herbivores and natural enemies. To collect data on floral traits and bee visitation, PhD student Anna Tatarko worked alongside Postdoctoral researcher Sarah Richman on a second round of data collection, during summer 2021. This dataset yielded intriguing connections between pesticide exposure, floral display production, and bee visitation, and we submitted an abstract to present our results at the 2022 Pacific Branch meeting of the Entomological Society of America meeting, as well as at the International Congress of Entomology (July 2022). As discussed in our last progress report, we made some progress on research to characterize not only how neonicotinoids impact the macronutrients available in pollen and nectar, but also the microbial communities found there that are consumed by bees. These microbes can change the nutritional content and volatile cues associated with nectar and pollen. PhD student Anna Tatarko and PI Leonard collected corbicular pollen loads from wild bumblebees, treated them with a small exposure to the neonicotinoid Imidiacloprid, and saved samples for both microbial and nutritional analyses. This project should yield a first-of-its-kind look into whether and how pesticide exposure might alter pollen nutrition, via effects on microbial activity.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Siviter, H., Richman, S. K., & Muth, F. (2021). Field?realistic neonicotinoid exposure has sub?lethal effects on non?Apis bees: A meta?analysis. Ecology Letters, 24(12), 2586-2597.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Richman, S. K., Muth, F., & Leonard, A. S. (2021). Measuring foraging preferences in bumble bees: a comparison of popular laboratory methods and a test for sucrose preferences following neonicotinoid exposure. Oecologia, 196(4), 963-976.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2021
Citation:
Richman, S. K., Maalouf, I. M., Smilanich, A. M., Marquez Sanchez, D., Miller, S. Z., & Leonard, A. S. (2022). A neonicotinoid pesticide alters how nectar chemistry affects bees. Functional Ecology, 36(4), 1063-1073.
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Progress 02/15/20 to 02/14/21
Outputs
Target Audience:Our primary has been the research community, whom we interfaced with through attending virtual conferences and preparing research for submission to peer-reviewed journals. A secondary audience has been members of the public and beekeepers, who we have reached via being interviewed for a podcast, and presenting a talk to the Marin County Beekeepers Association. Changes/Problems:COVID has forced us to approach some of our questions from different perspectives, for example writing reviews/meta-analyses during work-at-home restrictions, postponing intensive labwork, and focusing opportunistically on tractable dimensions of our driving questions that we felt have the highest potential for contributing new knowledge (e.g. exploring how floral microbial communities that help shape the nutritional value of nectar and pollen are potentially altered by pesticides). The sensory physiology portion of our grant research has been the most delayed by the events of the past year. We are hopeful we can still make some progress in this area if time allows, and plan to seek a no-cost extension in part to do so. What opportunities for training and professional development has the project provided?Postdoctoral researcher Sarah Richman presented three talks about her work on the interactive effects of neonicotinoid pesticides and nectar chemistry, at the 2020 Botany meeting (virtual), 2020 Ecological Society of America meeting (virtual) and 2020 Entomological society of America meeting (virtual). This project has involved numerous undergraduates in data collection, and ultimately two of these students (Denyse Marquez Sanchez and Sharon Miller) became co-authors on the resulting manuscript. Both of these students are members of groups traditionally underrepresented in the sciences, and through their involvement, they have gotten hands-on experience with troubleshooting protocols, interpreting results, and preparing results for publication. Sharon Miller was recently accepted to a graduate program in Entomology at Michigan State. PhD student Anna Tatarko mentored one undergraduate student (Ramneek Dhami), who was in the early stages of preparing a poster for the UNR Undergraduate Poster Session, when it was unfortunately cancelled due to COVID. Anna also prepared a dissertation proposal and NIFA Predoctoral Fellowship application that builds on some of the themes of the grant, exploring how agricultural chemicals alter floral and bee microbial communities. More broadly, PhD student Anna Tatarko had previously no experience with electrophysiological assays, and has had the chance to train extensively under Dr. Mathew's mentorship. Likewise, Dr. Sarah Richman joined the lab with limited behavioral/lab-based experience, and has had the chance to run two lab-based projects involving physiology and behavior. Additionally, she developed a collaboration with UNR Biology Associate Professor Angela Smilanich to design and complete the immune component of her study into the combined effects of imidacloprid and nectar secondary compounds on bee performance. Through this, she has gained hands-on experience in insect physiology techniques. Collaborating on a meta-analysis was another opportunity to gain a specific new set of analytic skills, and to network with other early career researchers doing cutting-edge work on the sublethal effects of pesticides on bees. How have the results been disseminated to communities of interest?In addition to Postdoctoral researcher Sarah Richman presenting three talks at virtual conferences (Botany, Entomological Society of America, Ecological Society of America) PI Leonard shared research as an invited speaker for the Marin County Beekeepers Association in May 2020 (audience ~60). Unfortunately, our plans to participate in public events (like our annual Pollinator Festival, held as part of UNR's Museum Day community open house) were curtailed by the pandemic. When we found opportunities to connect with interested audiences (e.g. PhD student Anna Tatarko was interviewed for a science-themed podcast, "Science! With Friends"), we did so. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Establish how neonicotinoids affect foragers' ability to discriminate among floral rewards that vary in composition using taste and scent. A final behavioral experiment to wrap up PhD student Anna Tatarko's dissertation chapter on how neonicotinoids impact insect olfaction is planned for Fall 2021; we plan to write up this project and submit it for publication in early 2022. We will continue to shepherd Postdoctoral Researcher Sarah Richman's manuscripts through the process of peer review (one has been resubmitted with revisions to Oecologia, one resubmitted with revisions to Ecology Letters, and one will be submitted shortly to PNAS). Objective 2: Explore whether neonicotinoids disrupt perception of chemical signals used to regulate colony-level foraging effort. This effort rests upon getting the electrophysiology rig up and modified for bees. This should be straightforward, but takes troubleshooting that was very much delayed by the pandemic. Hopefully we can continue to make progress on this goal as it seems achievable given time and access. We are preparing a no-cost extension request to allow us to complete the bee electrophysiology work that was disrupted by pandemic shut downs. Objective 3: Establish how neonicotinoid exposure scales up to affect: a) Bombus response to nutritional perturbations: We are still hopeful about this objective, but may need to scale down or rethink our efforts, as intensive behavioral experiments (which are difficult to run without undergraduate help) have been difficult to get going over the past year due to Covid disruptions. b) The nutritional value of wildflowers in agroecosystems. This is an area of pandemic-friendly research (plants can be grown at offsite facilities) where we think we have a chance to make a relatively large impact. Sample collection on an expanded iteration of this project will be complete by the end of Summer 2021, with analyses of samples planned for fall and winter. To complement the manipulative experiment we have planned (wherein some potted plants are dosed with pesticides, and others left as controls), we are considering an observational approach, whereby nectar and pollen samples would be collected from individual plants growing in sites exposed to high vs. low levels of pesticides. This approach has the advantage of (in theory) being a highly feasible way to test the effects of neonicotinoids on floral traits in "real world" settings, but will depend on our ability to locate enough plants of a focal species in different settings (e.g. landscaping, agricultural, pesticide-free conservation areas). PI Leonard plans to explore the feasibility of sampling Penstemon palmeri plants (a weedy species commonly found in a wide variety of settings) across a suburban-rural-agricultural gradient during summer 2021, and if the plants can be located, will collect samples for chemical analysis during the fall.
Impacts
What was accomplished under these goals?
The pandemic greatly affected our progress this year: The campus was closed on 3/18/20: "all non-essential employees will shift to working remotely and on-campus operations have ceased ...Other than operations deemed to be critical in nature, all on-campus research and laboratory operations should be suspended to the greatest extent possible or shifted to alternative, remote operations. We got approval to resume some research activities (largely solo and field-based) at the end of May 2020. Because running behavioral experiments on captive bee colonies requires at least two people to be present (to comply with lab safety guidelines), and trouble-shooting the electroantennagram experiments could not be done in a socially distant manner (they occur in a small room) we were forced to postpone much of the lab-based experiments. We pivoted to work-at-home and field-based projects, doing our best to make progress. ** Objective 1: Establish how neonicotinoids affect foragers' ability to discriminate among floral rewards that vary in composition using taste and scent. Project 1: Assessing how exposure to neonicotinoid pesticides impacts bees' ability to discriminate among nectar solutions. Status: Manuscript in revision at Oecologia. We began this project after realizing it was essential to validate the assumption that our tube-based preference assays would return results qualitatively similar to bees in free-flying assays (bolstering any potential findings of the effects of neonicotinoids on the expression of preferences). We compared two designs commonly used to assay bee foraging preferences and quantified the relative efficiency of each. We also developed a novel high throughput assay whereby bees in preference chambers are given repeated small amounts of each solution to sample, mimicking floral volumes. We used this new assay to compare the sucrose preferences of bees dosed with the neonicotioid imidacloprid vs. control, but found no strong effect. The revisions requested seemed (to us) relatively minor and so we are hopeful the paper is on the path to publication. Offshoot project 1.5: Exploring whether bumblebees' preferences for neonicotinoid solutions are based on taste or post-ingestive feedback, and whether nectar sugar concentration matters. Status: published (Royal Society Open Science). We asked whether the commercial bumblebee species used by US growers, Bombus impatiens, showed similar preferences as have been reported in European species, and whether they held across a varying range of realistic nectar sugar concentrations. We failed to detect any preference for neonicotinoids, either based on gustation or on post-ingestive feedback. We think this publication will be impactful because it suggests that the previous finding of preference relates either to methodological issues with previous research, or that there are species-specific responses to neonicotinoids found in floral nectar. Project 2: Using electrophysiological techniques to explore the impact of neonicotinoids on olfaction. Status: Data collection on electrophysiological portion involving Drosphila complete, planned extension to bees interrupted by pandemic. Co-PI Dennis Mathew has led this effort, which also comprises a chapter in PhD student Anna Tatarko's dissertation research. In order to learn the electrophysiological techniques required to address this question in bees, Anna completed a project to ask this question in Dr. Mathew's system of expertise, Drosophila. Starting on this model system made the most sense for training purposes; however, since it also offered the possibility of achieving much more fine-scale insight into the potential mechanisms of olfactory disruption (e.g. single neuron recordings) this work will stand on its own as a publication (currently in prep). Anna first developed a protocol to dose Drosophila with neonicotinoids, then collected single-neuron recordings from the antennae of flies exposed to a sublethal dose of the neonicotinoid imidacloprid. She then scaled up to look at whole-antenna activity in dosed vs. control flies using electroantennagrams (the technique she plans to also use on bees). We found that imidacloprid alters performance in single olfactory neurons, but may not disrupt antennal function as a whole. A planned behavioral experiment (Fall 2021) will strengthen this chapter of Anna's dissertation research. The project as a whole was greatly affected by the COVID research shut down (PI Leonard was in the process of adapting Co-PI Mathew's electrophysiology setup for use on bees when research was halted due to the pandemic. Though delayed, we are optimistic about submitting a publication on the Drosophila findings Winter 21-22. Project 3: How does a diet of realistic secondary nectar metabolites impact the performance of bees given an acute neonicotinoid exposure? Status: Manuscript complete. Inspired by her work exploring how neonicotinoid exposure impacts bees' preferences for nectar solutions (Project 1) Dr. Sarah Richman completed a second lab-based project looking at how nutrition interacts with neonicotinoids to impact behavior and health. She maintained bees on plain sucrose or sucrose containing a naturally-occurring nectar chemical: caffeine (in Citrus nectar); thymol, (in Thymus nectar and used against varroa mites in honeybees); or digoxin (in Digitalis nectar). Half of the bees within a diet treatment were given an acute exposure to the neonicotinoid imidacloprid, and we measured several outcomes to explore how pesticide exposure works alongside nutrition to affect survival and health. This paper will be impactful because nearly all work exploring the impacts of pesticides on bee health present them in a simple sucrose solution. By adding in nutritionally realistic dietary compounds, we gain insight into how well using nutritionally simplified sugar water as "nectar" estimates the impacts of these pesticides on bees. We uncovered patterns of effects on bee physiology, behavior, and survival that depended on phytochemical identity, and in many cases, show that adding in realistic nectar chemistry can alter our understanding of the effects of a neonoicotinoid on bees. Because we think these findings will interest a broad audience, we plan to submit this paper (target journal: PNAS) by 6/1/21. Objective 2 and 3a: Progress depends upon the modification to Co-PI Dennis Mathew's electrophysiology setup, both of which have been delayed by the pandemic. However, adapting to pandemic work-at-home conditions, Postdoctoral Sarah Richman was invited to co-author a meta-analysis regarding the effects of neonicotinoid pesticides on non-Apis bees, which is in line broadly with these objectives. The meta-analysis is currently in revision at Ecology Letters, and involves data extracted from 53 papers (212 effects sizes) focusing on Bombus and Osmia. Key findings include a pattern whereby neonicotinoid exposure negatively affected reproductive output across all bees and impaired bumblebee colony growth and foraging. Neonicotinoids also reduced bumblebee individual development but had no effect onOsmiadevelopment. As discussed in last year's progress report, we made some progress on a line of research to characterize not only how neonicotinoids impact the macronutrients available in pollen and nectar, but also the microbial communities found there that are consumed by bees. These microbes themselves can change the nutritional content and volatile cues associated with nectar and pollen. Invited to contribute to a special issue of Current Opinion in Insect Science, on the topic of floral rewards, microbes, and pollinator behavior, we took advantage of this opportunity to share some of our thoughts regarding how nectar and pollen microbes might alter the nutritional value of floral rewards, with possible consequences for bee foraging and plant performance.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Francis, J. S., Tatarko, A. R., Richman, S. K., Vaudo, A. D., & Leonard, A. S. (2020). Microbes and pollinator behavior in the floral marketplace. Current Opinion in Insect Science. 44: 16-22.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Richman, S.K., Muth, F. and Leonard, A.S. Measuring foraging preferences in bees: A comparison of popular laboratory methods and a test for sucrose preferences under neonicotinoid exposure. In revision at Oecologia.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Field-realistic neonicotinoid exposure has sub-lethal effects on non-Apis bees: A meta-analysis. Siviter, H., Richman, S.K., and Felicity Muth. In revision at Ecology Letters.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
Nectar secondary metabolites mediate bees physiological and behavioral responses to pesticides. Ecological Society of America Meeting, August 2020 (Virtual). Richman, S.K., Maalouf, I. M., Smilanich, A.M and Leonard, A.S.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
Nectar secondary metabolites and pesticide exposure synergistically affect pollinator survival, physiology and behavior. Entomological Society of America meeting (virtual) November 2020. Richman,S.K., Maalouf, I.M., Smilanich, A.M. and Leonard, A.S.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
Nectar secondary metabolites and pesticide exposure synergistically affect pollinator physiology and behavior. Botany 2020 meeting (virtual). July 2020. Richman,S.K., Maalouf, I.M., Smilanich, A.M. and Leonard, A.S.
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Progress 02/15/19 to 02/14/20
Outputs
Target Audience:Our primary has been the research community, whom we interfaced with through attending conferences (N = 4), giving invited seminars (N=1) and preparing research for submission to peer-reviewed journals (2 papers published or in press). A secondary audience has been members of the public in Northern Nevada, whom we have reached via a public open house in the UNR Pollinator Garden (May 2019). Changes/Problems:PI Leonard being out for a semester on maternity leave led to some predictable delays, but all things considered, we feel good about our progress, and the initiative grant personnel showed in working independently. Just as PI Leonard came back to campus to delve into a sabbatical's worth of research effort, the COVID-19 pandemic shuttered all research activities on campus. We thus plan to intiate a formal request for a no-cost extension. All experiments that are ongoing seem to us to be squarely in line with our proposed objectives. What we reported last year still stands: in a few cases, we realized that investing a bit more in foundational work seemed worthwhile. Thus, while we didn't anticipate collecting data for a publication on the neonicotinoid-olfaction connection in Drosophila, we realized that this just made the most sense for the training of PhD student Anna Tatarko (who had no previous experience with electrophysiology). Likewise, when approaching the design of our high throughput tube-based preference assays, we realized that it made sense to invest a bit of time refining our approach to how we measure preference in neonicotinoid-dosed bees. That way, we can be certain that any effects we report relate to the direct effects of the pesticide on preference, rather than, for example, bees' overall foraging motivation or motor system deficits. Thinking ahead, especially in light of the challenges of social distancing and uncertainty regarding campus closures, it strikes us as strategic to focus on the elements of the grant that involve relatively short-term experiments or those for which we could pause efforts and not lose large amounts of data. The electrophysiology-based experiments represent one category of this work, and are top priority once we regain lab access. A second line of research which we feel is in line with the grant objectives but which takes them in a new direction, would be to expand our Objective 3 (impacts of neonicotinoids on plants) to characterize not only how neonicotinoids impact the macronutrients available in pollen and nectar, but also the microbial communities found there that are consumed by bees. These microbes themselves can change the nutritional content and volatile cues associated with floral nectar, and are increasingly recognized as nutritionally critical component of pollen stores (e.g. Dharampal et al. 2020, Microbial diversity associated with the pollen stores of captive-bred bumble bee colonies Insects and references therein). We have had productive discussions with UC Davis Assistant Professor of Entomology Rachel Vannette, an expert in floral microbial ecology, who agrees that exploring how neonicotinoids impact nutrition via floral microbes could break new ground. Working in collaboration with Dr. Vannette, we could use funds originally budgeted to purchase the RFID setup for behavioral experiments (which now seem logistically challenging to execute in the face of pandemic conditions) to ask how neonicotinoid pesticides alter the microbial communities associated with floral nectar and pollen in addition to their macronutrient composition (as proposed in the grant). In principle, this pivot seems to us to be straightforward to execute in the face of social distancing constraints, and we think might actually be more impactful than the specific experiment (Ob j. 3a/ Exp.4) outlined in the grant, given recent publications that have some overlapping coverage (e.g. Crall et al. 2018, Science "Neonicotinoid exposure disrupts bumblebee nest behavior, social networks, and thermoregulation"; ). However, we would of course welcome input on this (i.e. the inclusion of a microbial component, prioritizing the plant-based projects in Obj. 3b, Exp. 5a and 5b over the behavioral/RIFD based work) from the Program Officer. What opportunities for training and professional development has the project provided?In November 2019, PhD student Anna Tatarko presented her talk, "Impacts of neonicotinoid pesticides on insect olfactory processing", at the Entomological Society of America's annual meeting, and was awarded first place in the Student Competition for the President's Prize in her session. Postdoctoral researcher Sarah Richman traveled alongside Anna to present their grant-funded research as posters the 2019 International Conference on Pollinator Biology Health and Policy (Davis, CA) and also presented a talk, "Consumption of nectar secondary compounds drives differences in survival probability in bumble bee exposed to dietary imidicloprid" at the American Society of Naturalists meeting (Asilomar, CA) and Evolution (Providence, RI) where she was an invited speaker at a symposium on mutualisms in a changing world. More broadly, new PhD student Anna Tatarko had previously no experience with electrophysiological assays, and has had the chance to train extensively under Dr. Mathew's mentorship. Likewise, Dr. Sarah Richman joined the lab with limited behavioral/lab-based experience, and has had the chance to run two lab-based projects involving physiology and behavior. Additionally, she developed a collaboration with UNR Biology Associate Professor Angela Smilanich to design and complete the immune component of her study into the combined effects of imidacloprid and nectar secondary compounds on bee performance. Through this, she has gained hands-on experience in insect physiology techniques. Additionally, Dr. Richman presented a departmental seminar on her research to the UNR Ecology, Evolution, and Conservation Biology program which served as good practice for a future job talk. Finally, we have involved four undergraduate students and one post-grad technician in this research, four of whom have all been mentored by Postdoctoral researcher Sarah Richman, and one mentored by Anna Tatarko. All the undergraduates are female, two are members of groups underrepresented in the sciences (Hispanic and African-American), and two worked towards preparing poster presentations describing elements of the larger experiment they assisted with. How have the results been disseminated to communities of interest?In addition to Dr. Sarah Richman and PhD student Anna Tatarko presenting two posters at the International Conference on Pollinator Biology Health and Policy and one talk at the Entomological Society of America's annual meeting, PI Leonard traveled to Penn State University to give a seminar which described some of our ongoing work. Likewise, Dr. Sarah Richman presented her research on neonicotinoids and nectar compounds at both the Evolution meeting as well as at the American Society of Naturalists meeting. We have also shared our work with the public via an exhibit at the UNR Natural History Museum's Museum Day event in the UNR Pollinator Garden which PI Leonard helped create. Grant personnel exhibited a live bumblebee colony, museum specimens, and facts about bee behavior and nutrition to families (total attendance ~750) attending this community open house event. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Establish how neonicotinoids affect foragers' ability to discriminate among floral rewards that vary in composition using taste and scent. The next steps for the unpublished Projects (1, 2 and 3) is to prepare our findings for publication and share them at international conferences. Specifically, postdoctoral Researcher Sarah Richman plans to share her Project 3 findings at the August 2020 (virtual) meeting of the Ecological Society of America; if possible, we would like to share work at the 2020 Entomological Society meeting if it occurs, likely virtually. PI Leonard is hopeful that her work (and that of graduate student Anna Tatarko) moving the bee electrophysiology forward can continue during Summer 2020, but we will stay flexible given the pandemic uncertainty. It is in large part due to the pandemic delay, combined with PI Leonard's parental leave that we plan to request a no-cost extension to complete this critical aspect of Objective 1. Objective 2: Explore whether neonicotinoids disrupt perception of chemical signals used to regulate colony-level foraging effort. As noted above, this effort rests upon getting the electrophysiology rig up and modified for bees. This should be straightforward, but takes troubleshooting and we are currently not allowed on campus due to the pandemic. Hopefully we can continue to make progress on this goal as it seems achievable given time and access. a) Bombus response to nutritional perturbations: Investigating whether neonicotinoid-exposed colonies show an impaired ability to respond to nutritional perturbations, and whether such impairments result in performance differences, measured via colony growth: this effort will likely be led by Dr. Richman, who is interested in moving in this direction (potentially involving colonies of our local Bombus vosnesenskii) after she completes her two ongoing projects described above in Objective 1. We are still hopeful about this objective, but given the current restrictions on lab-based research, we may need to scale down or rethink our efforts, as intensive behavioral experiments (which are difficult to run without undergraduate help-- for logistics and safety, people must work in teams which is challenging with social distancing) may or may not be feasible when the university reopens for research operations. See note in "Changes/Problems" section. b) The nutritional value of wildflowers in agroecosystems. This is an area of pandemic-friendly research (plants can be grown at offsite facilities) where we think we have a chance to make a relatively large impact. PI Leonard is currently running an experiment on two species of Penstemon and plans to scale it up to consider more than one neonicotinoid and additional floral traits (e.g. potentially nectar microbial communities, see note in "Changes/Problems" section). Sample collection on this initial run will be complete by the end of Summer 2020, with analyses of samples planned for fall 2020 (assuming on campus research activities are allowed).
Impacts
What was accomplished under these goals?
PI Leonard was on maternity leave from June-Dec, but was able to help move projects forward during the start of her sabbatical semester (Dec-Feb for this report). Objective 1: Establish how neonicotinoids affect foragers' ability to discriminate among floral rewards that vary in composition using taste and scent. Postdoctoral Researcher, Sarah Richman, and PhD student Anna Tatarko have focused on this question using both behavioral assays and electrophysiology. Their findings have dovetailed with PI Leonard's work which has uncovered unexpectedly complementary findings on the impact of neonicotinoids on olfactory processing. Project 1: Assessing how exposure to neonicotinoid pesticides impacts bees' ability to discriminate among nectar solutions varying in composition. Status: Manuscript complete. Postdoctoral Researcher Sarah Richman has led this project, in collaboration with PI Leonard and collaborator Dr. Felicity Muth. Dr. Richman is close to submitting a manuscript in which she compared three different preference assays and explored whether neonicotinoid consumption altered bees' sucrose preferences. We began this project after realizing it was essential to validate the assumption that our tube-based preference assays would return results qualitatively similar to bees in free-flying assays (bolstering any potential findings of the effects of neonicotinoids on the expression of preferences). We compared two designs commonly used to assay bee foraging preferences (free-flying assays and ad libitum feeding assays in preference tubes), and quantified the relative efficiency of each (e.g. by comparing person-hours per data point). We also developed a novel high throughput assay (the Restricted Volume Preference Assay RVPA) whereby bees in preference chambers are given repeated small amounts of each solution to sample, mimicking floral volumes. This technique represents an advance over ad libitum feeding assays because bees do not satiate before having the opportunity to sample options-- instead, RVPA returns results that, compared to ad libitum feeding assays more closely match the more realistic (but inefficient) free-flying preference assay. We used RVPA to compare the sucrose preferences of bees dosed with the neonicotioid imidacloprid vs. control, but found no strong effect. We aim to have this manuscript submitted to Methods in Ecology and Evolution by 6/1/20. Offshoot project 1.5: Exploring whether bumblebees' preferences for neonicotinoid solutions are based on taste or post-ingestive feedback, and whether nectar sugar concentration matters. Status: in press (Royal Society Open Science). At the intersection of neonicotinoids and bee nutrition, one high impact finding that many people are familiar with is that of Kessler et al. 2015 Nature who showed that the European bumblebee, Bombus terrestris, prefers sucrose solutions containing neonicotinoids. We asked whether the commercial bumblebee species used by US growers, Bombus impatiens, showed similar preferences, and whether they held across a varying range of realistic nectar sugar concentrations. We failed to detect any preference for neonicotinoids, either based on gustation or on post-ingestive feedback. We think this publication will be impactful because it suggests that the previous finding of preference relates either to methodological issues with the previous study, or that there are species-specific responses to neonicotinoids found in floral nectar. Project 2: Using electrophysiological techniques to explore the impact of neonicotinoids on olfaction. Status: Data collection complete. Co-PI Dennis Mathew has led this effort, which also comprises a chapter in PhD student Anna Tatarko's dissertation research. In order to learn the electrophysiological techniques required to address this question in bees, Anna completed a project to ask this question in Dr. Mathew's system of expertise, Drosophila. Starting on this model system made the most sense for training purposes; however, since it also offered the possibility of achieving much more fine-scale insight into the potential mechanisms of olfactory disruption (e.g. single neuron recordings) this work will stand on its own as a publication (currently in prep). Anna first developed a protocol to dose Drosophila with neonicotinoids, then collected single-neuron recordings from the antennae of flies exposed to a sublethal dose of the neonicotinoid imidacloprid. She then scaled up to look at whole-antenna activity in dosed vs. control flies using electroantennagrams (the technique she plans to also use on bees). Preliminary analyses suggest that imidacloprid alters performance in single olfactory neurons, but may not disrupt antennal function as a whole. PI Leonard was in the process of adapting Co-PI Mathew's electrophysiology setup for use on bees when research was halted due to the pandemic. Offshoot project 2.5: Assessing how acute exposure to neonicotinoids alters olfactory performance using learning assays. Status: Published (Biology Letters). PI Leonard collaborated with Dr. Felicity Muth to explore the relative impact of neonicotinoid exposure on visual vs. olfactory learning in bees. This work encouraged us to expand the electrophysiological work described above. Namely, we found that when we directly compared bees' ability to learn color vs. scent from multimodal "flowers" (in a lab setting), olfactory, but not visual, learning appeared to be impaired. This is consistent with olfactory processing being disrupted (as will be followed up by the electrophysiological work described above). Project 3: How does a diet of realistic secondary nectar metabolites impact the performance of bees given an acute neonicotinoid exposure? Status: Data collection complete. Inspired by her work exploring how neonicotinoid exposure impacts bees' preferences for nectar solutions (Project 1) Dr. Sarah Richman completed a second lab-based project looking at how nutrition interacts with neonicotinoids to impact behavior and health. She maintained bees on plain sucrose or sucrose containing a naturally-occurring nectar chemical: caffeine (in Citrus nectar); thymol, (in Thymus nectar and used against varroa mites in honeybees); or digoxin (in Digitalis nectar). Half of the bees within a diet treatment were given an acute exposure to the neonicotinoid imidacloprid, and we measured several outcomes to explore how pesticide exposure works alongside nutrition to affect survival and health (sucrose consumption; activity levels; immune function). This paper will be impactful because nearly all work exploring the effects of pesticides on bee health present them in a simple sucrose solution. By adding in nutritionally realistic dietary compounds, we gain insight into how well using nutritionally simplified sugar water as "nectar" estimates the effects of these pesticides on bees. Objective 2 and 3a: Exploring the physiological and behavioral effects of neonicotinoids on the social regulation of foraging. Progress depends upon the modification to Co-PI Dennis Mathew's electrophysiology setup, both of which have been delayed by PI Leonard's parental leave and the pandemic. Objective 3b: Establish how neonicotinoid exposure scales up to affect the nutritional value of wildflowers in agroecosystems. PhD student Anna Tatarko began a study comparing clover seedlings during Fall 2019, half of which were dosed with the neonicotinoid imidacloprid, and half of which were left as control. Data analysis is ongoing on this project, which served as a pilot run for a larger effort planned for Spring 2020. PI Leonard has taken on this research objective as a sabbatical project Spring semester 2020, and plans to compare floral traits, reward chemistry, and ecological interactions with herbivores and pollinators in two species of Penstemon, half of which will be dosed with the neonicotinoid imidacloprid and half left as a control.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Tatarko, A., Mathew, D., Leonard, A., 2019 International Conference on Pollinator Biology Health and Policy, "Impacts of neonicotinoid pesticides on insect olfactory processing", UC Davis. (July 17, 2019).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Richman, S., Smilanich, A., Marquez Sanchez, D. S., Leonard, A., Evolution 2019, "Consumption of nectar secondary compounds drives differences in survival probability in bumble bee exposed to dietary imidicloprid", Society for the Study of Evolution, Providence (RI) (June 21, 2019).
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Muth, F., Francis, J.S., and Leonard, A.S. 2019. Modality-specific impairment of learning by a neonicotinoid pesticide. Biology Letters. 15: 20190359. https://doi.org/10.1098/rsbl.2019.0359.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2020
Citation:
Muth, F., Gaxiola, R.L. and Leonard, A.S. In press. No evidence for neonicotinoid preferences in the bumblebee Bombus impatiens. Royal Society Open Science.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Tatarko, A., Mathew, D., Leonard, A., Entomological Society of America Annual Meeting, "Impacts of neonicotinoid pesticides on insect olfactory processing", Entomological Society of America. (November 15, 2019).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Richman, S., Smilanich, A., Marquez Sanchez, D. S., Leonard, A., 2019 International Conference on Pollinator Biology Health and Policy "Consumption of nectar secondary compounds drives differences in survival probability in bumble bee exposed to dietary imidicloprid", UC Davis. (July 17, 2019).
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Progress 02/15/18 to 02/14/19
Outputs
Target Audience:Our primary target during this first year of the grant has been the research community, whom we interfaced with through attending an international conference and preparing research for submission to peer-reviewed journals. A secondary audience has been members of the public in Northern Nevada, whom we have reached out to via a variety of different public lecture opportunities (e.g. public lectures at a local brewery about bee health; UNR Natural History Museum exhibit; presenting a talk at the Nevada Beekeepers Association meeting). Changes/Problems:We have no major changes to report; all experiments that are ongoing seem to us to be squarely in line with our proposed objectives. In a few cases, we realized that investing a bit more in foundational work to have our new graduate student and postdoc master the skills they will need to answer the ultimate question seemed like a worthy investment. Thus, while we didn't anticipate collecting data that could lead to a publication on the neonicotinoid-olfaction connection in Drosophila, we realized that this just made the most sense for the training of PhD student Anna Tatarko (who came in without previous experience in electrophysiology). Likewise, when approaching the design of our high throughput tube-based preference assays, we realized that it made sense to invest a bit of time refining our approach to how we measure preference in neonicotinoid-dosed bees. That way, we can be certain that any effects we report relate to the direct effects of the pesticide on preference, rather than, for example, bees' overall foraging motivation or motor system performance. Finally, a change that is on the horizon that we are planning for relates to PI Leonard's Family/Medical Leave during Summer 2019. We have discussed how this will affect communication about ongoing research and products with team members; while some disruption is going to be inevitable, we are hopeful that we can continue to make progress on all fronts. What opportunities for training and professional development has the project provided?In November 2018, PI Leonard and Postdoctoral Researcher Sarah Richman travelled to the 2018 Entomological Society of America Meeting in Vancouver, Canada. At this meeting, we attended (among others) a symposium on "Understanding and Mitigating the Risks of Pesticide Exposure for Pollinators and Other Beneficial Insects" which drew international leaders working on the intersection of bee pesticide exposure, nutrition, and behavior. In March 2019, Dr. Richman travelled to Davis, CA to visit the labs of Drs. Rachel Vannette and Neal Williams, to assist with and gain skills at rearing Bombus colonies. This enhanced skillset will position her to conduct research on colonies of our local species Bombus vosnesenskii this summer, potentially expanding the taxonomic scope of our work. More broadly, new PhD student Anna Tatarko had previously no experience with electrophysiological assays, and has had the chance to train extensively under Dr. Mathew's mentorship. Likewise, Dr. Sarah Richman joined the lab with limited behavioral/lab-based experience, and has had the chance to run two lab-based projects involving physiology and behavior. Additionally, she has taken a graduate seminar on ecoimmunology, and is engaging in a pedagogy workshop during Spring semester 2019. Finally, we have involved four undergraduate students in this research, who have all been mentored by Postdoctoral researcher Sarah Richman. All four students are female, and two are working towards preparing poster presentations describing elements of the larger experiment they assisted with. How have the results been disseminated to communities of interest?Since we are still in the data collection phase for most of our projects, results have not yet been disseminated widely (apart from the Scientific Reports paper that is published). However, PI Leonard has led efforts to share information about the kinds of questions we are asking broadly, by: 1) Preparing a talk and being featured as an invited speaker at the Nevada Beekeepers Association annual meeting (Yerington, NV). PI Leonard shared basic information about the complex interaction between nutrition and pesticide exposure on bee health. 2) Engaging in multiple public outreach events, including a Valentine's Day exhibit at the UNR Natural History Museum, which focused on threats to the stability of interactions between plants and pollinators. PI Leonard also participated in a public lecture series held at the Great Basin Brewing Company, "Operation Pollination" during which she highlighted the lab's current work on pesticides. 3) Speaking locally at the UNR Chemical Ecology Center's annual retreat about the goals of this project, an effort that helped identify some of the research synergies described above (e.g. collaboration with Dr. Smilanich). What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Establish how neonicotinoids affect foragers' ability to discriminate among floral rewards that vary in composition using taste and scent. We anticipate that the ongoing Projects 1-3 described above should wrap up data collection during Spring/early Summer 2019, and the next steps for each will be preparing our findings for publication and sharing them at international conferences. Specifically, postdoctoral Researcher Sarah Richman plans to share her findings on the interaction between neonicotinoids and natural nectar chemicals at the July 2019 Evolution Conference, as an invited speaker in the symposium "Origins, stability & benefits of interspecific cooperation in a changing world". PhD student Anna Tatarko will likewise present a poster at the July 2019 International Pollinator Conference (Davis, CA), "Impacts of neonicotinoid pesticides on insect olfactory processing". Objective 2: Explore whether neonicotinoids disrupt perception of chemical signals used to regulate colony-level foraging effort. In order to make progress on this objective, we have arranged to hire a full-time postgraduate research technician, whom we plan to have work closely with PhD Student Anna Tatarko and Dennis Mathew on electrophysiological investigation of how responses to social signals might differ in neonicotinoid-exposed bees. This effort will build on the progress that Dr. Mathew and Anna T. make over the next few months in refining the electroantennagram protocol. Additionally, we envision that the technician will also partly work closely with Dr. Richman on behavioral experiments investigating potential disruptions to chemical communication that may occur in neonicotinoid-exposed colonies. Objective 3: Establish how neonicotinoid exposure scales up to affect: a) Bombus response to nutritional perturbations: Investigating whether neonicotinoid-exposed colonies show an impaired ability to respond to nutritional perturbations, and whether such impairments result in performance differences, measured via colony growth: this effort will likely be led by Dr. Richman, who is interested in moving in this direction (potentially involving colonies of our local Bombus vosnesenskii) after she completes her two ongoing projects described above in Objective 1. b) The nutritional value of wildflowers in agroecosystems. This project is likely to be taken on as a dissertation chapter by a current student in PI Leonard's lab group during the 2019-2020 academic year; the timing seems ideal, as the student recently received a fellowship to work directly with the UNR Chemical Ecology Center, which would streamline analyses of floral rewards and chemistry. Alternately, elements of this objective may be pursued directly by PI Leonard as part of her sabbatical activities during Spring 2020.
Impacts
What was accomplished under these goals?
Objective 1: Establish how neonicotinoids affect foragers' ability to discriminate among floral rewards that vary in composition using taste and scent. Our efforts this past year have largely focused on this Objective, driven by the research interests of our new Postdoctoral Researcher, Sarah Richman, and first-year PhD student Anna Tatarko, who both joined the lab during Fall 2018. Their efforts have begun to tackle this question using both behavioral assays and electrophysiological approaches. Their findings have dovetailed with PI Leonard's ongoing work with collaborator Dr. Felicity Muth, which has uncovered some unexpectedly complementary results about the impact of neonicotinoid pesticides on olfactory processing and preference expression. Here we detail the status of these three ongoing lines of research: Project 1: Assessing how exposure to neonicotinoid pesticides impacts bees' ability to discriminate among nectar solutions varying in composition. Postdoctoral Researcher Sarah Richman has led this project, in collaboration with PI Leonard and collaborator Dr. Felicity Muth. Dr. Richman has finished data collection on a first phase, in which she compared the designs of three preference assays (free-flying vs. ad libitum feeding tubes offered to bees in preference chambers vs. bees in preference chambers given repeated small amounts of each solution to sample, mimicking floral volumes). We felt this comparison was an essential first step, as we realized that establishing that our tube-based preference assays return results qualitatively similar to bees in free-flying assays would bolster the support for any neonicotinoid-related impairments we might discover. Our results so far indicate that a tube-based preference assay that delivers multiple ecologically-realistic (small) volumes of each nectar option returns results that best match the preferences bees show in free-flying assays (which are ideal but low-throughput to run). While we expect that this finding, on its own, will itself be useful to the bee research community, it positions Dr. Richman to complete the second phase of the experiment, looking at how neonic exposure affects the expression of preferences for these nectar chemicals, during Spring 2019. Project 2: Using electrophysiological techniques to explore the impact of neonicotinoids on olfaction. Co-PI Dennis Mathew has led this effort, which also comprises a chapter in new PhD student Anna Tatarko's dissertation research. Anna is halfway through her first semester as an RA working on this project, and so far has been working to gain skills and familiarity with electrophysiological techniques first using Drosophila as a model system. She has spent time refining and troubleshooting a protocol to dose Drosophila with neonicotinoids, and is proceeding to conduct single-neuron recordings from the antennae of flies exposed to a sublethal dose. Starting on this model system made the most sense for training purposes; however, since it offers the possibility of achieving much more fine-scale insight into the potential mechanisms of olfactory disruption, we feel this work will stand on its own as a likely publication. This project should wrap up by the end of Spring 2019 semester, after which Anna will be transferring her new skillset to conduct electroantennagram studies of the effects of neonic exposure on the antennal function of Bombus and local solitary bees. Offshoot project 2.5: As part of a collaboration with a second postdoctoral researcher in the lab, Dr. Felicity Muth, PI Leonard has collaborated on research exploring the relative impact of neonicotinoid exposure on visual vs. olfactory learning in bees. Although motivated by a different question, this work is returning findings that are encouraging us to delve into the electrophysiological work described above. Namely, we have found that while visual (color) discrimination learning is not impaired by acute neonicotinoid exposure (Muth & Leonard, 2019), olfactory learning appears to be impacted specifically (Muth, Francis & Leonard, in prep for submission to Biology Letters). This would be consistent with olfactory processing being disrupted either at the peripheral level (as will be followed up by the electrophysiological work described above) or more centrally (e.g. in the antennal lobe of the bee brain, a prospect that grad student Anna T. may pursue in her dissertation work). Project 3: Inspired by her work exploring how neonicotinoid exposure impact bees' preferences for nectar solutions varying in composition (e.g. sucrose, amino acid composition, secondary metabolites), Dr. Sarah Richman has begun a second lab-based project to ask how exposure to ecologically realistic secondary metabolites in nectar impacts the performance and survival of bees exposed to neonicotinoids. She has begun work asking how bees maintained on alkaloid-rich nectar fare when given an acute dose of a neonicotinoid in terms of survival and immune function. This project takes a slightly different angle on the potentially complex interactions between nectar nutrition and pesticide exposure, and we hope it will be an impactful study, as nearly all work exploring the impacts of neonics on bee health deliver them in a simple sucrose solution. By adding in nectar secondary metabolites, Dr. Richmans' project hopes to assess whether we are estimating these pesticides' impacts on bee survival and physiology accurately in relation to ecologically-realistic nectar chemistries. This project represents a new and exciting collaboration with an insect eco-physiologist at UNR, Dr. Angela Smilanich.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Muth, F. and Leonard, A.S. 2019. Exposure to a neonicotinoid pesticide negatively affects foraging behaviours, but not learning, in free-flying bumblebees. Scientific Reports. 9: 4764. https://doi.org/10.1038/s41598-019-39701-5
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